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Chem. Rev.

2004, 104, 17117 17

The Assignment of Absolute Configuration by NMR

Jose Manuel Seco, Emilio Quinoa, and Ricardo Riguera*
Departamento de Qumica Organica and Unidad de RMN de Biomoleculas Asociada al CSIC, Universidad de Santiago de Compostela,
E-15782, Santiago de Compostela, Spain

Received November 18, 2002

Contents 3.9. Application to Chiral Polyhydroxylated 92

1. Introduction and Scope 17 3.9.1. MPA and 9-AMA 94
2. Methods Based on Chiral Derivatizing Reagents 19 3.9.2. MTPA 96
(CDAs): General Characteristics
4. Methods Based on a Single Derivatization 99
2.1. Description of the Method 19
4.1. Application to R-Chiral Secondary Alcohols 101
2.2. Characteristics of the Auxiliary Reagents 20
4.1.1. 9-AMA Esters: Esterification Shifts 101
2.3. The NMR Spectra of the Derivatives 20
4.1.2. Glycosidation Shifts 103
2.4. Importance of the Conformational Equilibrium 21
4.1.3. Low-Temperature NMR of MPA Esters 105
2.5. Substrates and Derivatives 22
4.1.4. Complexation of MPA Esters with 107
3. Methods Based on Double Derivatization 22 Barium(II)
3.1. Application to R-Chiral Secondary Alcohols 22 4.2. Application to R-Chiral Primary Amines: 108
3.1.1. Methoxytrifluoromethylphenylacetic Acid 22 Complexation of MPA Amides with Barium(II)
(MTPA): Moshers Reagent 5. New Trends 109
3.1.2. Methoxyphenylacetic Acid (MPA) 33 5.1. Hyphenated High-Pressure Liquid 109
3.1.3. 9-Anthrylmethoxyacetic Acid (9-AMA) and 40 Chromatography (HPLC) NMRMS
Related AMAAs Techniques for Automatization and
3.1.4. Other Reagents 45 Microscale Analysis
3.2. Application to -Chiral Primary Alcohols 57 5.2. Resin-Bound Auxiliaries: The Mix and 111
3.2.1. MTPA 58 Shake Method
3.2.2. 9-AMA 61 6. Concluding Remarks: A Critical Assessment 113
3.3. Application to R-Chiral Tertiary Alcohols 63 7. Acknowledgment 114
3.3.1. MTPA 63 8. References 114
3.3.2. Methoxy-(2-naphthyl)acetic Acid (2-NMA) 63
3.3.3. The Fucofuranoside Method 64 1. Introduction and Scope
3.4. Application to R-Chiral Primary Amines 65 The interest in determining the absolute stereo-
3.4.1. MTPA 65 chemistry of a chiral organic compound stems from
3.4.2. MPA 68 the widely recognized fact that the stereochemistry
3.4.3. AMAAs 70 often determines important properties in the chemi-
3.4.4. Boc-phenylglycine (BPG) 70 cal, physical, biological, and pharmaceutical aspects
of the compounds. The need to obtain enantiomeri-
3.4.5. Other Reagents 74
cally pure pharmaceuticals and chemicals generally
3.5. Application to Secondary Amines: MTPA 77 has produced a fantastic growth of fields such as
3.6. Application to R-Chiral Carboxylic Acids 79 asymmetric synthesis, asymmetric catalysis, and
3.6.1. Ethyl 2-(9-anthryl)-2-hydroxyacetate 80 other processes where the availability of simple and
(9-AHA) and Related Aryl Alcohols reliable methods for the determination of enantio-
3.6.2. Arylcyclohexanols 82 meric purity and absolute configuration is a must.
3.6.3. (S)-Methyl Mandelate. 84 Several instrumental methods exist for the deter-
3.6.4. Phenylglycine Dimethyl Amide (PGDA) 85 mination of absolute configuration. The most widely
and Phenylglycine Methyl Ester (PGME) known are X-ray crystallography, followed by chiro-
3.6.5. 1,1-Binaphthalene-8,8-diol (BNDO) 88 optical methods (e.g., circular dichroism1a (CD), opti-
3.7. Application to -Chiral Carboxylic Acids 89 cal rotatory dispersion (ORD), or specific optical
3.7.1. Arylethylamines 89 rotation); however, their use is not devoid of some
3.7.2. PGME 90 inconveniences and limitations related to the equip-
3.8. Application to Chiral Sulfoxides: MPA 92 ment, which is very specific to the method and
requires special training for operation, and to the
sample, which, in the case of X-ray diffraction (XRD),
* Author to whom correspondence should be addressed. E-mail: requires monocrystals of good quality. Other methods
R.R. dedicates this review to the memory of his beloved wife M. include specific rotation,1b infrared (IR) vibrational
Carmen. CD,1c and vibrational Raman optical activity.1d
10.1021/cr000665j CCC: $48.50 2004 American Chemical Society
Published on Web 01/14/2004
18 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Jose Manuel Seco was born in Dodro (La Coruna, Spain) in 1968. He Professor Ricardo Riguera was born in Lugo (Spain) in 1948. He received
received his B.Sc. (1991), M. Sc. (1992), and Ph.D. (1997) degrees in its M. S. degree in Chemistry from the University of Santiago de
Chemistry from the University of Santiago de Compostela, the latter under Compostela in 1971 and the Ph.D. degree in 1973 under the supervision
the supervision of Profs. Ricardo Riguera and Emilio Quinoa. He was of the late Prof. I. Ribas. From 1974 to 1976, he stayed at the University
awarded with the Ph.D. Extraordinary Prize in 1998. In 2001, he became College London as a postdoctoral fellow under the supervision of Prof.
Assistant Professor in Organic Chemistry. His current research interest Peter J. Garratt. After returning to the University of Santiago de
is focused in the development of new reagents and methods for the Compostela, he was appointed Lecturer of Organic Chemistry in 1978
determination of absolute stereochemistry of organic compounds by NMR. and became Full Professor in 1990. As academic, he has served the
university as Chairman of the Department, Dean of the Faculty of
Chemistry, and Vice-Chancellor. He also has authored textbooks for
students. His research interests have involved the fields of bioactive natural
products from terrestrial and marine organisms, in regard to new synthetic
methods and some aspects of medicinal chemistry. In the past few years,
he has focused on the development of methods for determination of the
absolute configuration by NMR.

zation of the substrate whose absolute configuration

is studied is not necessary. The sample (i.e., a pure
enantiomer) is analyzed by NMR in a chiral environ-
ment that is provided by a chiral solvent or by the
addition of a chiral solvating agent2 (CSA) to a
nonchiral standard NMR solvent. In this approach,
there is no covalent linkage between the substrate
and the chiral reagent, and this advantage is the
Emilio Quinoa, a native of Lugo (Spain), obtained his Ph.D. degree (1985) origin of its main limitation: The chiral environment
in Chemistry from the University of Santiago de Compostela, for work on produces very small differences in chemical shifts for
marine natural products from the Galician Coast, under the supervision
of Prof. Ricardo Riguera. From 1985 to 1988, he worked as a postdoctoral the two enantiomers whose NMR are very similar;
fellow with Prof. Phillip Crews at the University of California, Santa Cruz, many times, the two enantiomers must be available
where he participated in the study of bioactive metabolites from marine for comparison and no clear-cut correlations between
organisms of tropical waters. He returned to Santa Cruz in 1989 and the absolute configuration and the NMR spectra can
1990 as Visiting Associate Professor. After his return to the University of be established. For those reasons, the usefulness of
Santiago, he became Associate Professor in Organic Chemistry in 1989 this method is practically restricted to the determi-
and Full Professor in 1998. His current research interests include marine
natural products, solid-phase synthesis, and the stereochemistry of organic nation of enantiomeric purity.3
compounds. Among other recognition, he has received the Real Academia A clear distintinction between those two objectives
Gallega de Ciencias Award. should be established at this point. For the enantio-
meric purity measurement, we usually work with a
Recently, different approaches to the problem of mixture of both enantiomers in unknown ratio and
determining absolute configuration have emerged, just need to obtain separate signals in NMR (or peaks
based on NMR spectroscopy. These techniques are in high-pressure liquid chromatography (HPLC), gas-
very appealing, because of the undoubted advan- liquid chromatography (GLC), or similar techniques)
tages, which include the following: (a) the instru- that are amenable to quantification. Determination
ment is available in most laboratories; (b) an in-depth of the absolute configuration is a much more difficult
understanding of the fundamentals of the method is task, because it is performed on a single enantiomer
not necessary to apply this method; (c) a small and, usually, we have no other enatiomer for com-
amount of sample is needed, and this can be recov- parison; in addition, even if we had both and could
ered; and (d) because the analysis is conducted in produce different spectra, we must have criteria to
solution, it is applicable to both solid and liquid assign the absolute configuration separately for each
samples. enantiomer, not just its ratio.
Two general approaches are known. The first The second approach involves derivatization of the
approach involves those procedures where derivati- substrate (i.e., a pure enantiomer) with the two
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 19

Figure 1.

enantiomers of a chiral derivatizing agent4 (CDA), 2. Methods Based on Chiral Derivatizing

producing two diasteromeric derivatives. In this case, Reagents (CDAs): General Characteristics
the chiral environment is provided by the auxiliary
reagent; the association with the substrate is covalent 2.1. Description of the Method
and leads to much-greater differences in chemical
shifts than those obtained when CSAs are used. All the NMR-based methods for the determination
Although, in certain cases, a combination of the two of absolute configuration require the transformation
approaches has been used5 (i.e., the addition of of the chiral substrate to two different species that
lanthanide shift reagents to the CDA derivatives), the can be differentiated by NMR spectroscopy (i.e.,
derivatization with chiral auxiliary reagents is, by diastereoisomers or conformers). This is achieved by
far, the method of choice for the assignment of derivatization of the substrate with the CDA, fol-
absolute configuration by NMR and constitutes the lowed by comparison of the 1H NMR spectra of those
main object of this review. two species. Two main procedures exist, depending
on the number of derivates that should be prepared:
Many efforts to develop CDAs that are useful to
(a) those in which the substrate (a pure enantiomer)
assign the absolute configuration of different sub-
is separately derivatized with the two enantiomers
strates have been described;6 however, since its of the chiral auxiliary (Figure 1) and (b) those that
implementation in 1973, the so-known Moshers require the preparation of a single derivative, using
method, which uses MTPA (methoxytrifluorphenyl- one enantiomer of a chiral auxiliary only (Figure 2).
acetic acid) as the reagent in its original description, In the first case, the chiral substrate [(?)-A] is
has been the most successful, giving way in its coupled separately with the two enantiomers of the
evolution to many new and more-efficient reagents chiral auxiliary ((R)- and (S)-CDA), and the NMR
that are useful for different substrates and are based spectra of the resulting diastereoisomers ((?)-A-(R)-
on the same principle.
CDA and (?)-A-(S)-CDA) are compared.
The importance of this method can be evaluated In the second possibility, the chiral substrate ((?)-
from the ever-increasing number of papers where it A) is combined with only one enantiomer of the chiral
is mentioned; however, in contrast to the determi- auxiliary (either (R)- or (S)-CDA). In this way, one
nation of enantiomeric purity by NMR, which has diastereoisomer is obtained and the spectrum of this
been widely covered in the literature,3 exhaustive compound is recorded. A certain conformational
reviews of this topic are absent. This absence of a change is then induced (e.g., by a change in temper-
comprehensive coverage means that the researcher ature of the probe or by formation of a complex) and
often uses inadequate reagents for the problem being a second spectrum is taken (Figure 2). Analysis of
addressed or performs a nonrigorous evaluation of the differences and comparison with the conforma-
the experimental data, making the assignment of tional changes allows the configuration of the sub-
absolute configuration a risky exercise. strate to be correlated with that of the reagent.
In this review, we will cover the principles and Another option that does not require the aforemen-
foundations of the use of CDAs and the practical tioned conformational change relies simply on com-
aspects that are most relevant for its application to parison of the spectrum of the derivative with that
the different substrates. It is intended that this of the substrate prior to derivatization.
information will be of particular use to researchers Regardless of the chosen procedure, two different
who are not necessarily specialists in this field, and spectra always must be obtained for comparison. The
the purpose of this study is to provide a critical view assignment of configuration is based on the use of
of the methods available. the NMR to correlate the absolute stereochemistry
20 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 2.

of the chiral center of the auxiliary (CR; configuration ensure that the chemical shifts of L1 and L2 are
known) with that of the substrate (C(1), configura- different in the two species (diastereoisomers or
tion unknown). To this end, the changes in the conformers) that are used for the determination of
chemical shifts of the substituents of the asymmetric the conformation.
carbon of the substrate (L1 and L2) in the two For example, in the case of MPA, R1 is a methoxy
derivatives (or conformational species) are consid- group, Z is a carboxylic acid, and Y is a phenyl group
ered. (Figure 4).
These differences in the chemical shifts are repre-
sented by , and it is the sign of this parameter
(+ or -) that provides information about the config- 2.3. The NMR Spectra of the Derivatives
uration. For a particular substituent (e.g., L1), is As we have said, comparison of the NMR spectra
defined as the difference of chemical shifts of a given of the two derivatives and evaluation of the differ-
signal of the substituent (L1) in the two spectra ences in the chemical shifts of the signals for the
under consideration (diastereoisomers or conformers, substituents L1 and L2 (i.e., RS) must be performed.
depending on the routine chosen). As will be de- The signs of those parameters contain the informa-
scribed in this paper, there is a variety of ways to tion necessary for the configurational assignment,
express the differences in chemical shifts, their because they indicate the relative position of L1/L2,
significance, and their characteristics. For example, with respect to the anisotropic group Y; therefore, the
in Moshers method, a chiral secondary alcohol (i.e.,
observed differences must not be uncertain and must
2-pentanol; see Figure 3) is derivatized with the two
be clearly predictable.
enantiomers of an arylmethoxyacetic acid (AMAA)
i.e., (R)- and (S)-R-methoxy-R-phenylacetic acid (MPA), In the methods that are based on the formation of
the 1H NMR spectra of the two derivatives are two derivatives, it is important that both diastereo-
recorded, and the RS value is calculated for the isomers show a certain preference for a particular
substituents L1 (RSL1) and L2 (RSL2). In each conformation (NMR-significant conformer) indepen-
case, RS is the difference between the chemical dent of the particular substrate, and the Y group
shift in the (R)-MPA ester derivative ((R)) and in must project its magnetic anisotropy in a selective
the (S)-MPA derivative [(S)] (see Figure 3). way, i.e., on L1 in one derivative and on L2 in the
2.2. Characteristics of the Auxiliary Reagents For example, in the MPA esters (Figure 3), it is
In general, the structure of the chiral auxiliary assumed that the representative conformer in terms
reagent (B) must incorporate groups with specific of NMR is the one in which the methoxy, carbonyl,
functions: and C(1)H groups are situated in the same plane.
(a) A polar or bulky group (R1) to fix a particular In this way, in the (R)-MPA derivative, the phenyl
conformation. group shields L1 (-CH2CH2CH3), whereas, in the (S)-
(b) A functional group (Z) (e.g., carboxylic acid) that MPA derivative, the shielded group is L2 (-CH3). The
provides a site for covalent attachment of the sub- comparison of both spectra leads to RSL1 < 0 and
strate. RSL2 > 0 (-0.14, -0.23, -0.09, and +0.013 ppm,
(c) A group (Y) that is able to produce an efficient respectively).
and space-oriented anisotropic effect (e.g., aromatic, If the absolute configuration of the alcohol were the
carbonyl) that selectively affects substituents L1 and opposite, the signs of RS will also be reversed (i.e.,
L2 of the substrate. This shielding/deshielding will RSL1 > 0, RSL2 < 0).
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 21

Figure 3.

the resulting NMR chemical shift. Therefore, to

correlate the information gained from the NMR
spectra (the values) with the absolute configura-
tion, a detailed understanding of the structure and
energy of the main conformers, as well as the
strength and direction of the anisotropic effect of Y
on L1 and L2 in each conformation is necessary.
Naturally, for the direct use of the method, models
that simplify the equilibrium, showing only the most
representative conformer, are preferred (i.e., sp con-
former for MPA esters; see Figure 5).
Figure 4.
In general, the models described in the literature
have been formulated as empirical rules. That is, the
2.4. Importance of the Conformational NMR behavior of the CDA derivatives from several
Equilibrium substrates of known absolute configuration are ana-
Naturally, the key for the success of these methods lyzed and a conformational model that could explain
lies in the assumption that a certain conformer is the the experimental NMR shifts is devised. In a few
most representative in both derivatives and that it cases, the model proposed is claimed to be supported
remains so when different substrates are derivatized. by XRD data; however, the differences between
In solution, the CDA-derived diastereoisomers exist solution and solid state can be so big that not much
in a conformational equilibrium (Figure 5). This support can come from that.
equilibrium is often very complex, because several It is only in the past decade that extensive studies,
bonds are involved and the final balance has a fundamentally theoretical calculations of structure
substantial influence on the ability of the anisotropic and energy, and experimental dynamic NMR and
group Y to shield/deshield the substituents L1/L2 shielding effect calculations have been performed to
selectively. know the contribution of each conformer and that of
The balance between the relative populations of the average to the NMR shift that is observed.
each conformer and the strength of the effect of Y on This understanding of the fundamentals of the
each conformer will be reflected (as an average) in methods has allowed us to rationally explain not only
22 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 5.

the way in which the CDAs work but also led to the ever since Mosher first reported7,8 its use in 1973.
development of new and more-efficient CDA reagents The procedure starts with the esterification of the
and methodologies giving a plus of reliability to the alcohol with the two enantiomers of MTPA. After the
absolute configuration assignment process that is two diastereomeric esters have been prepared, their
now based on both empirical and theoretical results. NMR spectra are acquired and compared. MTPA is
available both as the acid and the acid chloride, and
2.5. Substrates and Derivatives users must remember that, although derivatization
The ranges of substrates whose configuration can of the alcohol with the (R)-MTPA acid leads to the
be assigned by these methods have increased signifi- corresponding (R)-MTPA ester, the (R)-MTPA chlo-
cantly recently and include primary, secondary, and ride leads to the (S)-MTPA ester, which has led to
tertiary alcohols, diols, carboxylic acids, primary and confusion and some erroneous assignments.9
secondary amines, and sulfoxides. Appropriate chiral In his first study, Mosher used 19F NMR spectros-
auxiliary reagents have been developed for each case copysparticularly, the chemical shifts of the CF3
that produces derivatives such as esters, amides, groupssfor the assignment. In later studies, how-
hemiacetals, phosphonates, etc. This review covers ever, 1H and 13C NMR were utilized, and the chemical
all those chiral substrates, the reagents and methods shifts of the protons and C atoms of the chiral alcohol
most commonly used and attempts to offer a critical under examination were used, respectively. The
assessment of each method. absolute configuration of the substrate is deduced by
interpretation of the signs of values, using certain
empirical models. A description of those models,
3. Methods Based on Double Derivatization classified according to the NMR technique used,
3.1. Application to r-Chiral Secondary Alcohols The Use of 19F NMR. 19F NMR was used
by Mosher to determine the absolute configuration
3.1.1. Methoxytrifluoromethylphenylacetic Acid (MTPA): of secondary alcohols.8a The procedure is based on the
Moshers Reagent anisotropic effect that the carbonyl group of the
auxiliary MTPA exerts on the CF3 group. For this
R-Methoxy-R-trifluoromethyl-R-phenylacetic acid purpose, Mosher assumed that the (R)- and (S)-
(MTPA; see Figure 6) has been the most commonly MTPA esters of secondary alcohols exist in a confor-
used derivatizing reagent for the determination of the
mation in which C(1)H, the carbonyl, and the CF3
absolute configuration of secondary alcohols by NMR
groups are situated in the same plane (Figure 7) in
the derivative where the phenyl ring is opposite to
the least bulky substituent of the alcohol. In the
derivative in which the phenyl ring is situated on the
same side as the most bulky substituent, the copla-
narity is lost and, as a consequence, the CF3 moves
from the deshielding zone to the shielding zone of the
carbonyl group.
When the bulkier substituent (e.g., L1) is on the
same side as the phenyl group, the CF3 resonates at
a higher fieldsbecause of greater shielding (Figure
7b)sthan if L1 were on the side of the methoxy group
Figure 6. (Figure 7a). These differences in chemical shifts are
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 23

Figure 7. Model for the correlation of configuration/NMR for 19F and NMR spectra of MTPA esters.

Figure 8. Models proposed by the Mosher model for (a, b) the assignment of configuration by 1H NMR and (c, d) the
expected sign of SR.

expressed in terms of the parameter SR(19F)CF3, made, regarding the spatial position of L1 and L2,
which is defined as the difference in the chemical with respect to the phenyl group of the MTPA moiety
shift of the trifluoromethyl signal in the (S)-MTPA on the basis of the signs of SR of the substituents.
ester (CF3(S)) and the chemical shift of the same In this case, Mosher assumed that the most repre-
signal in the (R)-MTPA ester (SR(19F)CF3 ) CF3- sentative conformation is that in which C(1)H, the
(S) - CF3(R)). carbonyl group, and the CF3 group are situated in
For example, an alcohol with the configuration the same plane (Figure 8a).
represented in Figure 7, in which L1 is more bulky Accordingly, the protons of substituent L2 are
than L2, gives a negative value of SR(19F)CF3 (<0). shielded by the phenyl ring in the (R)-MTPA ester,
If the configuration of the alcohol were the opposite, whereas those on L1 remain unaffected (Figure 8a).
or the size of the substituent L2 was much greater In the (S)-MTPA derivative, on the other hand, it is
than that of L1, the sign of SR would be positive. L1 and its protons that are shielded while L2 is
An advantage of this procedure lies in the clean- unaffected (Figure 8b). Therefore, the substituent L1
ness of the 19F NMR spectra, which generally only will be more shielded in the (S)-MTPA ester than in
contain signals that are due to the CF3 groups and the (R)-MTPA ester and the substituent L2 will be
eliminates the possibility of overlapping signals that more shielded in the (R)-MTPA than in the (S)-MTPA
often complicate proton spectra. Its application for ester derivative.
the configurational assignment will be discussed later These selective shieldings are expressed using the
in this review. parameter SR, which is defined as the difference The Use of 1H NMR. The use of 1H NMR between the chemical shift of a certain proton in the
spectroscopy is undoubtedly more common for the (S)-MTPA ester and the chemical shift of the same
assignment of the configuration of secondary alco- proton in the (R)-MTPA derivative. All the protons
hols10 than the use of 19F NMR that has been shielded in the (R)-MTPA will present a positive SR
described previously. This approach is based on the value, whereas those shielded in the (S)-MTPA
anisotropic effect that the phenyl group of the chiral derivative will present a negative SR value.
auxiliary MTPA exerts on the substituents (L1/L2) of In the case of the alcohol shown in Figure 8c, for
the alcohol. This effect allows a correlation to be example, the signs for each proton in L1 and L2 are
24 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

as follows:

SRL1 ) L1(S) - L1(R) < 0

SRL2 ) L2(S) - L2(R) > 0

If the configuration of the alcohol shown in Figure

8c were opposite, then the signs of SRL1 and SRL2
would also be opposite (see Figure 8d). An analysis
of the results obtained with this technique, its scope,
and its limitations are included in the corresponding
section of this review. The Use of 13C NMR. This approach for
the assignment of the absolute configuration of the
alcohol is similar to that previously described for 1H
NMR spectroscopy but using the 13C NMR chemical
shifts11 of the substituents L1 and L2 ((C)L1, (C)L2)
in the (S)- and (R)-MTPA esters. In the example
shown in Figure 8c, the substituent that results in
negative signs of SR occupies the place of L1,
whereas the substituent whose C atoms result in a
positive SR value is L2. Scope and Limitations in the Use of
F, 1H, and 13C NMR of MTPA Esters. The NMR
method for assignment of the absolute configuration Figure 9. Selection of compounds that do not conform to
of alcohols using MTPA, as described by Mosher,8d the 19F NMR Mosher model for assignment.12
is entirely empirical, and the models represent no
more than a convenient way to correlate the experi-
mental NMR with the absolute configuration of the
compounds. This means that the quality of the
assignment is indicated by the number and struc-
tural variety of the substrates of known absolute
stereochemistry used as test compounds. In addition,
the NMR data used for assignment should adhere to
several prerequisites:
(a) The SR values must be sufficiently large and
be above the level of experimental error,
(b) The distribution of the signs of the parameter
SR must be uniform for a given substituent (i.e.,
all the protons on the same side of the plane of the
MTPA ester should have the same sign), and, finally,
(c) If the sign of SR is negative for one substituent
(e.g., L1), then the sign of SR for the other sub-
stituent (i.e., L2) must be positive.
Unfortunately, MTPA derivatives frequently do not
seem to comply with those criteria, leading to a loss
of confidence in the assignment that clearly overcome
the alleged advantages of MTPA as an auxiliary
reagent. In this section, we will present a critical
account of the models described, the scope of each
procedure, and its anomalies and limitations.
In regard to using 19F NMR spectroscopy, the
researcher must remember that the assignment is
made on the basis of a single piece of data: SR(19F)- Figure 10. SR values from the 13C (and 1H) NMR
CF3 is the difference in the chemical shifts of the CF3 spectra of different MTPA esters. Anomalous values are
group in the two derivatives. In contrast, the use of underlined.
the proton or C chemical shifts from the two substit- Indeed, many configurations that had been as-
uents of the alcohol involves the recording of many signed on the basis of 19F NMR have since been
signals and allows any anomaly to be easily detected, corrected.12 A selection of these cases is given in
because it will produce a nonhomogeneous distribu- Figure 9.
tion of SR signs. This check is not possible using Apart from that phenomenon, 19F NMR of the
F NMR, because only one data point is obtained; MTPA esters is frequently performed with complete
the data point can be either positive or negative and, success for the enantiomeric excess calculation of
as such, will always give a certain configuration. alcohols.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 25

Figure 11.

Application of 13C NMR for the assignment of remember the following points that greatly limit its
configuration of alcohols presents some advantages application on a general basis:
but also some important limitations. (a) The number of alcohols of known configuration
Although the use of 13C NMR requires a greater (Figure 10) that have been used to test the validity
amount of sample than does 1H NMR, this technique of the correlations is rather small; therefore, the
could be especially interesting in cases where 1H accuracy of the configuration assigned to a certain
NMR is not useful because one of the substituents substrate has some uncertainty.
L1/L2 has no protons. Nevertheless, those wishing to (b) The SR values obtained are very small, in
use it as a general method for assignment should comparison to the 13C chemical shift scale.
26 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 13. (a) Structure of tanabalin (13.1) and SR

Figure 12. R-Aryl-substituted secondary alcohols that values for the MTPA esters; (b) SR values for the MTPA
present anomalies in the SR signs of the -protons (data esters of N-acetyladrenalin.

(c) Nonhomogeneous distributions of the signs of

SR have been obtained for some secondary alcohols
(data underlined in compounds 10.2 and 10.4 in
Figure 10).
The most numerous examples of application of
MTPA for the assignment of configuration of alcohols
have been described using 1H NMR. The method has Figure 14. Structure of quadrangularic acid L and SR
been tested with a fair number of alcohols of known values of the MTPA esters.
absolute configuration; nevertheless, numerous cases
have been described in which the SR values ob- group modifying the conformation of the MTPA
tained from the MTPA esters do not allow a safe esters. However, similar cases are known in which
assignment, in accordance with the criteria discussed this explanation is not applicable, such as sinulari-
previously,13 either because (a) the magnitude of the olide and its epimer 11-episinulariolide,15 which both
shifts is very similar to the experimental error or (b) produce anomalous SR sign distributions and, thus,
the distribution of signs is not consistent. cannot be assigned a configuration with any degree
Both the small shifts and the irregular distribution of certainty by this method.
of signs can lead to erroneous assignment of config- Given the frequency with which MTPA esters lead
uration. This has been demonstrated in certain cases to irregular sign distributions, Kakisawa and co-
by XRD12 or other methods; however, in other cases, workers10,12b,16 proposed a new way to evaluate the
the configuration that is assigned has remained SR data; this methodology is called the Modified
unchecked. Mosher Method. This procedure involves the analy-
For example, the MTPA derivatives of siphlenol sis of all (or the vast majority) of the protons in the
A14a (Figure 11) result in SR values with the same molecule, so that a representative sign for SR of
sign for both substituents (L1 and L2), which is a fact substituents L1 and L2 can then be adopted based on
that makes the assignment impossible, whereas its the majority of the protons for each substituent. In
epimer, epi-sipholenol A14b (Figure 11), gives opposite our opinion, however, the elimination of those reso-
signs for each substituent. Similar situations have nances that produce anomalous signs can only be
been described for other cyclic alcohols12b that give justified if (a) those proton(s) are located at a long
irregular distribution of the signs of SR, including distance from the chiral center and (b) there is a
3-R-cholesterol and friedelan-3-ol, which possess sufficiently large number of protons close to the
axial hydroxyl groups. In contrast, the epimers of asymmetric C atom producing the expected homoge-
these two compounds, cholesterol and friedelan-3R- neous sign distribution.
ol, with equatorial hydroxyl groups give a perfectly Another class of compounds in which irregular SR
homogeneous distribution of signs for SR (Figure signs have been observed is alcohols with aromatic
11). rings in the R-position.17 This situation is illustrated
The anomalous behavior of siphlenol A, 3-R- in Figure 12 by the MTPA esters of the alcohols
cholesterol, and friedelan-3-ol has been explained derived from tautomycin,17a in which irregular SR
in terms of the steric hindrance of the axial hydroxyl values are found exclusively for the -protons (un-
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 27

Figure 15. SR values obtained for the MTPA esters


derlined in Figure 12) whereas the rest of the protons

give regular sign distributions. The authors suggest17a
that the cause of these irregular sign distributions
could be the fact that these protons are affected not
only by the phenyl ring of the MTPA but also by the
aromatic ring at the R-position in the alcohol, which
is a view supported by Molecular Mechanics calcula-
Other examples include tanabalin17b (13.1, Figure
13), which is an insect antifeedant isolated from
Tanacetum balsamita, and N-acetyl L-adrenalin (13.3,
Figure 13).
Irregular distributions in the signs of SR have
also been found in substrates that do not contain Figure 16.
aromatic groups competing with MTPA. Quadran- have no polar groups or neighboring aromatic rings
gularic acid L18 (Figure 14) is one such example that in their structures. Some examples are shown in
produces negative signs for SR of both substituents Figure 15.19
L1 and L2. The fact that the dehydro-derivatives of Many compounds have been reported whose MTPA
these compounds give the correct sign distribution esters produce sign distributions in good agreement
has led to the belief that the presence of a neighbor- with Moshers model, despite the presence of hin-
ing polar group should be responsible for that anoma- dered hydroxyl groups, polar groups, or other aro-
lous sign distribution. matic rings. Their structures are shown in Figures
Unfortunately, the explanation for that incoherence 16,20 17,21 and 18.22
is not so simple, and there are many other compounds One specific class of compounds in which irregular
that produce nonhomogeneous sign distribution but sign distributions are obtained from the MTPA esters
28 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 18.

will be discussed in the section covering polyfunc-

Figure 17.
tional compounds. Special Models for Special Cases. It is
warrants special mention. Those are the polyalcohols obvious that no prediction of configuration is possible
that have been completely derivatized with MTPA. by the method described if one of the substituents of
Examples of such systems include penaresidin A and the substrate has no resonances to examine or if
B,23 pyripyropene A,24 and other compounds25 shown those peaks cannot be clearly distinguished because
in Figure 19. The anomalies observed in these of overlapping.
compounds have been identified as being due to the Alternatives have been proposed26 to overcome
presence of several MTPA units whose effects inter- these limitations in two particular classes of com-
fere with each other. Such crossed effects cause sign pounds.
distributions that bear no relation to Moshers pre- Thus, for fluoro-substituted secondary alcohols
dictions for monoalcohols where only the effect of a such as the 1,2-difluoroallyl alcohols shown in Fig-
single aromatic ring must be considered. The assign- ures 20 and 21, where one substituent contains
ment of the configuration of polyalcohols by NMR protons and the other contains only F atoms, the
spectroscopy must be analyzed in a different way and combined use of 1H and 19F NMR spectroscopy has
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 29

Figure 20.

Figure 21.
Figure 19.
most of the SR signs are in good agreement with
been described.26a 1 19
The H and F NMR spectra of the configuration, some compounds (21.3 and 21.6)
the two MTPA esters are recorded and the SR exhibit signals (underlined in the figure) that are
values calculated for the protons on one substituent inconsistent with their configuration and put a limit
and for the fluoro-substituents of the other substitu- on the validity of the method.
ent. The model proposed assumes that if the config- The purpose of the second alternative described26b
uration of the alcohol is that shown in Figure 20b, is to solve the difficulties presented by overlapping
then the protons in L1 must produce a negative SR signals for L1/L2 in aromatic and heteroaromatic
value, whereas the fluoro-substituents in L2 must alcohols. It is based on the combined use of the
have a positive SR value. When the configuration anisotropic effect caused by the aromatic ring of the
is the opposite, the signs of SR then will be reversed alcohol on the methoxy group of the auxiliary MTPA
(see Figure 20c). and that of the phenyl ring of the auxiliary on the
Figure 21 shows the alcohols of known absolute substituents L1/L2 of the alcohol.
configuration that have been used as support of the According to the author, in the (R)-MTPA ester of
mixed proton-fluorine NMR approach. Although the alcohol shown in Figure 22a, both aryl rings are
30 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 22.

on the same side of the plane of the MTPA, and,

consequently, neither the signals of the MeO nor
those of substituent (R) of the alcohol are affected
by the anisotropy of the rings. For its part, in the
(S)-MTPA ester (Figure 22b), the aromatic groups are
on opposite sides and both the MeO and substituent
(R) are under their shielding influence. Application
of this method requires the derivatization of the
alcohol with the two enantiomers of MTPA and Figure 23. Generation of conformers in MTPA esters and
comparison of their NMR spectra. the most-significant characteristics of each.
Negative SR values for the MeO group result
when the configuration is that indicated in Figure
22, and positive SR values are to be obtained if the real position of the phenyl ring, with respect to the
configuration of the alcohol were the opposite. substituents, and stressed the need to study the
conformational composition and the exact direction
This model has been tested with 50 alcohols of
of the anisotropic effect in MTPA esters.
known absolute configuration. The prediction of the
configuration was correct in 46 cases and incorrect In fact, theoretical calculations on the conforma-
in just 1. In 3 examples, the SR values were tions (molecular mechanics, semiempirical and ab
negligible and did not allow the assignment of con- initio) and shielding/deshielding contributions (aro-
figuration. matic ring current increments) were performed27 in
In summary, the data presented indicates that conjunction with variable-temperature NMR and
MTPA frequently presents severe limitations in its dynamic NMR experiments that allowed the full
use for the assignment of configuration of secondary understanding of the fundamentals of the MTPA
alcohols: small SR values, which are very much action and of the correlation between NMR spectra
affected by the presence of anisotropic groups in the and absolute configuration.27
substrate, and irregular sign distributions. The results indicate that the conformational com-
In addition, the explanations that have been put position of MTPA esters is very complex and that the
forth for these inconsistenciessusually centered on situation cannot be fully represented by the empirical
the particular characteristics of the substratessare model proposed by Mosher (Figure 8). According to
not satisfactory. They are neither general nor do they those studies,27 the main conformational processes
allow the researcher to predict whether this method involve rotation about the CR-CO and CR-Ph bonds
could be used to assign, with complete certainty, the (Figure 23). The first generates two conformers: the
absolute configuration of a new substrate. We will sp conformer, in which the CF3 has a syn-periplanar
show in the next section that the problem is not with disposition, with respect to the carbonyl group, and
the substrate but rather with the characteristics and the ap conformer, in which these groups have an anti-
limitations of the MTPA itself. periplanar disposition. Rotation about the CR-Ph The Mode of Action of MTPA: A Revi- bond generates three conformers that differ in the
sion of the Model. As we have seen, the assignment orientation of the phenyl ring (coplanar with the CR-
of absolute configuration by 1H NMR of MTPA esters CO, CR-CF3, and CR-OMe bonds, respectively).
rests with the detection by NMR of the relative Consideration of their relative energies indicates that
position of substituents L1 and L2, with respect to the only three of the six possible forms (Figure 24) are
phenyl ring of the auxiliary MTPA. It is this spatial really representative: ap1, sp1, and sp2. Their order
information that is contained in the SR signs of the of stability is ap1 > sp1 > sp2, although the energy
substituents. differences are so small (0.40 and 0.63 kcal/mol,
The anomalies observed in the use of MTPA sug- respectively) that they are present in similar popula-
gested that perhaps the empirical model described tions, and, therefore, significant contribution of each
by Mosher in 1973 is not fully representative of the one to the average spectrum mus be expected.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 31

Figure 24.

Conformer ap1 is the most stable conformer and

has the CF3 group anti-periplanar, with respect to
the carbonyl group; its phenyl ring produces a
deshielding effect on the substituents of the alcohol.
The next conformer, in terms of energy, is sp1; it has
the CF3 and carbonyl groups in a syn-periplanar
disposition, as in the empirical Moshers model, and
its phenyl ring produces a shielding effect on the
alcohol part. The third conformer is sp2, and this
conformer also has a syn-periplanar disposition that
results in deshielding on the alcohol substituents.
The conformers are shown in Figure 24.
In accordance with that composition, the final
chemical shift of substituents L1 and L2 in the
average spectrum will be the result of the combined
action of those three similarly populated forms. Each
conformer contributes a different shielding/deshield- Figure 25. Shielding/deshielding effects in the three most-
ing effect; therefore, some cancellations may result, representative conformers of the MTPA esters.
causing small SR values.
For example, in the ester that is derived from (R)- partial cancellation of effects and this results in
MTPA (Figure 25a), substituent L1 is deshielded in substituent L1 being slightly shielded/deshielded or
conformer ap1, whereas substituent L2 is shielded in remaining unaffected by the phenyl ring of the
conformer sp1 and deshielded in conformer sp2. reagent.
Therefore, the net effect on substituent L2 will range The final results for substituent L1 will be deshield-
from slight shielding/deshielding to a mutual cancel- ing in the ester-derived form (R)-MTPA, and either
lation of effects. slightly shielding or just a null effect in the (S)-MTPA
For its part, in an ester that is derived from (S)- derivative. Substituent L2, on the other hand, is
MTPA (Figure 25b), substituent L2 is deshielded in deshielded in the (S)-MTPA derivative but only
conformer ap1, whereas substituent L1 is shielded in slightly shielded or unaffected in the (R)-MTPA
conformer sp1 but deshielded in conformer sp2. As derivative. Negative signs for SR are then expected
in the previous example, the net result is a total or for all the protons on substituent L1 and positive
32 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 26. Moshers model8 and the revised model27 for the assignment of absolute configuration of MTPA esters.

signs are expected for those on substituent L2:

SRL1 ) L1(S) - L1(R) < 0

SRL2 ) L2(S) - L2(R) > 0

As indicated, experimental support for those con-

clusions has been obtained from the evolution of the
NMR spectra, with temperature, of the MTPA esters
that showed the shift of the equilibrium toward
conformer ap1 at lower temperatures. Similarly, good
agreement was observed between the experimental
chemical shifts and those expected from the calcu- Figure 27. Spatial distributions of (a) the shielding effect
lated aromatic shielding effect of the phenyl ring on on a MTPA ester and (b) the resulting signs of SR in the
conformers of a MTPA ester.
L1 and L2 in each individual conformer and in the
average mixture.27 Interestingly, both calculations Another consequence of this same fact is reflected
and experiments demonstrate that the effect on in the case of the R aryl alcohols in Figures 12 and
substituents L1 and L2 of the phenyl ring of MTPA 13, where anisotropic groups in the alcohol substit-
is net deshielding rather than shielding, as suggested uents effectively overcome the reduced shielding/
by Mosher in its empirical rule.8 deshielding produced by the MTPA, leading to SR
On that basis, a simplified model representative signs that do not necessarily reflect the configuration
of the NMR behavior of the MTPA esters and useful of the substrate.
for the assignment has been formulated.27 It has the Figure 27a illustrates how the magnetic field of the
CF3 group, the carbonyl group, and the H atom on phenyl ring affects the space occupied by the alcohol
the chiral C atom of the alcohol in the same plane substituents. When we consider the three main
and the CF3 anti-periplanar to the carbonyl group. conformers of a MTPA ester, the space distribution
In this way, the phenyl ring deshields the substituent of the magnetic field is different for each conformer
on the same side of the plane, i.e., L1 in the (R)-MTPA and leads to the signs of the SR value that are
ester and L2 in the (S)-MTPA analogue (Figure 26). indicated in Figure 27b. The Conformations of MTPA Esters. As can be observed, it is perfectly possible for a
The existence of three main conformers that have substituent such as L1 to possess protons located in
similar energy and contribute to the chemical shifts the space determined by quadrant 2 and other
with both shielding and deshielding effects serves to protons in quadrant 3. If this were the case, different
explain the origin of the two most important limita- SR signs will result in the average spectra and,
tions of this reagent: the small SR values and the therefore, an irregular distribution of signs will result
frequent surge of irregular distributions of the signs for the protons in L1.
of SR. Furthermore, if one considers the role of the
The origin of the reduced SR values has been conformational equilibrium in the average spectra,
already advanced and resides in the eventual cancel- it is easy to understand that small changes in the
lation in the average spectrum of the shielding/ balance of conformers may produce certain protons
deshielding that is associated with each individual to pass from a net shielding area to a net deshielding
conformer. area. In fact, even if we assume that conformer ap1
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 33

Figure 28. Isopropyl esters of MPA, MA, and atrolactic acid.

remains the most significant conformer in all cir-

cumstances, small variations in the ratio of the other
two forms could eventually lead to a different shield-
ing/deshielding balance, because each conformer has
a different shielding/deshielding effect. Summary. In summary, the use of MTPA
for the assignment of the absolute configuration of
alcohols is restricted by the conformational limits
imposed by the reagent. Three conformations of
similar population are present, and these simulta-
neously cause shielding/deshielding effects on the
same substituent. This situation explains many of the
observations regarding this method,13 including (a)
the frequency with which small values of SR are
found that are of little use for the assignment of
configuration and (b) irregular sign distributions.
The conformational flexibility of MTPA esters and
the magnetic field distribution facilitates small struc-
tural or experimental changes (e.g., NMR solvent or Figure 29. Model for configurational correlation of MPA
temperature) to change the balance between the esters.
conformers, causing the sign distributions of SR to
follow patterns that are different from those pre- The procedure for the determination of absolute
dicted by the original Mosher model. configuration with this reagent is the same as that
Overall, Moshers reagent, although still useful for already described for MTPA and can be summarized
the determination of enantiomeric purity by NMR as follows (Figure 29): (a) derivatization of the
spectroscopy, is not recommended for the determi- alcohol in question with the two enantiomers of the
nation of the absolute configuration of secondary reagent, (b) examination of the NMR spectra of the
alcohols by NMR. Such assignments should be made (R)- and (S)-MPA ester derivatives and evaluation
using other, more-reliable reagents. of the corresponding differences in the chemical shifts
of the substituents of the alcohol (RSL1 ) L1(R)
3.1.2. Methoxyphenylacetic Acid (MPA) - L1(S) and RSL2 ) L2(R) - L2(S)). As noted Classical Models for MPA Esters. previously, the configuration is deduced from the
R-Methoxy-R-phenylacetic acid (MPA; see Figures 4 signs of these differences and their comparison with
and 28), along with MTPA, has been one of the most an empirical model (see Figure 29b).
frequently used auxiliary reagents for the assignment In this model, the most representative conformer
of the absolute configuration of secondary alcohols has the methoxy group of MPA (the hydroxyl group
by NMR. The application of this reagent, together in MA and atrolactic acid) and the carbonyl and
with the use of mandelic acid (MA) and atrolactic acid C(1)H proton of the alcohol in the same plane (see
(Figure 28), was described by Mosher in 1973.8c Figure 29a). These groups are arranged in such a way
However, the utility of this reagent was not fully that, in the (R)-MPA ester, substituent L1 is shielded
accepted at the time, mainly because of problems by the phenyl ring while L2 is unaffected, whereas,
associated with racemization found during the de- in the (S)-MPA ester, substituent L2 is the shielded
rivatization of the alcohol. It was later found that this group and L1 remains unaffected (Figure 29a).
problem could be easily circumvented if different In this way, the alcohols in the figure show RSL1
derivatization conditions are used.28 < 0, because substituent L1 is more shielded in the
34 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

theoretical structure calculations (molecular mechan-

ics, semiempirical (AM1) and ab initio), (b) shielding-
effect calculations on the contribution of the phenyl
group to the chemical shift, and (c) dynamic NMR to
elucidate the conformational characteristics of the
As a result, the main conformers of the MPA esters
were identified. Their populations and the role of the
phenyl ring were determined, and this determination
allowed a complete understanding of the fundamen-
tals of the method and provided the basis for the
design of more-efficient reagents.30
It was found29 that the MPA esters of secondary
alcohols are mainly composed of two conformers that
are formed by hindered rotation around the CR-CO
bond (see Figures 30 and 31). The most stable one
Figure 30. Main conformers of the MPA esters.
(by 0.6-1.0 kcal/mol) is the sp conformer, in which
the methoxy group, the CR carbon, and the carbonyl
(R)-MPA ester than in the (S)-MPA ester, and RSL2 group of the MPA portion and the C(1)H hydrogen
> 0, because substituent L2 is more shielded in the of the alcohol portion are in the same plane, with the
(S)-MPA ester than in the (R)-MPA ester (see Figure methoxy and the carbonyl groups in a syn (syn-
29b). periplanar) disposition. In this conformation, the
The relative position of the phenyl group of the phenyl ring is coplanar with the CR-H bond, which
auxiliary, with respect to L1 and L2 of the same is the best arrangement to effectively transmit aro-
alcohol, is opposite in the MPA and MTPA esters, matic shielding to the substituent (L1 or L2) located
thus producing opposite signs for the same chirality in the same side of the plane (see Figure 31).
(see Figure 8). To obtain the same signs with the two The next conformer, in terms of energy (the ap
reagents, the differences in chemical shifts are cal- conformer), has the same coplanar arrangement of
culated as RS in MPA esters, whereas SR is used the aforementioned groups, but, in this case, the
with MTPA esters. methoxy and carbonyl groups are in an anti-peri- Conformational Analysis. Historically, planar relationship and the phenyl group is rotated
the first model used to correlate the absolute config- so it cannot transmit the shielding effect to substit-
uration of alcohols with the NMR shifts and signs of uents L1/L2 as efficiently as in the example discussed
the RS values of the MPA esters was proposed by previously (see Figure 31). Thus, in the (R)-MPA
Mosher8c and is, as in the case of MTPA derivatives, ester derivative, substituent L1 is shielded by the
empirically generated from the study of several phenyl ring in the sp conformation while substituent
alcohols of known stereochemistry. For this reason, L2 is unaffected (see Figure 32a). In contrast, sub-
the validity of the method is related to the represen- stituent L2 should be slightly shielded in the ap
tativity and scope of the series of alcohols initially conformation while substituent L1 remains un-
studied.8c affected (see Figure 32a). The opposite situation
To evaluate the limitations of this model, studies occurs in the (S)-MPA ester: substituent L2 is
have been conducted29 that are comprised of (a) shielded and substituent L1 remains unaffected in the

Figure 31. Conformers sp and ap of the isopropyl (R)-MPA ester.

Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 35

Figure 32. Conformational equilibrium in MPA esters.

sp conformer, whereas substituent L1 is slightly equilibrium, which is also the most important, from
shielded and substituent L2 is unaffected in the ap the standpoint of NMR, and is therefore suitable to
conformer (Figure 32b). predict the correct configuration. Figure 33 shows a
As a result of the characteristics discussed above, selection of compounds studied using that model.31
the aromatic group of the reagent will modify the Note that this is not the case for MTPA esters,
chemical shifts of substituents L1 and L2 in a very where the model used by Mosher is not truly repre-
selective way: In the (R)-MPA ester, it is expected sentative of the conformational composition.27
that strong shielding will be experienced by substitu- MPA versus MTPA. In general, research-
ent L1 but only on the proportion of the molecules in ers have not been very critical when choosing be-
which substituent L1 is beside the phenyl group tween MPA and MTPA for the assignment of sec-
(conformer sp). On the other hand, in the (S)-MPA ondary alcohols. Quite frequently, they have over-
ester, substituent L1 will be slightly shielded, but only looked the anomalies detected with MTPA13 that
for those molecules in the ap conformation. Given were mentioned in the previous chapter.
that the sp conformer is more abundant than the ap In the case of MTPA esters of secondary alcohols,
conformer, substituent L1 is more shielded in the (R)- two factors contribute to these limitations:27,32 (a) the
MPA ester than in the (S)-MPA ester. Similar presence of three main conformers with similar
reasoning for substituent L2 leads to the conclusion populations and (b) the fact that some of these
that it will be more shielded in the (S)-MPA ester conformers produce shielding effects and others
than in the (R)-MPA ester. Thus, the differences in produce deshielding effects on substituents L1/L2 (see
chemical shifts (RS) will be positive in one case and Figure 25), the combination of which produces very
negative in the other (see Figure 32c). If the opposite small SR values and sometimes unexpected signs.
configuration is considered for the alcohol (i.e., sub- In contrast, the MPA esters present a simpler
stituent L1 in place of substituent L2), then the conformational composition (see Figure 32) with only
opposite distribution of signs is obtained (see Figure two conformers (sp/ap) and a clearer preference for
32d), thus demonstrating that the sign of RS is a one of those (sp), which, in turn, transmits a shielding
reliable indicator of the spatial arrangement of the effect to the substituents to give higher RS values
substituents. that are more reliable and homogeneous in terms of
distribution of the signs (Figure 34).
RSL1 ) L1(R) - L1(S) < 0 On the basis of the aforementioned discussion, the
choice between MTPA and MPA as a derivatizing
RSL2 ) L2(R) - L2(S) > 0 agent for secondary alcohols should be clearly in favor
of MPA.
The model proposed by Mosher8c,28a for the assign- New MPA Analogues: The Effect of
ment of the configuration of MPA esters is, in fact, a Structural Modifications. In the search for more
representation of the most stable conformer of the efficient and reliable reagents, a variety of modifica-
36 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 34. RS and SR values (in ppm) for a selection

of MPA and MTPA esters of secondary alcohols obtained
in (a) Cl3CD and (b) CS2:CD2Cl2 (4:1). MTPA data are noted
by an underline.

Figure 35. O-Substituted Mandelic Acids. Table 1

Figure 33. shows the structures of several O-substituted MAs
tions of the structure of MA have been performed and the ability of their enantiomers to differentiate
(Figure 35), leading to a series of arylmethoxyacetic the NMR signals34a of (-)-menthol expressed as RS.
acids30,33 (AMAAs). Some of these compounds are As can be seen, the values are very similar in all
interesting from the practical standpoint and others cases, being somewhat greater in MA and its acetyl
are interesting because of the information they derivative (see Figure 36). In addition to similar RS
provide regarding our understanding of the method. values, the same distribution of the signs as that
The most revealing modifications have been (a) the observed with MPA itself is obtained, indicating that
introduction of different O-substituents on MA,34 (b) their mode of action and conformational composition
the introduction of different substituents on the is quite probably the same, suggesting that no real
phenyl ring30,33 of MPA, and (c) the replacement of improvement can be expected by changing the O-
that phenyl ring by other aryl ring systems.29,30,33 substituent in MA.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 37

Figure 37. RS values of O-substituted mandelic esters.

Data for R ) MPA is given in boldface type; underlined
data present anomalous signs.
Table 2. RS Values Obtained for Phenyl-Substituted
MPA Esters of (-)-Menthol
Figure 36. RS values obtained for O-substituted man-
delic esters with a selection of secondary alcohols. Data for
R ) AcMA is given in italic type, and data given for R )
MA is given in boldface type.
Table 1. RS Values of O-Substituted Mandelic
Esters of (-)-Menthol

derivative, the RS values are, in fact, smaller than

those obtained with MPA and similar to those
obtained with MTPA, whereas the methoxy-substi-
tuted reagents give RS values that are greater than
A different case is constituted by the THP ether of those for MTPA and equivalent to those of MPA.
MA, which has been investigated34b as a reagent for Conformational studies were performed and re-
the assignment of the absolute configuration of cyclic vealed that the introduction of those substituents
alcohols (see Figure 37). Unfortunately, an irregular shifts the equilibrium between the sp and ap con-
distribution of RS signs were obtained in the two formers and changes the orientation of the phenyl
compounds tested, which makes the reagent useless ring to a less favorable arrangement for shielding.
for the intended purpose. Only two open-chain sec- It is the combination of these two facts that result in
ondary alcohols (Figure 37) were studied with this smaller RS values.
reagent and showed the same distribution of signs Arylmethoxyacetic Acids (AMAAs). The
as that for MPA. structural modifications discussed in the preceding The Role of the Substituents in the lines have shown that no improvement of can be
Aromatic Ring. Similar studies have been con- expected by introduction of substituents on MPA. As
ducted30,33 to ascertain whether the introduction of will be fully discussed in the corresponding section
substituents into the phenyl ring of MPA will produce (Section 3.1.3), the replacement of the phenyl ring of
an increase in the RS values. MPA by a naphthyl or anthryl ring has been
The results obtained for some of these new re- shown to produce the most efficient reagents to
agents with (-)-menthol as the substrate are shown date.29,30,33,35-37 More specifically, R-(2-naphthyl)-R-
in Table 2. These data indicate that, in general, the methoxyacetic acid (2-NMA), R-(1-naphthyl)-R-meth-
presence of fluoro or methoxy groups in several oxyacetic acid (1-NMA), R-(9-anthryl)-R-methoxy-
different positions does not produce any improvement acetic acid (9-AMA), and R-(2-anthryl)-R-methoxy-
in the capability of MPA to separate the signals of acetic acid (2-ATMA) are effective reagents (see
enantiomers. In the case of the pentafluorphenyl Figure 38).
38 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 38. Molecular structure of the arylmethoxyacetic acids (AMAAs).

Figure 39. Conformational composition of the MPA esters of (-)-menthol. The Effect of the Temperature on the alcohols, when their NMR spectra were recorded at
NMR of MPA Esters. A different and successful different temperatures, the changes in chemical
approach to obtain the best RS values without shifts consistently produced higher RS values38 of
modification of the structure of the auxiliary reagent practical interest.
consists of the controlled modification of the relative Thus, in the (R)-MPA ester of (-)-menthol, the
populations (conformer sp/ap) of the MPA esters by Me(8) and Me(9) protons are strongly shielded by
directly acting on the thermodynamic parameters the phenyl ring in the sp conformer (see Figure 39a,
governing the equilibrium,38 namely the polarity of resonances at 0.651 and 0.418 ppm, respectively),
the NMR solvent and the temperature of the NMR whereas Me(10) is slightly shielded in the ap con-
probe. former (0.888 ppm). A decrease of the probe temper-
Although changes in the NMR solvent30 did not ature should produce an increase in the number of
lead to any significant improvement of the RS molecules in the sp conformation (the most stable
values obtained for MPA esters of several secondary one) and a corresponding decrease in the ap popula-
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 39

Table 3. Chemical Shifts of MPA Esters of have their Me(8) and Me(9) groups shielded (in-
(-)-Menthol at Different Temperaturesa creased shielding in the average spectrum; reso-
chemical shift nances at 0.561 and 0.165 ppm) and fewer molecules
ester Me(8) Me(9) Me(10) have their Me(10) shielded by the phenyl group
T ) 296 K (increased shielding in the average spectrum; reso-
(R)-MPA 0.651 0.418 0.888 nance at 0.898 ppm).
(S)-MPA 0.846 0.642 0.822
RS -0.195 -0.224 0.066
Similarly, in the (S)-MPA ester, the Me(8) and
T ) 153 K
(R)-MPA 0.561 0.165 0.898
Me(9) groups become less shielded at lower temper-
(S)-MPA 0.895 0.681 0.785 atures and the signals are shifted from 0.846 and
RS -0.334 -0.516 0.113 0.642 ppm to 0.895 and 0.681 ppm, respectively,
CS2:CD2Cl2 ratio ) 4:1. whereas Me(10) is more intensely shielded at lower
temperatures, with a change in chemical shift from
tion (the least stable one). For this reason, as shown 0.822 to 0.785 ppm for the corresponding signal (see
in Table 3, at lower temperatures, more molecules Figure 39b).

Figure 40. Evolution with temperature of the RS values of MPA esters of secondary alcohols.
Table 4. RS Values for a Selection of MPA Esters at Different Temperaturesa
temp, RS (ppm)
T (K) H(1) H(3) H(4) H(5) H(6a) H(6e) H(6ax) H(7) H(8) Me(8) Me(9) Me(10)
298 0.050 0.085 -0.210 -0.250 -0.216 0.039
213 0.100 0.175 -0.319 -0.373 -0.348 0.049
183 0.100 0.175 -0.346 -0.403 -0.40 0.049
298 -0.45 -0.195 -0.224 0.066
153 -1.170 -0.334 -0.516 0.103
298 0.050 0.255 0.080 -0.200
213 0.104 0.499 0.138 -0.312
183 0.125 0.596 0.162 -0.331

298 0.101 -0.095 -0.161
263 0.120 -0.127 -0.207
230 0.138 -0.137 -0.259
203 0.156 -0.166 -0.316
CS2:CD2Cl2 ratio ) 4:1.
40 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

mental RS value. Nevertheless, and despite the

aforementioned limitations, the method may be use-
ful to analyze structures that lack protons in one of
the substituents.
3.1.3. 9-Anthrylmethoxyacetic Acid (9-AMA) and Related
The results described to date for MPA, along with
the discovery of the role played by the phenyl ring
in influencing the NMR shifts, have led to a search
Figure 41. Alcohols tested with this method.
for more-efficient reagents by replacement of the
phenyl ring by other aryl systems that are able to
produce a more intense aromatic shielding effect on
the substrate.29,30,33,35-37 The most widely studied
systems in this respect are the arylmethoxyacetic
acids (AMAAs; see Figures 38 and 42), which contain
naphthyl or anthryl systems. In general, all these
compounds produce more-intense shielding than
MPA and, consequently, better separation of the
signals is observed for the enantiomers of the sub-
strate. This effect is particularly important when
9-AMA is used as the reagent with secondary alco-
Figure 42. Structure of the arylmethoxyacetic acids hols: the RS values obtained are 3-4 times higher
(AMAAs). than those with MPA.29,30 In fact, this more pro-
nounced separation of the signals for the substrate
In accordance with those results, the RS values results from the combination of two factors: the
of MPA esters of secondary alcohols become higher intense aromatic magnetic field of the anthracene
as the probe temperature is reduced (see Table 4 and system and the greater conformational rigidity of
Figure 40). 9-AMA, which is a factor that leaves the anthracene
This trend reflects a general behavior that is well ring particularly well-oriented, with respect to the
illustrated by the variety of structures shown in substrate.29
Figure 40. This technique is particularly useful when
the RS values obtained at room temperature are
so small that reliable e.e. or absolute configuration
cannot be derived. In these cases, simply repeating
the spectrum at a lower temperature can be sufficient
to obtain well-separated signals and large RS
values. Other Models for MPA Esters. A new
application of MPA in the determination of the
absolute configuration of secondary alcohols has been
described recently,39 based in the exclusive use of the
chemical shifts of the CRH protons of the substrate
in the corresponding (R)- and (S)-MPA esters. To
assign the configuration, interactions between the
reagent and substrate moieties of the diasteromeric
esters must be estimated in the two main forms in
the equilibrium (sp and ap) and compared with the
chemical shifts of the CRH protons. The diastereomer
with the greater relative population of the most Figure 43. Main conformational processes in AMAA
stable conformer in the equilibrium (sp form) should esters.
resonate at lower field. This behavior was experi-
mentally proven for six alcohols and two amines (see
Figure 41).
This method presents some limitations. One of
them lies in the fact that it is based on the analysis
of data from a single group (RS of the CRH),
analogous to Moshers Method for 19F NMR (SR of
the CF3). The sign will always be correlated to a
certain configuration, so if an anomaly arises, it will
not be detected. In some cases, another restriction
comes from the need of a combined use of NMR data
and theoretical studies (i.e., semiempirical calcula-
tions) to evaluate the difference of energy between
both conformers to compare them with the experi- Figure 44. Main conformers of the 9-AMA esters.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 41

(no such information is available on 2-ATMA37) have

revealed that, in a way similar to that for the MPA
esters, the main conformational process is rotation
around the CR-CO bond, and this process generates
the same types of conformers as already described
for MPA esters:29 conformer sp, with the MeO group
syn to the carbonyl group, is the most stable con-
former and conformer ap, where the MeO group is
anti, with respect to the carbonyl group, is the least
stable (see Figures 43 and 44).
In regard to rotation around the CR-Ar bond,
calculations indicate that the most stable conformer
is that in which the CR-H bond is coplanar with the
aryl plane, as shown in Figure 44. In those reagents
where the aromatic substitution is asymmetric (e.g.,
1-NMA, 2-NMA, and probably 2-ATMA), the main
portion of the aryl ring is anti, with respect to the
CR-H bond (see Figure 45).
Therefore, in the more stable conformer (sp), the
CR-Ar bond is almost perpendicular to the CdO
Figure 45. Main conformers generated by rotation around bond and the plane of the aromatic ring is parallel
the CR-Ar bond (a) for 1-NMA and (b) for 2-NMA. to the CdO bond. This arrangement makes the
orientation of the aryl ring particularly effective, in Conformational Analysis. Conforma- terms of transmission of the shielding effect to the
tional studies29 (theoretical and experimental) based alcohol substituents L1/L2.
on structure minimizations (molecular mechanics, From the conformational standpoint, the equilib-
semiempirical and ab initio, and shielding-effect rium between the sp and ap conformations is, in all
calculations) and NMR studies (variable tempera- cases, shifted toward the sp conformer, which is the
ture) on the auxiliaries 9-AMA, 1-NMA, and 2-NMA most stable conformer and the most representative

Figure 46. Conformational equilibrium in the AMAA esters.

42 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 47. (a, b) NMR spectra of the (R)- and (S)-9-AMA esters of a marine metabolite and (c) comparison of the RS
values of the 9-AMA esters with those of the MPA esters.

in the NMR process. However, the difference in stituent L1 is shielded in conformer sp and substitu-
energy29 varies with the nature of the aryl ring and ent L2 remains unaffected, whereas in the ap con-
follows the order 9-AMA > 1-NMA > 2-NMA > MPA. former, substituent L2 is shielded and substituent L1
Thus, changing the aromatic ring plays a double is not affected (see Figure 46a). In the ester derived
role, because it affects both the intensity of the from the (S)-AMAA auxiliary, the opposite situation
aromatic magnetic current and the conformational occurs: L2 is shielded in the sp conformer and L1 is
orientation of the ring, with respect to the substrate. shielded in the ap conformer (see Figure 46b).
In fact, the larger the ring, the more intense the Comparison of the spectra of the (R)- and the (S)-
aromatic shielding effect and the greater the result- AMAA esters, and considering the greater population
ing difference of energy between the sp and ap of the sp conformer in comparison to that of the ap
conformers. The combined effect of these two factors conformer, leads to the following signs for RS (these
makes 9-AMA the most useful reagent of all, produc- are the same as those obtained in the MPA esters
ing RS values that are much greater than those with the same configuration):
caused by other reagents.
The substitution pattern of the ring is also impor- RSL1 ) L1(R) - L1(S) < 0
tant because the rotational barriers for the CR-Ar
and CR-CO bonds will be different. For example, the RSL2 ) L2(R) - L2(S) > 0
barrier in 1-NMA is greater than that for 2-NMA and
this, in turn, affects the NMR results. Naturally, if the absolute configuration of the alcohol NMR Spectra of AMAA Esters. The is opposite to that shown above, then the signs should
NMR spectra of the AMAA esters are similar to those be reversed (see Figure 46c and d).
of the MPA esters, and the contribution of each Figure 47 illustrates the power of 9-AMA as a
conformer to the average NMR spectra also follows reagent for the assignment of the absolute configu-
the same trend. Thus, in the (R)-AMAA ester, sub- ration. The NMR spectra of the (R)- and (S)-9-AMA
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 43

esters of a marine metabolite40 show that the signals

for protons H(1), H(2), H(4), H(5), H(6), and
Me(14) result in negative RS values and, therefore,
take the place of substituent L1 in the model shown
in Figure 47. On the other hand, protons H(9),
H(10), Me(12), and Me(13) give positive RS values
and are therefore represented by substituent L2. As
can be seen in Figure 47c, the RS values obtained
with 9-AMA are far superior to those obtained using
MPA as the auxiliary (in some cases, by a factor of
>4), although the signs are the same.
An experimental demonstration of the reliability
of the configurational assignment obtained with these
AMAAs, and, consequently, of the model described
in Figure 46, was achieved by examination of a series
of alcohols of known absolute configuration. In all the
cases described41 in Figures 48 and 49, the RS signs
are in accordance with the stereochemistry of the
alcohol examined. The magnitude of the RS values
follows the order 9-AMA > 1-NMA > 2-NMA > MPA
The number of alcohols of known absolute config-
uration used to validate 2-ATMA37 as the reagent is
much lower than for the other reagents. In addition,
neither structure calculations nor conformational
analyses have been presented,37 although it does not
seem unreasonable to believe that the model should
be the same as that presented for the other AMAAs.
On the basis of the nature and orientation of the
aromatic ring in the AMAAs and its associated
magnetic field, it was proposed that some of these
reagents should be particularly suited for study
linear alcohols and others should be suited for cyclic
systems.37b In this respect, the reagents 2-NMA and
2-ATMA were proposed36,37 as being the most suitable
for the study of long-chain alcohols, on the basis of
the expectation that, in the esters, the aromatic rings
of the reagent would lie over the chain of the
substrate and thus cause significant shift changes on
the protons of a large portion of the chain (see Figure
50). Reagent 9-AMA, on the other hand, was expected
to be more efficient when used in conjunction with
cyclic alcohols.30
Nevertheless, comparison of the RS values ob-
tained30,36,37 for the esters of 9-AMA, 2-NMA, and
2-ATMA with the linear long-chain alcohols shown
in Figure 51 demonstrates that the highest RS
values are always obtained when 9-AMA is used as
the reagent, whereas 2-NMA is the least-efficient
reagent of the three.
Figure 48. RS values for selected 9-AMA and MPA
A graphical representation of the RS values esters.
obtained for protons at different distances from the
asymmetric C atom for the 9-AMA esters42 derived In the esters formed with 2-NMA and 2-ATMA, the
from 51.1, 51.2, and 51.3, and of the 2-NMA and variation of shielding intensity with distance is much
2-ATMA esters derived from 51.4 and 51.5, is shown more uniform and the maximum shielding zone
in Figure 51. The spatial zone of maximum shielding covers the region from approximately two carbon
is much more concentrated in the 9-AMA esters than bonds away from the asymmetric C atom up to
in the other. In the 9-AMA esters, it is experienced approximately six bonds away.
by protons 2-3 carbon bonds away from the asym- In practice, when the assignment of the configu-
metric C atom, decreases rapidly with distance, and ration of an alcohol is made difficult by substantial
at approximately the fifth bond away shows RS overlap of the NMR signals, 9-AMA is better than
values equivalent to those produced by 2-NMA and both 2-NMA and 2-ATMA, because the intense, yet
2-ATMA. rapidly decaying, shielding zone ensures a wide
44 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 49. RS values for selected 9-AMA, 1-NMA, 2-NMA, 2-ATMA, MPA, and MTPA esters of secondary alcohols.

signal distribution across the spectrum for the rel- The 500 MHz NMR spectrum of cis-androsterone30
evant protons and eliminates signal overlap. The is shown in Figure 52b and those of its (R)- and (S)-
reagents 2-NMA and 2-ATMA37 are less effective in 9-AMA ester derivatives in Figure 52a and c, respec-
this respect, because a large number of protons in tively. It is clear that an unambiguous assignment
the chain will be shielded to approximately the same of the signals in Figure 52b is impossible, because
extent and, therefore, considerable signal overlap is most of the signals are overlapped in a narrow range
likely. of 1 ppm. In contrast, the 9-AMA esters present an
This ability of 9-AMA to separate the signals of the extraordinary dispersion of these signals, and this
alcohol portion is essential for the correct assignment phenomenon allows the complete assignment of all
of the protons and calculation of the RS values and the protons in the molecule.
is not limited to linear substrates but can also be of As predicted, the maximum shielding is located in
use for cyclic alcohols.30 An illustrative example of the region near to the asymmetric center (rings A and
such a case is represented by cis-androsterone. B of the steroid) and is so intense that the chemical
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 45

Figure 50.

Figure 51. Variation of RS with the distance to the asymmetric C atom in 9-AMA, 2-NMA, and 2-ATMA esters of
lineal alcohols.

shifts of protons H(1), H(9), H(6), H(5), and H(7), sp conformer in its esters is greater than that in the
which are indistinguishable in cis-androsterone itself, other AMAAs.
are moved to the right of TMS and is enough to move
protons in the C ring. 3.1.4. Other Reagents
In conclusion, replacement of the phenyl ring of In addition to the reagents described thus far,
MPA by naphthyl or anthryl rings is shown to be a many other compounds have been proposed as CDAs
useful way to modify these reagents. Of all the for the assignment of configuration of secondary
AMAAs investigated, 9-AMA is the one that produces alcohols by NMR. Most of the methods reported in
the most marked changes in shifts and RS values, this section use 1H and 13C NMR, with only a fews
because (a) its aromatic system produces the most mostly those devoted to e.e. calculations rather than
intense magnetic field and (b) the population of the absolute configuration assignmentsinvolving 19F
46 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 52. 1H NMR spectra of cis-androsterone (b) and its (R)-9-AMA (a) and (S)-9-AMA esters (c).

NMR. Generally, these reagents contain a magneti-

cally anisotropic group (e.g., phenyl, naphthyl), which
is responsible for the shifts experienced by substit-
uents L1/L2, a polar group (e.g., OMe, F) that favors
a certain conformation, and an additional functional
group for bonding to the substrate. This linkage is,
in most cases, an ester bond, although there are
examples where an ether, a glycoside, or a phospha-
mide bond have been used.
In contrast to the cases described for MPA, MTPA,
and 9-AMAA, only a few of the procedures described
in this section involve conformational analysis and/
or theoretical calculations to support the results. For
this reason, the reliability of the configuration as-
signed by these procedures relies essentially on the
number and structural variety of the alcohols of
known absolute configuration used to validate the
method/reagent. 2-Phenylbutyric Acid: Helmchens
Method. The commercially available (R)- and (S)-2-
phenylbutyric acids ((R)- and (S)-PBA, respectively;
see Figure 53a), which are well-known in the kinetic
method described by Horeau,43 have been proposed
by Helmchen et al.44 as CDAs for secondary alcohols,
and this method has since been extended to assign Figure 53. (a) Structure of (R)-PBA and (b) model for the
assignment of the configuration of secondary alcohols from
the stereochemistry of amines and thiols. the NMR spectra of the PBA esters.
Helmchens procedure is similar to that described
by Mosher and consists of the preparation of the NMR spectra of the diastereomeric esters. The signs
esters of the alcohols with the two enantiomers of the of the resulting RS values are then used to locate,
reagent (PBA) and subsequent comparison of the 1H in space, the substituents L1 and L2, in accordance
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 47

Table 5. RS Values Obtained for the (R)- and

(S)-PBA Esters of a Selection of Secondary Alcohols
of Known Configuration
C6H13a 1.11 CH3 1.16 R
1.28 1.05 S
-0.17 0.11
C6H5 CH3 1.45 R
1.35 S
COOEt 1.09/4.04 CH3 1.41 R
1.23/4.15 1.36 S
-0.14/-0.11 0.05
bornylb 0.63 R
0.81 S
Shift of the CH2 group. b Signal corresponding to Me(10).

with an empirical model (see Figure 53b). The

empirical model was designed to correlate the NMR
results of many secondary alcohols of known absolute
configuration with the stereochemistry. Table 5
shows a selection of secondary alcohols of known
absolute configuration, along with the resulting RS
values, that have been used to validate this proce-
dure. This model has also been applied, without
modifications, to the assignment of the configurations
of amines45a and thiols.45b
Despite the very small number of substrates of
known absolute configuration originally used for the
validation of PBA as a CDA,45 as well as the absence
of other studies that could lend support to the model,
PBA has been frequently used to deduce the config-
uration of natural products.46 Examples (Figure 54)
include the following: the fungus metabolites chryso- Figure 54. Natural products of microbial origin studied
gine, which is isolated from Fusarium sambucinum;46a by Helmchens method.
ripostatin A, B, and C, which is isolated from Sor-
angium cellulosum;46b massarinolin B methyl ester46c The authors assume that the most representative
(from Massarina tunicata); and streptoketol A46d conformation is the one in which the H atoms at the
(from an Streptomyces sp). -carbon of the acid, the carbinol, and the carbonyl
The applicability of this reagent for the assignment group are syn-periplanar. In accordance with this
of the configuration of secondary alcohols using 13C model, substituent L1 should be shielded in the (R)-
NMR instead of 1H NMR has also been investigated.47 ester, while substituent L2 is shielded in the (S)-ester;
However, in this case, the number of alcohols of therefore, RS must be negative for substituent L1
known absolute configuration studied as test com- and positive for substituent L2.
pounds is very limited and more examples should be In conclusion, the applicability of both PBA and
analyzed before the procedure is recommended. 3-PBA as CDAs is limited by several considerations:
More recently, the possibility of using the isomer (a) the small number of substrates of known absolute
3-phenylbutyric acid (3-PBA) as a CDA has been configuration that have been used to validate the
evaluated48 (Figure 55a). However, the presence of assignment, (b) the absence of other experimental or
the phenyl group and asymmetric center at position theoretical studies that might support the correlation
, rather than at position R (as in PBA, MPA, MTPA, models, and (c) (in the case of 3-PBA) the small RS
or 9-AMA), and the higher conformational freedom values.
inherent in this system leads to a reduction of the Naphthylpropionic Acids. In the past
aromatic shielding/deshielding on substituents L1/L2, few years, several naphthylpropionic acids have been
which is a situation that gives smaller RS values. investigated as CDAs.49 One of the most commonly
In fact, comparison of the RS values obtained for used is 2-methoxy-2-(1-naphthyl)propionic acid
the ester derivatives of diacetone-D-glucose and 2-bu- (MRNP; see Figure 56), which is already known as a
tanol with 3-PBA, MPA, and MTPA as reagents derivatizing agent for HPLC resolution of alcohols.50
showed that the smallest shifts were obtained with In the (R)-MRNP ester of the alcohol shown in
3-PBA. Figure 57, substituent L1 experiences a shielding
The model presented in Figure 55b is empirical and influence from the naphthyl group, whereas, in the
was designed to correlate the NMR spectra of the (S)-MRNP ester, the shielded group is substituent L2.
3-PBA esters of just five alcohols of known absolute Therefore, substituent L1 results in a negative RS
configuration with their stereochemistry. value and substituent L2 yields a positive RS value.
48 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 55. (a) Structure of (R)-3-PBA and (b) model for the assignment of the configuration of secondary alcohols from
the NMR spectra of the 3-PBA esters.

Figure 56. Structure of the 2-methoxy-2-(1-naphthyl)-

propionic acids.

Figure 58. Alcohols of known configuration studied by

NMR of their MRNP esters and the resulting RS values.
the model shown in Figure 57 allows the configura-
tion to be assigned.49a
In addition to MRNP, the potential of some other
naphthylpropionic acids to act as CDAs has been
evaluated using (-)-menthol as a test compound, on
Figure 57. Model for the assignment of configuration by the assumption that the correlation model of MRNP
NMR of MRNP esters.
is still valid.
This model was derived from the study of just five Table 6 shows the RS values obtained for the (-)-
alcohols of known absolute configuration, and the menthol esters of 2-hydroxy-2-(1-naphthyl)propionic
structures and RS values for these are shown in acid49c (HRNP), 2-hydroxy-2-(2-naphthyl)propionic
Figure 58.49a acid (HNP), and 2-methoxy-2-(2-naphthyl)propionic
The authors proposed a variant using the racemic acid49d (MNP), as well as, for comparative purposes,
mixture of the auxiliary (()-MRNP. In this case, the those obtained with several AMAA reagents de-
mixture of MRNP esters first should be separated and scribed previously in this reviewsincluding MPA and
a small sample of one diastereoisomer transformed MTPA.
to the corresponding methyl ester. Inspection of the Examination of the data shows that the RS
CD spectra allows the identification of the (R)- and values of the naphthylpropionic acids mentioned in
(S)-MRNP esters (the (S)-MRNP has a negative CD this section follow the order HRNP > MRNP > HNP
band and the (R)-MRNP has a positive band at 280 > MNP, and, in fact, the values for MNP are very
nm). After the (R)- and the (S)-MRNP esters have small and the distribution of the signs is irregular
been identified, their NMR spectra are recorded and (for example, see H(10)).
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 49

Table 6. Structure of the Naphthylpropionic Acids

Evaluated as CDA Reagents and the RS Values
Obtained for Their Esters with (-)-Menthol and
Those of Other AMAAs

Figure 59. Structure of axially chiral acids MBNC and


Figure 60. Model for the assignment of the configuration

of alcohols using the NMR of their MBNC and MNCB
Comparison of these reagents with the AMAA esters.
systems discussed previously shows that the anthryl examples are 2-methoxy-1,1-binaphthyl-2-carboxylic
reagent 9-AMA produces the highest RS values of acid (MBNC) and 2-(2-methoxy-1-naphthyl-)-3,5-
all, with the second-highest values resulting from the dichlorobenzoic acid (MNCB). These compounds have
naphthyl acid, 1-NMA. In addition, comparison be- been evaluated for the assignment of the configura-
tween the 1-naphthyl-substituted reagents MRNP tion of secondary alcohols by means of 1H and 13C
and 1-NMA indicates that 1-NMA is the more effec- NMR spectroscopy51a,b (see Figure 59).
tive reagent. Similarly, among the 2-naphthyl- This protocol is not unusual in any respect and
substituted systems, it is evident that 2-NMA is more consists of the derivatization of the substrate with
effective than MNP and this clearly suggests that the two enantiomers of the reagent and subsequent
the replacement of the H atom in the R-position by a comparison of their 1H or 13C NMR spectra.
methyl group (as in MRNP and MNP) leads to a The interpretation of the NMR data follows the
significant loss of efficacy of the reagents sprobably model shown in Figure 60. Thus, in the case of the
a consequence of a reduction in the energy difference esters of MNCB (Figure 60a), the most representative
between the conformers. conformer is assumed to be that which the phenyl
In conclusion, the use of naphthylpropionic acids group of the acid (Ar1), the carbonyl group, and the
as CDAs is limited by (a) the small number of hydrogen bond to the chiral center of the alcohol are
substrates of known absolute configuration used to in the same plane (plane CB). In addition, the Ar1-
validate the method and (b) the absence of informa- Ar2 bond and the CdO group should be anti-peri-
tion about their conformational characteristics. The planar, with the aromatic rings (Ar1, Ar2) perpen-
shifts obtained are smaller than those observed for dicular to each other.
other, better-known naphthyl-substituted reagents This geometry allows the naphthyl ring to transmit
and, therefore, do not provide any advantage. its shielding to the substituents of the alcohol but, Axially Chiral CDAs (MBNC and unfortunately, not in a very selective manner, be-
MNCB). Several compounds of the binaphthyl and cause both substituents in each conformer are
biphenyl classes with axial chirality have been shielded, although one is slightly more affected than
proposed as CDA reagents.51 The most important the other.
50 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 63. Conformational equilibrium in the esters of


irregular distribution of signs for SR (see 61.8, 61.9,

and 61.12).
In regard to the 13C NMR data (Figure 62), the
resulting SR values are too small to ensure a safe
assignment. A similar situation had been observed
previously with other reagents (MTPA; see Section and this is not surprising, given the low
sensitivity of 13C chemical shifts to the anisotropic
The values shown in Figure 61 are clearly
smaller than those obtained with other naphthyl-
containing reagents described in this review, such as
the AMAAs (1-NMA and 2-NMA). The explanation
for this less-marked effect is evident after performing
calculations51c on the structure and conformation of
the compounds in question. For example, molecular
mechanics (MM), semiempirical (PM3 and AM1), and
ab initio calculations have revealed that these com-
pounds exist in solution as two main conformers in
equilibrium: the sp conformer (with the Ar1-Ar2
bond and the CdO group syn-periplanar; see Figure
63) and the ap conformer (where the aforementioned
groups are anti-periplanar). In both cases, the Ar1
Figure 61. Secondary alcohols and (1H NMR) values ring, the CdO group, and the C(1)-H bond remain
obtained from their MNCB (plain, SR) and MBNC (in coplanar, whereas Ar2 is perpendicular to Ar1.
parentheses, RS) esters. Although the ap conformer has a better geometry
than the sp conformer, in terms of transmission of
the effect of Ar2 to the substituents of the alcohol
(R1/R2), the calculations reveal that the most stable
form is the sp conformer, in which the aromatic ring
is poorly oriented for transmission purposes.
Despite the fact that the ap conformer is less
abundant, it is considered to be the most important
conformer, in terms of NMR shifts, and its use in the
correlation model shown in Figure 60 is justified by
the results of NMR studies at low temperature and
by aromatic shielding calculations. A second aspect
that must be considered concerns the fact that in
neither of the two conformers can the aromatic ring
produce selective shielding on just one of the sub-
Figure 62. Secondary alcohols and (13C NMR) values stituents (R1/R2), which is a situation that is contrary
obtained from their MNCB (SR) and MBNC (RS) esters.
MBNC data given in parentheses. to that observed for the AMAA derivatives. In the
case in question here, both substituents are moder-
The signal for Ha appears in the (aS)-MNCB ester ately shielded in the ap conformer, whereas in the
at higher field than in the (aR)-MNCB ester; the sp conformer, they are only slightly shielded.
reverse situation is observed for Hb. In this way, the These two points explain the small shifts obtained
resulting differences ( ) aS - aR) are HbSR with these reagents and the need for more efforts to
> 0 and HaSR < 0. improve their performance. With this intent in mind,
Figures 61 and 62 show the structures of the the reagent 1-(10-bromo-1-anthryl)-2-naphthoic acid
alcohols of known configuration used to validate the (BANA, Figure 64) was prepared and its ability to
model and the corresponding SR values (1H and 13C produce separate signals for diastereomeric (-)-
NMR data). The proton NMR data of the lineal menthol esters was evaluated. Although the SR
alcohols are in good agreement with the model; values obtained are slightly better than those ob-
however, the use of cyclic alcohols results in an tained with MNCB, they are still too small to justify
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 51

Figure 64.

Figure 66. Conformational model and predicted signs

for the assignment of the configuration of secondary
alcohols from the NMR of their alcohol-DPEA derivatives.

Figure 65. Derivatization procedure for secondary alco-

hols. Figure 67. Secondary alcohols of known absolute config-
uration and values obtained from their alcohol-DPEA
the extensive use of these reagents for the assign- derivatives.
ment of secondary alcohols. Difluorodinitrobenzene. An empirical
method based on the use of 1,5-difluoro-2,4-di-
nitrobenzene (FFDNB) as a CDA for the assignment
of secondary alcohols and primary amines has been
described.52 In the case of alcohols52 (the application
to amines is reviewed in Section, the sub- Figure 68. Preparation of N-(alkoxymethyl)-2-pyrrolidi-
strate and the difluorodinitrobenzene reagent are none derivatives.
linked through an ether bond and a second auxiliary
reagent is then introduced. Therefore, the procedure This model has been experimentally assessed with
for derivatization entails the reaction of the chiral five alcohols of known absolute configuration (Figure
alcohol with FFDNB and the resulting fluoro-dini- 67). The signs of are in good agreement with the
trobenzene (FDNB) ether is subsequently made to stereochemistry and the shifts are large. However,
react separately with the two enantiomers of 1-phe- note that the number and structural variety of
nylethylamine (second auxiliary). The resulting di- compounds used to validate the method are too small
astereomeric alcohol-DPEA derivatives are then to guarantee its general applicability to other sub-
examined by NMR (Figure 65). strates. This fact, together with the two-step deriva-
Interpretation of the NMR spectra of the deriva- tization procedure, represents the main limitation of
tives and the assignment of the configurations of the this reagent.
alcohols are based on a model proposed by the 5(R)-Methyl-1-(chloromethyl)-2-pyrro-
authors (based on fast atom bombardment (FAB), UV lidinone. 5(R)-Methyl-1-(chloromethyl)-2-pyrrolidi-
and 2D NMR spectra, and NOE experiments) in none (68.1) has been proposed53 as an auxiliary
which the proton on the asymmetric C atom of the reagent for absolute configuration (and % ee) deter-
alcohol and H(6) on the benzene ring are coplanar minations of chiral secondary alcohols by the forma-
(see Figure 66). According to this conformation, the tion of the corresponding N-(alkoxymethyl)-2-pyrro-
DPEA derivative containing (R)-1-phenylethylamine lidinone derivative 68.2 (see Figure 68).
has substituent L2 shielded by the phenyl group of Theoretical calculations have shown that those
the amine, whereas, in the derivative containing the derivatives exist in equilibrium between two confor-
(S)-1-phenylethylamine fragment, it is substituent L1 mational forms obtained by rotation of the CH2OR
that is shielded. Consequently, the difference in the unit around the C-N bond. The more stable one has
chemical shifts ( ) R - S) for an alcohol with an NCOC angle of 90, whereas the conformation
the stereochemistry shown in Figure 66 is negative with a linear arrangement (NCOC angle of 180)
for substituent L2 and positive for substituent L1. has greater energy. In both, the lactam ring is
52 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 69. Alcohols of known absolute configuration

studied by this method.

Figure 71. Structure and correlation models of -D- and


the NMR data are very limited: Only two alcoholss

one primary and the other secondarysand seven
amines have been studied, and, even then, only the
shift of one of the substituents of the substrate (L1
or L2) is considered.
A model for the correlation of the 31P NMR shifts
Figure 70. Structure of the diazacyclophosphamide chlo- with the configuration is not proposed by the authors;
rides and correlation model for configurational assignment
of alcohols. therefore, the best application of this method is in
the field of e.e. calculation rather than the assign-
essentially planar and the methyl group has a ment of configuration.
pseudo-axial orientation. Tetra-O-benzoylglucosyl Bromides: The
In accordance with that prediction, experimental Glucosylation Method. There have been several
data indicated that the NMR shifts of the NCH2O attempts to use sugars as CDAs for the assignment
protons are determined by the anisotropic effect of of the configuration of alcohols that are derivatized
the CdO bond and the polar effect of the O atom in as glycosides55-57 and examined by 13C and 1H NMR.
each conformer. Under those conditions, a relation- In a few cases, a single derivative is used55 (compari-
ship between the chemical shifts and the relative son of the spectra of the alcohol with that of the
position of those protons, with respect to the carbonyl derivative) and these will be discussed in the corre-
group, and the absolute stereochemistry of the de- sponding section (Section 4.1.2). In this part of the
rivatives can be established. In fact, application of review, however, we are only concerned with cases
the proposed model to six chiral alcohols (Figure 69) in which the assignment is performed by comparison
predicted the correct configuration. of the L and D glycosides.56
The authors warn about the convenience of work- The most commonly used of these procedures
ing with both diastereomeric derivatives; that is to consists of the preparation of the tetra-O-benzoyl--
say, both enantiomers of the chiral alcohol are D-glucopyranoside and tetra-O-benzoyl--L-gluco-
necessary. This is a clear limitation of the method, pyranoside of the alcohol and comparison of their 1H
and the report explicitly says that if only one enan- NMR spectra.56
tiomer of the alcohol is available for derivatization, The interpretation of the spectra and the configu-
assignment of the correct absolute configuration is rational assignment is based on the observation that,
risky. Nevertheless, the procedure works well when in both derivatives, protons anti to the endocyclic
substituents L1 and L2 are clearly different in size. oxygen (substituent L2 in -D-glucopyranoside; see Diazacyclophosphamides. The use of Figure 71) are shielded, probably because of the effect
diazacyclophosphamide chlorides as reagents for of the benzoyl group at C-2, whereas those with the
secondary alcohols and primary amines has been syn arrangement (substituent L1 in -D-glucopyra-
described54 (see Figure 70a). In this procedure, the noside) are deshielded, presumably because they are
linkage between the substrate and the reagent is a close to the endocyclic O atom (O-5).
phosphate or phosphamide bond and the configura- In this way, the differences in the chemical shifts
tion of the substrate is determined by 1H NMR on ( ) D - L) for an alcohol with the configuration
the basis of the models indicated in Figure 70b. shown in Figure 71 are negative for substituent L2
This method is totally empirical and conforma- and positive for substituent L1.
tional studies have not been performed to support the Nine alcohols of known absolute configuration were
model shown. In addition, the number of substrates studied, and these are shown in Figure 72. The
used to validate this model is extremely small and results obtained are in agreement with the predic-
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 53

Table 7. Relationship between the Absolute

Configuration of the Carbinylic Carbon and the Sign
carbinyl carbon
alcohol configuration (C1) D - L
(-)-2-octanol R +0.09
(+)-2-octanol S -0.09
(-)-menthol R +0.16
(+)-menthol S -0.16
(-)-neomenthol R +0.16
(+)-neomenthol S -0.16
(-)-borneol R +0.23
(+)-borneol S -0.23
(3b)-cholesterol S 0.00
(3b)-cholestanol S 0.00
(17b)-testosterone S -0.03
dimethyl-D-malate R -0.14
dimethyl-L-malate S +0.14
(-)-methyl 3-hydroxy-butyrate R +0.02
(+)-methyl 3-hydroxy-butyrate S -0.02

Figure 74.

These rules are based on the study of seven examples

of known absolute configuration (Figure 75), although
Figure 72. Secondary alcohols of known absolute config-
uration studied as tetra-O-benzoyl--glucopyranosyl de- the range of structures is not very diverse (six are
rivatives and the corresponding values. cyclic alcohols and just one is an open-chain alcohol)
and, as such, the use of this reagent for the assign-
tions and, therefore, validate the method. Only in the ment of the configuration of a secondary alcohol is
methylene group of (-)-methyl-3-hydroxybutyrate not yet fully established. In addition, the preparation
are the signs unexpected, and this is probably due of the auxiliary reagent and the derivatization of the
to the anisotropic action of the two carbonyl groups. alcohol are more complex and time-consuming than
The authors also mention that a correlation ap- that with glucopyranosides or AMAAs. Finally, the
parently exists between the chemical shift of the shifts obtained with the fucofuranosides are smaller
proton at C(1)H, the carbinylic carbon, and the than those obtained with the tetra-O-benzoylgluco-
absolute configuration: For the derivatives with the sides, and, for this reason, its use does not offer any
R configuration, is positive, whereas is nega- advantages.
tive when the configuration at that C atom is S (see If the configurational assignment is to be per-
Table 7). Unfortunately, cholesterol and cholestanol formed using 1H NMR spectroscopy, the spectra of
are two exceptions to this rule, and the authors the -L-fucofuranoside and the -D-fucofuranoside are
explain this phenomenon as being due to the absence taken in pyridine and the differences between the
of substituents in the position. chemical shifts (Hp) is calculated ( ) L - D). Fucofuranose Tetra-O-acetate: The In the case where the alcohol has the configuration
Fucofuranoside Method. A procedure has been shown in Figure 74, substituent L1 should result in
described57 that involves comparison of the NMR a positive Hp and substituent L2 should result in a
spectra of the -D- and -L-fucofuranosides (Figure negative value. In this procedure, only the signs for
73) in chloroform or pyridine as the solvent and the the protons on the -carbon of substituents L1/L2 are
application of totally empirical rules (see Figure 74). considered.

Figure 73.
54 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 76. Hp for the carbinyl protons.

Figure 75. Hp values observed for -D- and -L-fuco-

furanoside derivatives.

Figure 75 shows the structures of the compounds

studied by this method, along with the corresponding
Hp data. It can be seen that, with the exception of
furospongin 1, the signs of Hp for the protons on
the -carbons correlate well with the stereochemistry.
However, the other protons do not show any correla-
tion and different signs are obtained for protons in
the same substituent.
A study by the same authors on a series of just four
compounds (Figure 76) led them to suggest that there
could be a correlation between the configuration and
the signs of Hp for the protons on the asymmetric
carbon of the alcohol (carbinyl protons, C(1)H): the
sign of Hp is positive for those classified as S-type
alcohols and negative for R-type alcohols. The small
number of examples investigated, along with the lack
of studies on the fundamental aspects of this method, Figure 77. Chemical-shift differences (13C NMR, C) in
represents a clear limitation for the general ap- pyridine-d5 (Cp) and CDCl3 (Cc) of -D- and -L-
plicability of this approach. fucofuranosides. CDCl3 data given in parentheses.
A procedure very similar to that previously de-
scribed but involving 13C NMR shifts has been -position are considered; because the signs for the
described, and the results are represented in Figure other C atoms are unreliable, the absolute values are
77. Once again, only the shifts of the C atoms at the very small and, furthermore, the degree of substitu-
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 55

Table 8. The b-pl Rules for the Assignment of

Configuration from Noe Lactols
pl 2-H 7a-H C*-H

Figure 78. aryl >0 <0 <0

CN, CO, CtC <0 >0 <0
tion at that C atom should be taken into consider- - AA) of protons H(7a), H(2) in the auxiliary portion
ation for the assignment. and H-C* in the alcohol portion (Figure 79). Alter- Noe Lactols. The lactols derived from natively, the corresponding C-atom signals can be
camphor are known as Noe lactols, and these have considered. The chemical-shift differences should be
been used as CDAs for secondary alcohols58 in which calculated and the signs interpreted, using the so-
one of the substituents (L1 or L2) is a phenyl, nitrile, called b-pl rules (where b denotes bulky and pl
ethynyl, or formyl group. One example of this type denotes plane/lineal) indicated in Table 8.
of reagent is (+)- and (-)endo-octahydro-7,8,8-tri- For example, starting from the lactol MBF-OH of
methyl-4,7-4,7-methanobenzofuran-2-ol ((+)- and type A and a secondary alcohol of unknown config-
(-)-endo-MBF-OH), the structure of which is shown uration, we can obtain two derivatives, arbitrarily
in Figure 78. designated as A-A and A-B (see Figure 79a). If the
The method is totally empirical and consists of the differences ) AB - AA obtained are as shown
derivatization of the alcohol with the two enanti- in Table 8, derivative AA is formed from an alcohol
omers of the reagent MBF-OH, followed by analysis with configuration type A and the derivative denoted
of the 13C or 1H NMR spectra of the resulting as AB comes from the enantiomeric alcohol of con-
diastereomeric lactols. figuration B. If the signs obtained were opposite
The stereochemistry at carbon 2 (Figure 78) in the to those shown in Table 8, the configuration of the
starting hemiketal is not fixed; therefore, when single alcohol would also be opposite.
enantiomers of the auxiliary and the substrate are The chemical shifts (1H and 13C NMR) and their
made to react, two epimers at C-2 result (types A and differences () are shown in Table 9 for the three
B). The predominant epimer in the mixture is the most-relevant signals of a series of alcohols of known
type-A lactol (see Figure 79). absolute configuration. The results obtained validate
The four stereoisomers obtained from a single the rules for the assignment previously discussed and
enantiomer of the auxiliary and the two enantiomers indicate that the method works well with secondary
(named A and B) of the alcohol are represented in alcohols containing aryl, heteroaryl, nitrile, carbonyl,
Figure 79 and classified according to their origin as carboxyl, or ethynyl groups directly bonded to the
AA, AB, BB, and BA. asymmetric center (the pl substituent), as well as in
The assignment of the configuration involves de- cases where the substituents can be easily classified
termining the differences in the chemical shifts (AB according to their b-pl character.

Figure 79.
56 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Table 9. Selected 1H and 13C NMR Data for the Noe

Derivatives of Secondary Alcohols of Known Absolute
hydrogen carbon
2H 7a-H C*-H C-2 C-7a C*
b ) CH3, pl ) CN
5.38 4.42 4.30 108.9 90.1 60.5
5.51 4.19 4.54 106.5 90.3 59.0
-0.13 0.23 -0.24 2.4 -0.2 1.5
b ) C6H5b, pl ) CN
5.35 4.51 5.33 108.1 90.6 66.2
5.71 4.23 5.51 107.0 90.8 65.6 Figure 80. Structures of R-fluoroacetic acids explored as
-0.36 0.28 -0.18 1.1 -0.2 0.6 CDAs.
b ) CH3, pl ) CtCH
5.45 4.38 4.40 106.6 89.3 61.4
5.61 4.21 4.46 105.5 89.3 60.1
-0.16 0.17 -0.06 1.1 0.0 1.3
b ) C6H5b, pl ) CtCH
5.23 4.40 5.37 105.9 89.7 66.9
5.82 4.22 5.45 105.7 89.7 66.2 Figure 81. Main conformations of the esters of AFPA.
-0.59 0.18 -0.08 0.2 0.0 0.7
b ) CH3, pl ) CHdO
5.39 4.31 3.94 108.7 90.2 78.0
5.44 4.27 4.17 107.9 89.9 76.3
-0.05 0.04 -0.23 0.8 0.3 1.7
b ) C6H5b, pl ) CHdO
5.38 4.37 5.00 107.7 90.4 83.2
5.58 4.07 5.14 107.6 90.1 81.6
-0.20 0.30 -0.14 0.1 0.3 1.6
b ) CH3, pl ) C6H5b
5.49 3.95 4.73 106.7 89.5 73.0
5.12 4.32 4.78 105.6 89.0 72.9
0.37 -0.37 -0.05 1.1 0.5 0.1
b ) i-Pr, pl ) C6H5b Figure 82. Secondary alcohols studied by NMR of their
5.40 3.61 4.07 109.1 89.1 84.7 AFPA esters.
5.08 4.30 4.26 105.3 89.3 82.1
0.32 -0.69 -0.19 3.8 -0.2 2.6
although conformer I is the most representative
In the table, b denotes bulky and pl denotes plane/ species from the standpoint of 1H NMR, only con-
lineal. The first-line value indicates Type AB, whereas the
second-line d value denotes Type AA; the third line is . b In former II is used as a model (see Figure 81) for the
this case, because both substituents are plane/lineal, the correlation of the 19F shifts with the configuration of
phenyl group at the position is considered to be the bulky the alcohol. This situation arises because it is this
group. conformer in which the F atoms are subjected to the
electronic effects of substituents L1/L2 of the alcohol
These limitations certainly restrict the use of Noes (the -gauche-type effect). Figure 82 shows a selection
method for other alcohols but, at the same time, of the substrates of known absolute configuration
makes this procedure advantageous, because it al- that produce 19F shifts coherent with the model
lows the assignment of the configuration of alcohols indicated.
that do not have protons in one of the substituents Another fluorine-containing compound used for
L1/L2sa case where the classical Mosher method this purpose is R-cyano-R-fluoro-p-tolylacetic acid60e
cannot be used (see Section 3.1.1). (CFTA). The structure of this compound is shown in Fluoroacetic Acids. As mentioned pre- Figure 80, and it is used for secondary alcohols (1H
viously in this review, with the exception of MTPA, NMR) and amines (19F NMR; see Section The
very few reagents that contain F atoms have been procedure requires the preparation of the esters from
explored59,60 as auxiliary reagents for the assignment the two enantiomers of CFTA and measurement of
of the absolute configuration of substrates through the SR value ((S) - (R)) for the substituents (L1/
F NMR. In general, the structures of these reagents L2) of the alcohol. The assignment is based on the
are derived from that of MTPA (Figure 80) and, fact that, in the ester derived from (S)-CFTA, sub-
although some have been successfully used for the stituent L1 is shielded by the fluorophenyl group
determination of the e.e. of alcohols, their application whereas in the (R)-CFTA ester, the shielded sub-
as CDAs has been hampered by the absence of a truly stituent is L2 (see Figure 83).
general model that can be applied to any other Figure 84 shows the structures of the compounds
substrate. of known absolute configuration used to validate this
In a study describing the application of AFPA (2- procedure, along with the SR values that are
fluoro-2-phenylacetic acid) to secondary alcohols,60a-d obtained. The data demonstrate that, in all cases, the
the authors indicate that the corresponding esters signs of SR are coherent with the configuration and
exist in two conformations (I and II). However, the model.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 57

Figure 83.

rotamer, denoted sp, has the C-F and CO bonds in

a syn-periplanar arrangement and the other, desig-
nated ap, contains the aforementioned bonds in an
anti-periplanar relation. According to the calcula-
tions, the most stable conformer is sp, and this is
represented in Figure 83.
The 19F NMR spectra of these esters have also been
reported; however, a convincing model that allows the
correlation of the absolute configuration of the alcohol
with the signs of SR has yet to be proposed. Chiral Silylating Reagents. Attempts
to use chiral silylating reagents to determine the
absolute stereochemistry of chiral allylic alcohols led
to the proposal of (+)- and (-)-chloromenthoxydiphen-
ylsilanes and (+)- and (-)-2-bromo-R-methylbenzyl-
oxychlorodiphenylsilanes as CDAs.61 Although they
showed promising results, the small number of
alcohols tested (two and one examples, respectively)
prevents their use as a general method.

3.2. Application to -Chiral Primary Alcohols

The extension of the NMR methods for the assign-
Figure 84. ment of the configuration of secondary alcohols
described previously to other substrates is particu-
Theoretical calculations on the geometry and con- larly appealing, because systems such as primary
formational composition of these CFTA esters, using alcohols with the chiral center in the position to
ab initio (GAUSSIAN 98, RHF/6-31+G(d)) methods, the OH group, are frequently found in nature in
revealed that there is an equilibrium between two chiral forms or are obtained by a reduction of the
main rotamers around the CR-CdO bond. One carboxylic acids and aldehydes.

Figure 85. Empirical models for the determination of the absolute configuration of primary alcohols by NMR of their
MTPA esters in the presence of Eu(fod)3. Rm represents the medium-sized group and Rl represents the large-sized group.
58 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

(1) The distance between groups L1/L2 of the

substrate and the aryl ring of the auxiliary reagent
is greater than that in secondary alcohols, because
the asymmetric center is at the -position, and
(2) The presence of an extra C-C bond between
the chiral center and the auxiliary reagent reduces
the conformational preference by increasing the
rotational freedom. As a result, the RS values for
primary alcohols would be expected to be smaller and
less reliable than those usually obtained for second-
ary alcohols.
Very few papers have been published on this
particular subject. MTPA and recently 9-AMA are the
reagents most amply used in the assignment of
absolute configuration of such substrates.
3.2.1. MTPA
A quarter of century ago, Yasuhara and Yama-
Figure 86. Lanthanide-induced shift (Eu(fod)3; LISOMe guchi62a developed a procedure that is based on the
) LISA - LISB) obtained for the OMe group of diatestereo- use of a shift reagent with MTPA derivatives for the
meric (R)-MTPA esters. determination of the absolute configuration of pri-
Two main difficulties are apparent when dealing mary carbinols with a chiral center at the -position.
with primary alcohols: This approach was an extension of a previous method

Figure 87. Natural compounds studied with MTPA and steroidal side chains obtained by synthesis and used as models.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 59

Table 10. Selected NMR Data (CD3OD) for Side-Chain Signals in Synthetic 24-Methyl-26-hydroxy Steroids and in
Natural Compounds 87.6 and 87.7
(R)-MTPA ester
compound 26-CHH2 27-CH3 28-CH3 C-24 C-27 C-28 (26-CH2)
22 Series
(87.8a) (24R,25S) 3.28 dd, 3.60 dd 0.90 d 0.95 d 39.7 13.8 17.1 4.19 dd, 4.31 dd
(87.9a) (24S,25R) 3.29 dd, 3.59 dd 0.90 d 0.97 d 39.5 13.8 16.8 4.13 dd, 4.38 dd
(87.9b) (24S,25S) 3.34 dd, 3.53 dd 0.87 d 1.02 d 39.2 13.6 19.0 4.21 br d
(87.8b) (24R,25R) 3.34 dd, 3.52 dd 0.88 d 1.02 d 39.3 13.6 19.2 4.16 dd, 4.21 dd
Saturated Series
(87.10a) (24R,25S) 3.38 dd, 3.55 dd 0.83 d 0.81 d 35.1 14.8 12.0 4.23 br d
(87.11a) (24S,25R) 3.38 dd, 3.49 dd 0.81 d 0.81 d 35.1 15.1 11.6 4.14 dd, 4.34 dd
(87.11b) (24S,25S) 3.36 dd, 3.58 dd 0.93 d 0.92 d 36.8 17.5 14.4 4.22 dd, 4.32 dd
(87.10b) (24R,25R) 3.37 dd, 3.57 dd 0.91 d 0.91 d 36.1 17.4 14.1 4.16 dd, 4.38 dd
Natural Compounds
(87.6) (24R,25S) 3.46 dd, 3.58 dd 0.91 d 0.97 d 40.4 14.5 17.5 (R)-MTPA: 4.17 dd, 4.33 dd
(S)-MTPA: 4.06 dd, 4.46 dd
(87.7) (24S,25S) 3.34 dd, 3.62 dd 0.92 d 0.92 d 36.7 17.4 14.3 (R)-MTPA: 4.24 dd, 4.32 dd
(S)-MTPA: 4.16 dd, 4.37 dd

Figure 88. Shape of the partial NMR spectra of the 26-methylene protons in (R)- and (S)-MTPA esters of (a) a 25S
steroid and (b) its 25R isomer.

used for secondary alcohols4a,62b,c and consisted of the esters of 2-phenylethanols with bulky Rm groups are
conversion of a mixture of the two enantiomers of a larger than those with smaller Rm groups is consis-
primary alcohol into the corresponding mixture of tent with the model. The procedure was tested on the
(R)-(+)-MTPA esters. The magnitude of the lan- 12 substrates shown in Figure 86, where the chiral
thanide-induced shift (LISOMe) produced by Eu(fod)3 center is not present as part of a ring in any of the
on the OMe group of the MTPA ester showed a cases.
correlation with the absolute configuration. The The application of this method to cyclic alcohols
diastereomeric (R)-(+)-MTPA esters with the larger was explored by Takahashi et al.63a with the terpenes
LISOMe value had configuration A and the diastere- 87.1 and 87.2 shown in Figure 87. The compounds
omer with the smaller LISOMe value had configuration were transformed to the corresponding (R)- and (S)-
B (see Figure 85). MTPA esters and the resulting LISOMe values
The results were rationalized in terms of the indicated configurations that were in agreement with
models shown in Figure 85, where the complexation the known configurations deduced by alternative
constant KA is larger than KB, because of the less- correlation procedures.
marked steric interaction between Eu(fod)3 and the The same method has also been applied to the
H atom at the chiral center in A than with Rm in B. determination of the stereochemistry of a variety of
The fact that the magnitudes of LISOMe for the steroidal side chains. Minale et al.63b,c assigned the
60 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

stereochemistry at C-25 of a series of polyhydroxy-

lated steroids, such as 87.3 or 87.4 (Figure 87), which
were isolated from different starfish species.
It has been observed empirically that the LISOMe
signal in the 1H NMR spectrum of a primary carbinol
(+)-MTPA ester with R chirality at the -carbon (or
a (-S) carbinol (-)-MTPA ester) is greater than that
of its diastereomer. This information was used in the
assignment of the C-25 configuration of steroid 87.5
(Figure 87), which was obtained after acid metha-
nolysis of pavoninin 1, isolated from the defense
secretion of the sole P. pavoninus.63d The addition of
an equimolar amount of Eu(fod)3 to a mixture of the
(+)- and (-)-MTPA esters (ca. 10:7) of 87.5 in CDCl3
shifted the two OMe signals by 0.68 and 0.66 ppm,
respectively, and the addition of 2.8 molar equiva-
lents Eu(fod)3 shifted them by 2.05 and 2.00 ppm,
respectively. The larger downfield shift observed for
the more intense OMe signal of the (+)-MTPA ester
showed the configuration at C-25 to be R.
For those cases where the steroidal side chains had
an extra chiral center at C-24, several compounds of
known stereochemistry were synthesized and used
as model systems. This was the case for two naturally
occurring marine steroids from a starfish and an
ophiuroid: 87.6 and 87.7, respectively64 (see Figure
Also, the stereoisomers of 22E and the saturated
24-methyl-26-hydroxy steroids 87.8-87.11 (Figure
87) were prepared by Claisen rearrangement and the
relative stereochemistries (threo or erythro) were
deduced by comparison of 1H and 13C NMR spectra
(see Table 10) and application of the Yasunara-
Yamaguchi method that established the configura-
tion at C-25.
Similar NMR studies of MTPA esters showed that
there was a correlation between the shape of the
signals due to the CH2OH (C-26) group in the (R)-
and (S)-MTPA esters and the absolute configuration Figure 89. Chiral primary alcohols used in these studies.
at C-25. This new approach (i.e., comparison of the
chemical shifts of the methylene protons at C-26 in
ences between the methylene group in the (R)- and
the spectra of the (R)- and (S)-MTPA esters) has the
(S)-MTPA esters very small ( 4.15/4.25 and 4.15/
advantage of avoiding the use of europium salts and
has been widely used by researchers interested in the 4.23, respectively). Removal of the sulfate group
stereochemistry of steroid side chains.65 produced the expected sets of doublet of doublets at
4.14/4.27 and 4.19/4.23, respectively. This case
It can be seen from Figure 88 that, in the spectra
illustrates the limitations imposed by the presence
of the MTPA esters of a 25S isomer, the C-26
of bulky substituents in ring D of steroids.66a
methylene protons resonate much closer to each other
in the spectrum of the (R)-MTPA ester than in the Other examples of the use of this technique include
(S)-MTPA derivative, whereas the reverse situation 2,6-dimethylheptyl sulfate, 89.1 (see Figure 89),
occurs for the MTPA esters of a 25R isomer. which was isolated from marine ascidians and its
The assignment of the configuration of the marine configuration at C-2 was inferred as R, from the
polyhydroxylated steroids 88.1 and 88.2 was con- application of the same method to its desulfated
ducted using this technique. In the case of 88.1, the derivative 89.2.66b Kobayashi et al. also used this
signals due to the methylene at C-26 in the (R)-MTPA technique on a variety of marine metabolites such
derivative appeared as two well-separated doublet of as the ecdysteroids palythoalones A (89.3) and B
doublets (dd) at 4.14 (J ) 6.6 and 10.8 Hz) and 4.28 (89.4) 66c and the amphidinolides T (89.5)66d and E
ppm (J ) 6.4 and 10.8 Hz) ( ca. 0.14 ppm). The (89.6)66e In these latter cases, the absolute configu-
(S)-MTPA derivative, on the other hand, exhibited ration was deduced after degradation of the natural
very similar dd signals, at 4.19 and 4.23 ppm ( ca. product (89.7, 89.8, 89.9, and 89.10), subsequent
0.04 ppm), thus indicating an R configuration for the derivatization with MTPA, and the use of compounds
compound in question. The same procedure was with known absolute configuration, obtained by
applied to 88.2; however, unfortunately, the presence synthesis, as models for comparison of the NMR
of the bulky sulfate group at C-15 made the differ- spectra.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 61

Figure 90. 19F NMR chemical shifts of MTPA esters of primary hydroxy-sulfonamides and empirical models.

In 1996, Seebach and co-workers67 proposed a has been shown29,30 to possess two very important
method based on 19F NMR analysis of the MTPA features: (a) a higher conformational preference (the
esters and, more specifically, directed toward the syn-periplanar form, sp), than other AMAA reagents,
determination of the absolute configuration of hy- and (b) the anthryl ring in the main conformer (sp)
droxy-sulfonamides. The study was comprised of six is particularly well-oriented, in terms of projecting
cyclic hydroxy-sulfonamides, one cyclic hydroxy-ester, its strong aromatic magnetic cone on substituent L1
and just one acyclic primary alcohol (2-methylbu- or L2.
tanol) of known configuration. A correlation between No such advantages are expected from MPA or
the relative configuration of the MTPA esters with MTPA, because they have lower conformational
the 19F NMR chemical shift of the CF3 group was preferences, less-favorable orientation of the phenyl
deduced. Figure 90 shows the structures, chemical ring (MTPA), and a weaker magnetic cone (phenyl
shifts, and the empirical model used to explain the versus anthryl). Indeed, when these three reagents
correlation. were tested using 2-methylpropan-1-ol as a model
compound, completely separate signals for the di-
3.2.2. 9-AMA astereotopic methyl groups were obtained only in the
The most general approach to the assignment of 9-AMA esters69 (RS ) 0.033 versus RS ) 0.002
the absolute configuration of primary alcohols is ppm for 9-AMA and MPA respectively, and no
based on the use of 9-AMA,68,69 because this reagent separation for MTPA).
62 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 91. Low-energy conformers (perspective and Newman projections) for (R)-9-AMA esters of primary alcohols. In
most cases, sp-a/a is the most populated and relevant for NMR studies.
Complex theoretical studies (including molecular Sixteen 9-AMA esters of primary alcohols were
mechanics, semiempirical, ab initio, and aromatic studied,69 and, in most cases (93.1-93.13; see Figure
shielding-effect calculations), complemented by D 93), the conformational preferences and the model
NMR, were performed69 with simple model com- previously described do, in fact, apply. This observa-
pounds to assess the viability of 9-AMA esters for this tion means that the NMR spectra of this set of
purpose. The results of these studies indicated that 9-AMA esters could be reliably interpreted on the
the main conformational processes involve rotation basis of the corresponding model, and NMR spec-
around 1 and 2 bonds (see Figure 91 a), generating troscopy can be used to correlate the absolute con-
three main low-energy conformations (syn-peripla- figuration at the asymmetric center of the 9-AMA and
nar-gauche+/anti (sp-g+/a), syn-periplanar-anti/ that of the alcohol.
anti (sp-a/a), and syn-periplanar-gauche-/anti (sp- Unfortunately, in three examples involving cyclic
g-/a); see Figure 91b), with one of these (sp-a/a) structures or bulky substituents (93.14-93.16), the
becoming the most significant, in terms of NMR calculations showed that the sp-a/a forms were not
spectroscopy in most cases. In all these conforma- the preferred forms and that the sp-g-/a and sp-g+/a
tions, the 9-AMA substructure maintains its most- forms make a very significant contribution. In these
stable sp form. cases, it is clear that the sp-a/a model is not valid
Because conformer sp-a/a is the more stable and for interpretation of the NMR spectra of the 9-AMA
representative, from the standpoint of NMR, for a esters.
primary alcohol with the stereochemistry shown in In conclusion, to assign the absolute configuration
Figure 92, substituent L1 will be more shielded in of a primary alcohol by this method, a preliminary
the (R)-9-AMA ester, whereas substituent L2 will be calculation must be performed (being the best force
in the (S)-9-AMA ester. Therefore, a negative RS field, PCff), especially when the chiral center is part
sign is expected for substituent L1 and a positive RS of a ring, or when strong steric interactions could be
sign is expected for substituent L2. in operation, to confirm that the anti form is the most
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 63

Figure 92. Conformational model for the determination of the absolute configuration of -chiral primary alcohols by
NMR of their 9-AMA esters.

stable one and, consequently, that the model shown number of substrates studied is small and their
in Figure 92 is representative and can be applied to structural variety is limited; therefore, the methods
the interpretation of the chemical shifts. are applicable only to the cases reported.
The use of the auxiliary reagents MTPA and MPA
did not generate either the required RS values or 3.3.1. MTPA
homogeneity in the sign distribution, and this situ-
ation confirmed that these compounds could not be The use of MTPA with tertiary alcohols71 was
used as auxiliary reagents with chiral primary alco- explored for just two examples: pinan-2-ols 95.1 and
hols. 95.2 (see Figure 95).
A good example of the application of this procedure A combination of MM2 calculations and NOE and
for the determination of the configuration of a anisotropic effects (measured as SR of the methyl
complex substance is shown in Figure 94, which groups) was used to predict the configuration of these
depicts oxazinin-1, a cytotoxic compound that has substrates. The authors state that MTPA can be used
been isolated from the digestive glands of an edible in combination with conformational analysis; how-
mussel (Mytilus galloprovincialis). That was recently ever, the method was not expanded to include other
published by Fattorusso et al.70 In that paper, MM alcohols.
calculations were initially performed to check the
reliability of the NMR method with that particular 3.3.2. Methoxy-(2-naphthyl)acetic Acid (2-NMA)
substrate, and the stereochemistry was determined A method for the prediction of the absolute config-
using the 9-AMA esters only after a positive result uration of acyclic R-methyl-substituted tertiary al-
had been obtained. cohols has been published and involves the use of
methoxy-(2-naphthyl)acetic acid (2-NMA) as an aux-
3.3. Application to r-Chiral Tertiary Alcohols iliary reagent.72
Although the determination of the configuration of The method is based on an analysis of the NMR
chiral secondary alcohols by NMR has been satisfac- spectra of the 11 compounds of known configuration
torily accomplished through the use of the various shown in Figure 96. According to the authors, the
alternatives already discussed (see Section 3.1), a RS signs obtained are systematically distributed as
general methodology for chiral tertiary alcohols has positive and negative for substituents L1 and L2.
not yet been established. This fact is mainly due to This arrangement can be represented by the model
intrinsic problems associated with these substrates, shown in Figure 97, and it was used by the authors
such as difficulties in making derivatives (i.e., esters) for the assignment of configuration. The -methylene
in high yields and their unsuitable conformational and methyl protons directly bonded to the tertiary
characteristics. A few attempts have been made to hydroxyl group should not be considered for the
find solutions to these problems but usually the assignment.
64 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 96. R-Methyl-substituted tertiary alcohols used by

Kobayashi et al.

Figure 97. Empirical model to determine the absolute

configuration of R-methyl-substituted tertiary alcohols.

3.3.3. The Fucofuranoside Method

Kobayashi73 applied a method that was devised for
Figure 93. RS values for the 9-AMA esters of primary the determination of the absolute configuration of
alcohols. secondary alcohols to the series of six chiral tertiary
alcohols (substituted with methyl and two methylene
groups) shown in Figure 98. The method involves the
derivatization of the alcohol as -D- and -L-fucofuran-
osides, comparison of the NMR spectra (1H and 13C)
of the derivatives taken in pyridine-d5, and evalua-
tion of the values (L - D) and signs for the
proximate protons (1H NMR) or -carbons (13C NMR).
Figure 94. Structure of oxazinin-1. The experimental data show that the values are
generally positive for the protons in the right-hand
segment (Rr) and negative for those in the left-hand
segment (Rl); however, in several cases, both positive
and negative signs coexist on the same methylene
Figure 95. The values from the 13C NMR study are positive
Other drawbacks associated with this method are for the right-hand -carbon and negative for the left-
the low yields reported for the esterification (i.e., hand -carbon, except for the five-membered ring
27%) and the racemization problems that are en- compound, which did not follow this trend.
countered. MTPA and MPA were also tested; how- These results can be interpreted on the basis of the
ever, they resulted in smaller values than those model shown in Figure 99. This model does not have
for 2-NMA. any proven conformational meaning, because NOE
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 65

absolute configuration of primary amines8c,74 that

have a chiral center at the R-position. The model used
to correlate the stereochemistry with the chemical
shifts is analogous to that proposed for secondary
alcohols and, although a procedure based on 19F NMR
studies of MTPA esters has been formulated for
alcohols (Figure 8), a similar application with amines
has very rarely been used.
The method is standard and involves the prepara-
tion of the corresponding MTPA amides from the two
enantiomers of MTPA and the amine of unknown
configuration. The NMR spectra of the two deriva-
tives are then recorded and the differences in the
chemical shifts (SR) are calculated. Mosher pro-
posed that the most-relevant conformer would be the
one in which the CF3, carbonyl, NH, and C(1)H
groups are in the same plane, with the CF3 and the
carbonyl units in a syn-periplanar disposition. In
accordance with this model (see Figure 100), sub-
stituent L2 is shielded in the (R)-MTPA amide and
Figure 98. Tertiary alcohols used in this study. substituent L1 is shielded in the (S)-MTPA amide.
For an amine with the configuration shown in Figure
100c, substituent L1 has negative SR values and
substituent L2 has positive values. In the case where
the amine has the opposite configuration, as in
Figure 100d, the signs are also opposite to those in
the example in Figure 100c.
Figure 99. Models for assignment of tertiary alcohols as In an effort to establish the scope and limitations
-D- and -L-fucofuranosides. of this method, theoretical calculations (MM, semi-
or low-temperature NMR studies or geometry calcu- empirical AM1) and NMR studies (aromatic anisot-
lations were not performed to establish the confor- ropy increments, dynamic NMR) have recently been
mational characteristics of the derivatives. conducted.75
Indeed, the author states that the method may not This conformational study showed that the main
be valid for tertiary alcohols with more-complex processes are those due to rotations around the CO-
substitution patterns. In addition, the very low yields NH, CR-CO, and CR-Ph bonds (see Figure 101).
obtained in the derivatization step (in the range of Rotation around the CO-NH bond generates two
2%-23%) or, in some cases, even the failure to obtain rotamers (Z and E). The Z rotamer is predominant,
the fucofuranosides, represents a major limitation to because of its greater stability, in terms of energy.
the use of this method. Rotation around the CR-CO bond produces the two
3.4. Application to r-Chiral Primary Amines usual sp (CF3 and carbonyl syn-periplanar) and ap
(CF3 and carbonyl anti-periplanar) conformers. Rota-
3.4.1. MTPA tion around the CR-Ph bond results in three main
Another of the applications of Moshers method orientations, with the phenyl group coplanar with
is the determination by 1H NMR spectroscopy of the either the CR-OMe, CR-CO, or CR-CF3 bonds.

Figure 100. (a, b) Moshers model for the correlation of the configuration with the 1H NMR shifts and (c and d) expected
signs of SR.
66 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 101. Generation of Z-conformers in MTPA amides and the most significant characteristics of each one.

The combination of these three orientations of the of the aromatic ring to the chemical shifts (aromatic
phenyl group with the two sp and ap conformers anisotropy increments) of each conformer.
generates six possible situations (as in the case of In the conformational equilibrium, each conformer
esters).27 These six possibilities can be reduced to just plays a different role (shielding or deshielding) and
three (sp1, ap3, and ap1; see Figure 101)sas in the overall result is the weighted average among the
MTPA esters27son the basis of theoretical calcula- conformer populations and their shielding/deshield-
tions (MM and AM1). The most stable conformer is ing effects. From the NMR standpoint, the L1 sub-
sp1, with the other two conformers (ap3 and ap1) stituent of an (S)-MTPA amide is shielded in con-
being energetically less stable (see Figure 102). former sp1 (see Figure 103a), while substituent L2
In the sp1 conformer, the CF3 group is syn- is virtually unaffected because it is deshielded in
periplanar to the carbonyl and the phenyl ring is conformer ap1 and shielded in conformer ap3 (Figure
coplanar with the CR-OMe bond. Overall, this ar- 103a). The magnitude of the shielding/deshielding
rangement leads to selective shielding on one of the effects on substituent L2 depends on the difference
substituents (L1/L2) of the amine (see Figure 102a). in the populations of the two conformers. Similarly,
In the other two conformers (ap1 and ap3), the CF3 in the (R)-MTPA amide, substituent L2 is shielded
is anti-periplanar to the carbonyl group. However, in conformer sp1 and substituent L1 is almost un-
in conformer ap1, the phenyl ring is coplanar with affected (deshielded in conformer ap1 and shielded
the CR-OMe bond, causing deshielding on one sub- in conformer ap3) (Figure 103b).
stituent of the amine (see Figure 102b), whereas in The overall result is that the L1 substituent is more
conformer ap3, the phenyl ring is coplanar to the shielded in the (S)-MTPA than in the (R)-MTPA
CR-CF3 bond, which produces shielding (see Figure amide. Conversely, substituent L2 is more shielded
102c). in the (R)-MTPA than in the (S)-MTPA amide. When
These shielding/deshielding effects were quantita- the differences in the chemical shifts are calculated
tively demonstrated by calculating the contributions for an amine with the configuration shown in Figure
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 67

100c, SR has a negative value for substituent L1

and a positive value for substituent L2. In the case
where the configuration is opposite, so too are the
signs for SR (see Figure 100d).
Further experimental evidence to support this
equilibrium and the structures of the conformers was
obtained from the study of the low-temperature 1H
NMR spectra of the MTPA amides of (+)-bornyl-
amine. The results (Figure 104a) are consistent with
the previously discussed predictions and illustrate
the effect of the phenyl ring on the substrate in each
Because of the greater significance of conformer sp1
in comparison to conformers ap1 and ap3, conformer
sp1 can effectively be used as a correlation model for
the assignment of the configuration of amines from
the NMR spectra. This simplified model is shown in
Figure 104b and is in full agreement with that
originally proposed by Mosher (see Figure 100a). For
instance, Me(10) is shielded by the phenyl group in
the (R)-MTPA amide of (+)-bornylamine and H(6)
is shielded in the (S)-MTPA amide, causing the SR
sign to be positive for Me(10) and negative for H(6).
The reliability of this model for the prediction of
the configuration was confirmed experimentally by
comparing the NMR spectra of the MTPA amides
derived from a series of R-substituted primary amines
of known absolute configuration.74,75 The signs of
SR are consistent with the model in all the ex-
amples studied, and a selection of data is shown in
Figure 105.
The MTPA derivatives of alcohols27 and amines75
both have a conformational equilibrium that is
considerably more complex than that of other re-
agents such as MPA (MTPA results in three main
conformers with close populations instead of two29).
However, there is a difference between MTPA esters
and amides that is of significant practical impor-
tance: the major conformer in esters causes deshield-
ing, whereas in amides, it causes shielding, and,
therefore, larger SR values are obtained for amides
than esters. For this reason, the use of MTPA as a
CDA for the determination of the configuration of
Figure 102. Main conformers for the MTPA amides. alcohols is frequently hampered by the small changes

Figure 103. Low-energy conformations of (a) (S)-MTPA and (b) (R)-MTPA, as obtained by MM and AM1 calculations.
68 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 104. (a) Low-energy conformations of the (R)-MTPA amide of (+)-bornylamine. Solid arrows show shielding, and
dashed arrows show deshielding effects. (b) Simplified models for (R)- and (S)-MTPA amides.

in shift obtained and the uneven distribution of SR Z and E rotamers result from rotation around the
signs;13,27 such problems for MTPA amides have not CO-NH bond, with the Z arrangement being the
been reported. predominant one. The CR-CO rotation generates the
two usual conformers for this type of system: sp
3.4.2. MPA (OMe and carbonyl in a syn-periplanar disposition)
and ap (OMe and carbonyl in an anti-periplanar
The application of MPA to the determination of the disposition) (Figure 106). In both the sp and ap forms,
configuration of R-substituted primary amines is a the OMe is anti, with respect to the CR-Ar bond,
very recent development.76,77 At present, there are and the phenyl group is coplanar to the CR-H bond.
two methods available for this reagent. The method
Theoretical calculations showed that the ap con-
described in this chapter involves the use of the two
enantiomers of MPA,76a whereas only one enantiomer former has a lower energy than the sp conformer (this
is necessary in the procedure described in section 4.2 is opposite to the case for MPA esters, where sp is
of this review.77 the most stable conformer). The evolution of the NMR
spectra of the MPA amide of (+)-bornylamine at
The method consists of the derivatization of the
different temperatures confirmed these conforma-
amine with the two enantiomers of MPA, acquisition
tional characteristics.
of the 1H NMR spectra of the corresponding (R)- and
(S)-MPA amides (preferably in a nonpolar solvent76a), The results of all these studies indicate that, in
and analysis of the RS parameters. solution, the MPA amides exist in a conformational
The RS signs for substituents L1/L2 are correlated equilibrium composed of two main conformers: ap-Z
to the absolute configuration of the amine through (MeO and carbonyl, anti-periplanar) and sp-Z (MeO
out a nonempirical model, which has been established and carbonyl, syn-periplanar; carbonyl and C(1)H
from a detailed study of aromatic shielding effects bond, syn-periplanar). In both cases, the methoxy
and structural calculations76a (molecular mechanics group, the carbonyl group, the N-H bond, and the
(MM), semiempirical (AM1, PM3) and ab initio), as CR-H bond are in the same plane and the phenyl
well as by NMR experiments76a (low-temperature and ring is almost coplanar to the CR-H bond (Figure
dynamic NMR). 106).
The conformational analysis shows that the main Accordingly, in (R)-MPA amides, the L2 substituent
conformational processes involve rotations around is shielded by the phenyl ring in the ap conformer
the CO-NH and CR-CO bonds (see Figure 106). Two whereas substituent L1 is shielded in the sp con-
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 69

Figure 105. SR values (ppm, CDCl3) of the (R)- and (S)-MTPA amides of a selection of amines of known configuration.

former (Figure 107a). In the (S)-MPA amides, how- The reliability of the assignment obtained from the
ever, substituent L1 is shielded in the ap conformer model shown in Figure 107c and d has been demon-
and substituent L2 is shielded in the sp conformer strated experimentally with a series of amines of
(see Figure 107b). Given that the ap conformer is known absolute configuration (see Figure 108); in all
more populated than the sp conformer, the L2 sub- cases, the signs of the RS values are consistent with
stituent is more shielded in the (R)-MPA than in the the predictions.
(S)-MPA amides and, conversely, substituent L1 is MPA versus MTPA. When determining
more shielded in the (S)-MPA than in the (R)-MPA the configuration of a substrate, it is important to
amides. Comparison of the spectra of the two deriva- select the most suitable CDA available. In the case
tives of an amine with the configuration shown in of amines, note that there are no significant differ-
Figure 107c shows that the RS value is positive for ences in the magnitude of obtained for MPA
substituent L1 and negative for substituent L2 (with (RS) and MTPA (SR) amides, indicating that
opposite signs for the enantiomeric amine) (see neither of these reagents has a clear advantage over
Figure 107d). In practice, this method effectively the other75 (see Figure 108). This situation may seem
considers the ap conformer as a simplified model for surprising initially, because the population of the
the assignment of configuration of the amine by predominant conformer in MTPA amides is smaller
correlation of the RS signs with the configuration. than that in MPA amides. However, in the former
70 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 106. Generation and main conformers of MPA amides.

case, the phenyl ring is better orientated for shielding 3.4.3. AMAAs
than in MPA amides and the combination of the two
factors leads to similar shift changes. Other than MPA and MTPA, other arylmethoxy- The Effect of the Polarity of the Sol- acetic acids with different aryl rings (9-AMA and
vent on the NMR of MPA Amides. MM calcula- 1-NMA) have been explored as CDAs for amines.76a
tions on MPA amides revealed that the dipolar The predominant conformer in the amides derived
moment of the predominant ap-Z conformer is smaller from both reagents was ap-Z (as in MPA amides).
than that of the sp-Z conformer.76a Thus, the popula- Despite the presence of an anthryl or naphthyl ring
tion of ap-Z (and, therefore, the RS values) should in these reagents, NMR experiments show that the
increase if the polarity of the NMR solvent is reduced. RS values obtained for 9-AMA and 1-NMA amides
This proposal has been experimentally proven76a are no higher than those obtained with MPA or
by acquiring NMR spectra in several solvents, which MTPA. This is probably due to a lower population of
all showed the expected trend for RS values: RS- the shielding conformer or to an unfavorable orienta-
CS2 > RSCDCl3 > RS(CD3)2CO (see Figure 109). tion of the aryl ring. In either case, the practical
Therefore, one way to increase the efficacy of MPA result is small RS values; therefore, the use of
as a CDA for amines (larger RS values) involves 9-AMA and 1-NMA as CDAs for amines does not
recording the spectra of the corresponding amides in provide any advantages over MTPA or MPA.
nonpolar solvents such as CS2.
3.4.4. Boc-phenylglycine (BPG)
A similar improvement in the RS data for MPA
esters was obtained by reducing the temperature of The use of Boc-phenylglycine (BPG; see Figure 110)
the NMR probe. This technique was described in as an auxiliary reagent78 fills a gap that neither
Section MTPA, MPA, nor the other AMAA reagents were able
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 71

Figure 107. (a, b) Conformational equilibrium for MPA amides. (c, d) Prediction of RS signs.

Figure 108. Selected RS values (ppm) obtained from the 1H NMR spectra of (R)- and (S)-MPA amides (in CDCl3) of the
illustrated amines. For the sake of comparison, SR values obtained with MTPA are shown in parentheses.
to cover due to the small magnitude of the differences The procedure consists of the derivatization of the
between the chemical shifts (RS or SR) they amine of unknown configuration with the two enan-
generate. tiomers of BPG, recording of the 1H NMR spectra of
72 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

The main conformational processes involved are

shown in Figure 111, and their study led to BPG
amides being characterized as follows:
(a) In the amide bond (CO-NH), the Z isomer is
more stable than the E isomer.
(b) Rotation around the 1 (CR-CO) bond gener-
ates the common sp and ap conformations (CR-H
bond and carbonyl group, syn-periplanar and anti-
periplanar, respectively).
(c) The phenyl group is coplanar with the CR-H
bond by rotation around 2 (CR-Ph).
(d) The CR-H, N-H, and CdO bonds have a
tendency to remain coplanar, the CR-H and N-H
bonds (3) have a tendency to remain in an anti
disposition, and the N-H bond have a tendency to
be in an anti disposition, with respect to the CdO
bond (4).
Both the theoretical calculations and the NMR
studies revealed that the most important conforma-
Figure 109. Plots showing the dependence of RS tional process is the equilibrium between the ap and
(expressed as a percentage of RS in (CD3)2CO) versus the sp conformers, with the former being the most stable
polarity of the solvent (as ( - 1)/(2 + 1), where  is the (see Figure 112).
dielectric constant): Line 1, Me(10) of (+)-bornylamine; In this way, substituent L1 is shielded by the
Lines 2 and 3, H(3) and H(4) of L-valine methyl ester,
respectively; and Lines 4 and 5, H(2) and H(7) of L-
phenyl group in the ap conformer and substituent L2
tryptophan methyl ester, respectively. is shielded in the sp conformer in the case of (R)-
BPG amides (see Figure 113a). Conversely, substitu-
ent L2 is shielded in the ap conformer and substituent
L1 is shielded in the sp conformer in (S)-BPG amides
(see Figure 113b). Because the ap population is larger
than the sp population, L1 is the most shielded
substituent in (R)-BPG amides and L2 is the most
shielded substituent in (S)-BPG amides.
Figure 110. Structures of (R)- and (S)-BPG.
Thus, when the differences in chemical shifts are
measured (RS), a negative value is obtained for
the corresponding diastereoisomeric BPG amides and substituent L1 and a positive value is obtained for
measuring the differences in the chemical shifts substituent L2 if the amine has the configuration
(RS) of substituents L1/L2. The signs of RS are shown in Figure 113c. Opposite signs are clearly
then correlated with the absolute configuration by obtained if the configuration of the amine is opposite
means of a nonempirical model that was established (see Figure 113d).
by detailed conformational analysis,78 including MM This prediction has been experimentally proven in
and semiempirical (AM1) calculations, and experi- a series of amines of known absolute configuration
mentally proven by exhaustive NMR studies. and with a variety of structures (see Figure 114).

Figure 111. Generation of conformers of BPG amides.

Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 73

Figure 112. Main conformers of BPG amides: (a) sp Figure 114. Selected RS values for (R)- and (S)-BPG
conformer and (b) ap conformer. amides.

The ap conformer is the major component in the For the sake of comparison, Figure 116 shows the
mixture and, therefore, is the most significant, from values observed in a selection of BPG (RS), MPA
the NMR standpoint. As such, it can be used as a (RS), and MTPA (SR) amides derived from amines
simplified model to correlate the RS signs with the of known configuration. The data show beyond doubt
absolute configuration of the amines. This simplified that BPG produces the largest values and, hence, the
model is shown in Figure 115 and can be used to most-accurate assignments.
place substituents L1 and L2 on the amine by simply The reason for the advantage that is inherent in
considering the relevant RS signs. BPG systems, in comparison to the other reagents,

Figure 113. Conformational equilibrium in BPG amides.

74 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 115. Simplified model for the assignment of the configuration of BPG amides.

two enantiomerically pure forms, and, in addition,

it is cheaper than any of the alternative reagents.
3.4.5. Other Reagents
In addition to the reagents already described in this
section, several other CDAs have been used with
amines, and these compounds are discussed in this
Several methods and reagents are specifically not
covered, because they have already been discussed
in other sections (i.e., Helmchens reagent44,45 (PBA)
and diazacyclophosphamides54 are used for both
alcohols and amines) or have been extensively de-
scribed in other review articles4a (e.g., 2-[1-(9-an-
thryl)-2,2,2-trifluoroethoxy]acetic acid79 (ATEA) and
R-methyl-R-methoxypentafluorophenylacetic acid80
(MMPA)) for which no new relevant contributions
have appeared since their last publication. 1-Fluoro-2,4-dinitrophenyl-5(R,S)-phen-
ylethylamine [(R,S)-FDPEA]. This is the same
reagent that is used in the difluoronitrobenzene
method for the determination of the absolute config-
uration of secondary alcohols.52 In its application to
amines,81 (R)- and (S)-FDPEA (Figure 118) are
reacted with the amine of unknown configuration to
give the corresponding diastereomers. On the basis
Figure 116. Comparison of the values of BPG (RS), of UV and NMR studies, a predominant conformation
MTPA (SR), and MPA amides (RS). Data for BPG given
in boldface type, data for MTPA given in normal typeface, has been proposed that has the R-protons of the
and data for MPA given in parentheses. amine and phenylethylamine moieties, as well as the
phenyl H6, located in the same plane (see Figure
119a). In accordance with this model, in the (R)-
lies in the coexistence of two factors that only work FDPEA derivative, substituent L2 is shielded by the
together in BPG amides: (a) the excellent position phenyl group of the phenylethylamine while L1
of the phenyl group to transmit its shielding effect remains unshielded. On the other hand, in the (S)-
to substituents L1/L2 of the amine in the most stable FDPEA derivative, substituent L1 is shielded whereas
conformer (ap; see Figure 117a) and (b) a conforma- L2 is not (Figure 119a). When the differences in the
tional equilibrium of just two conformers (sp/ap). The chemical shifts (RS) are calculated for an amine
good orientation of the phenyl ring is also present in with the configuration shown in Figure 119b, sub-
MTPA amides75 (Figure 117c); however, its effect is stituent L2 should show a negative RS value and
reduced by a three-conformer composition. In MPA substituent L1 should show a positive RS value.
amides, the equilibrium is simple76 but the orienta- Table 11 shows the RS values for the three
tion of the phenyl is not as good as that observed in FDPEA amides of known absolute configurations
the other two cases (see Figure 117b). that have been used to validate the previously
In conclusion, BPG has structural and conforma- described predictions. However, despite the fact that
tional features that make it the reagent of choice for the signs are as expected and the SR values are
the assignment of the absolute configuration of larger than those obtained with MTPA (included in
primary amines. It is commercially available in its Table 11 for comparison), the number of test com-
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 75

Figure 117. Main orientation of the phenyl ring and direction of maximum shielding in (a) BPG, (b) MPA, and (c) MTPA

amines. The O-aryllactic acids (ROAL) shown in

Figure 121 have been proposed for the determination
of the absolute configuration of R-substituted primary
amines, on the basis of theoretical and experimental
Figure 118. Structure of (R)- and (S)-FDPEA. MM and semiempirical calculations, together with
NMR data, suggest that the amides derived from the
pounds is too small to recommend the method for ROAL preferentially exist in the ap-Z conformation
application to amines in general. in solution. In this conformation, the aryl and car-
Despite this limitation, the method has been used bonyl groups are anti-periplanar (Figure 122), as in
to determine the configuration of microginin (Figure MPA amides, but are different from the main con-
120), which is a natural product from cyanobacteria.81 formation of their esters with secondary alcohols, (R)-O-Aryllactic Acid (ROAL). As dis- where the CR-H and CdO bonds are syn.
cussed previously, many O-aryllactic acids have been According to this conformational model, the ROAL
used as derivatizing agents for the determination of amide derived from an amine with the configuration
the enantiomeric purity3a,60d of alcohols and amines shown in Figure 122a has substituent L1 shielded
by 19F NMR, for the assignment of the absolute while substituent L2 is not. Conversely, if the amine
configuration of amines82 and as solvating agents83 had the configuration shown in Figure 122b, then the
in the determination of the enantiomeric purity of shielded group would be substituent L2.
76 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 119. Model used to correlate the configuration with the 1H NMR shifts and RS signs.
Table 11. Comparison of Values between FDPEA and MTPA Amides
L-isoleucinol L-phenylalaninol
R-CH 0.0045 -0.0420 -0.0725 0.0400 0.0240
-CH 0.3120 0.0000
-CH2 0.2410 -0.0270 -0.2880
0.2855 -0.0010 -0.3410
-CH3 0.2260 -0.0420
-CH2 -0.43750 0.0500 -0.4825 0.0570 0.4355
-0.5085 0.0700 -0.5360 0.0540 0.4880
-CH2 0.3910 -0.0840
0.3095 -0.0900
-CH3 0.2830 -0.0560
-NH 0.0020 -0.1420 -0.0230 0.1210 -0.0695

Figure 120. RS values for microginin-FDPEA.

Figure 122. Model for the assignment of the configuration

of (R)-O-aryllactic acid amides.
Figure 121. Structures of the O-aryllactic acids.
limitations are apparent: Only one acid (121.1) was
Therefore, determination of the configuration of tested with all the amines, whereas the others were
R-substituted primary amines or R-amino acids is tested with just one, two, or three amines.
possible by comparison of the chemical shifts of the In most cases, only the values for one substitu-
relevant substituents. ent (either L1 or L2) are reported. All the O-aryllactic
Experimental validation of the predictions given acids described have the (R)-configuration (i.e., they
by this method was provided by the ROAL deriva- are prepared from (S)-ethyl lactate) and examples of
tives of the amines shown in Figure 123. The avail- the application of both enantiomers of the reagent
able RS data is presented in Table 12, and some to one enantiomer of the amine have not been
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 77

Figure 123. Structures of the amines used in this study.

Table 12. Selected Values for (R)-O-Aryllactic Acid
Figure 124. Conformational equilibria of CFPA (Ar ) Ph)
ROAL amine Ha Hb Hc Hd and CFTA (Ar ) p-tol) amides.
121.1 123.1 0.09 -0.15
121.3 0.11 -0.21 Table 13. F Values for CFTA and MTPA Amides
121.4 0.04 -0.25
121.1 123.2 0.08
121.1 123.3 0.08
121.2 0.07
121.3 0.10
121.4 0.11
121.1 123.4 0.10
121.1 123.5 -0.05 0.08
121.1 123.6 -0.08 0.09 0.18 0.07
121.3 -0.11 0.1 0.24 0.22
121.1 123.7 -0.06 0.06
121.1 123.8 0.13 -0.18/-0.07 -0.25
RS) RR - RS.

performed. Thus, in its present form, this methodol-

ogy can only be used when the two enantiomers of
the chiral amine are available. R-Cyano-R-fluoro-p-tolylacetic Acid
(CFTA). R-Cyano-R-fluoro-p-tolylacetic acid (CFTA)
has been described as a CDA for the determination
of the absolute configuration of secondary alcohols61
(by 1H NMR) and e.e. calculation (by 19F NMR). Its
use for the determination of the absolute configura-
tion of R-amino esters by 19F NMR has also been 3.5. Application to Secondary Amines: MTPA
described84 and is based on the study of the 19F NMR A procedure for the assignment of the absolute
spectra of the CFTA amides of a series of (S)-amino configuration of cyclic secondary amines with asym-
esters. The spectra of these compounds showed that metric carbons in the R- or -positions has recently
the fluorine signals of the amides derived from (R)- been proposed by Hoye et al.,85 using MTPA as the
CFTA ((R,S)-amides) resonate at higher field by 2-6 CDA. This method is based on the standard compari-
ppm, compared to those derived from (S)-CFTA son of the 1H NMR spectra of the corresponding (R)-
((S,S)-amides). Molecular orbital calculations84 sug- and (S)-MTPA amides; however, in this case, the
gest a conformational equilibrium between two con- foundations of the correlation between NMR shifts
formers: ap (the most stable; CR-F bond anti- and configuration and the analysis of the spectra
periplanar to the carbonyl) and sp (both groups syn- warrant a more detailed explanation.
periplanar) (Figure 124). The calculations also help In general, the 1H NMR spectra of these MTPA
to explain the shift of the fluorine signal to higher amides show the presence of two rotamers around
field in the ap conformer, with respect to the sp. The the amide bond (syn and anti; see Figure 125) that
large difference in ap/sp populations between the are in equilibrium. The signals of these rotamers can
(R,S)- and (S,S)-amides also helps to explain the fact
that the fluorine signal resonates at higher field in
the (R,S) compound than in the (S,S) compound.
Table 13 shows the NMR data obtained for CFTA
and MTPA amides (included for comparative pur-
poses). In all cases, the fluorine chemical shifts (F)
and their differences () are coherent with the
trends discussed, with larger values obtained for Figure 125. Conformational equilibrium in the (R)-MTPA
the CFTA amides. amide of (R)-2-methylpiperidine.
78 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

be identified because the populations of each are

different, because of steric effects. The most abundant
rotamer has the anti configuration when only one
substituent is present.
The differentiation between the spectra of the two
rotamers is essential because, in this procedure, only
the signals of the rotamer with the asymmetric
carbon of the MTPA syn to the asymmetric carbon
of the amine under examination should be consid-
For instance, in the assignment of the configuration
of carbon-2 of (R)-2-methylpiperidine (Figure 125),
only the signals due to the syn rotamer (Figure
126a,b) in the (S)-MTPA and the (R)-MTPA amides
are relevant and only the SR values corresponding
to these rotamers are valid for assignment purposes.
In contrast, the anti conformers (Figure 126c, d)
should not be considered.
The second requirement of this procedure is that
only the chemical shifts of axial substituents in the
aforementioned syn rotamer should be compared to
obtain the SR value. This is due to the fact that
the axial substituents are more exposed to the
shielding/deshielding effects of the CDA.
Thus, in (R)-2-methylpiperidine, the resonances for
the syn rotamer in the spectra of the (S)- and (R)-
MTPA amides should be identified first and the
signals due to the axial methyl group at C-2 and the
axial proton at C-3 (Figure 126a and b) used to obtain
the SR value.
In this situation, the axial methyl group is more
Figure 126. (a, b) Syn and (c, d) anti rotamers for the shielded in the (S)-MTPA amide (Figure 126a) than
(S)- and (R)-MTPA amides of (R)-2-methylpiperidine. in the (R)-MTPA amide (Figure 126b), and the H-3

Figure 127. (a, b) Syn and (c, d) anti rotamers for the (S)- and (R)-MTPA amides of (2S,6S)-(2-carbomethoxy-
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 79

The assignment of the configuration at C-6 requires

examination of the spectra of the axial groups in the
anti rotamers (Figure 127 c,d) and, for the structure
shown, the axial substituent at C-6 gives a positive
SR and the axial hydrogen at C-5 a negative SR
value (Figure 127f).
The generality of this correlation between the SR
signs and the absolute stereochemistry has been
tested with the cyclic secondary amines of known
absolute configuration shown in Figure 128. In all
cases, the signs are as expected from the stereochem-
istry and, therefore, the procedure can be used for
the assignment of other amines.
In addition, the absolute values obtained for SR
are much larger than those observed for the MTPA
Figure 128. Cyclic secondary amines of known configu- esters or amides; this condition is probably related
ration examined by NMR of their MTPA amides. to the proximity of the groups under examination to
the phenyl group and also to the favorable orientation
proton is more shielded in the (R)-MTPA derivative of the phenyl ring for effective shielding.
(Figure 126b) than in the (S)-MTPA amide (Figure
126a). The resulting SR values are negative for the 3.6. Application to r-Chiral Carboxylic Acids
methyl at C-2 and positive for the proton at C-3.
Conversely, SR signs will indicate the reverse Carboxylic acids are frequently found in nature as
configuration for the amine. optically active compounds with the chiral center at
If the cyclic amine had more than one asymmetric the R-position, and general approaches for the de-
C atom near the N atom, e.g., (2S,6S)-(2-carbometh- termination of the absolute stereochemistry of these
oxymethyl)-6-methylpiperidine (Figure 127), the same systems are rare.
principles would apply; however, the situation is An important decision that had to be made in the
more complex and the spectra of both the syn and development of new reagents for this purpose con-
anti rotamers should be considered. cerned the selection of the type of bond used to link
For example, in the assignment of the absolute the auxiliary reagent to the substrate carboxylic acid.
configuration at C-2, the NMR spectra of the axial
Several studies were undertaken for the purpose of
groups in the syn conformers of the (R)- and (S)-
MTPA amides should be examined (see Figure 127a, obtaining suitable reagents, and ester29,30 and
b). For the structure shown, the axial methylene at amide75-78,86 bonds seemed to be the most appropriate
C-2 results in a negative SR value and the axial H candidates.
atom at C-3 results in a positive SR value (see The main issue in relation to the selection of chiral
Figure 127c). reagents for the assignment of absolute configuration

Figure 129. Conformers ap and sp in carboxylic acids.

Figure 130. Conformational preference in the ester and amide bonds.

80 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

empirical calculations (AM1, PM3) on more than 20

carboxylic acids, and their methyl esters and methyl-
amides, were performed87 and showed that the main
conformational process in all those compounds is the
rotation around the 1 bond (Ca-CdO; see Figures
129 and 130). This generates two main conformers:
ap (CR-H and CdO in anti-periplanar) and sp (syn-
periplanar) (see Figure 129). In the case of the esters,
the ap conformer is the more stable conformer;
however, this preference is absent in amides.
In addition, it is known29 that, although, in the
esters, the (CdO)-O fragment has a clear preference
for a Z orientation, the rotation around the (CdO)-
NH fragment in amides is more complex75-78 and
ratios between the two main rotamers from 90:10 to
50:50 have been reported86 (see Figure 130).
From that data, one can conclude that the ester
Figure 131. bond is more convenient than the amide bond to link
the auxiliary to the substrate, because the last one
is conformationally less reliable and, as a conse-
of carboxylic acids is to demonstrate the existence of quence, the best auxiliary reagents for carboxylic
an NMR significant conformational preference that acids should be found among secondary alcohols and
remains the same in the R and S derivatives and is not among amines.
independent of the nature of the carboxylic acid.
In accordance with previous experience, alcohols 3.6.1. Ethyl 2-(9-anthryl)-2-hydroxyacetate (9-AHA) and
and amines can be considered potentially good can- Related Aryl Alcohols
didates to that purpose. With that information on hand and using the
To determine the conformational characteristics of AMAAs,29,30 as models, many aryl alcohols have been
carboxylic acids, molecular mechanics and semi- evaluated as CDAs for R-chiral carboxylic acids, using

Figure 132. Conformational equilibrium in the esters of (R)- and (S)-9-AHA with (a, b) an R-chiral carboxylic acid and
(c) predicted RS signs. Energies calculated by AM1 for (S)-2-methylbutiric acid.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 81

Figure 133. RS values for the 9-AHA esters of compounds 133.1-133.16.

Figure 134. Conformational model for the determination of the absolute configuration of R-chiral carboxylic acids by
NMR of their 9-AHA esters.

(S)-2-methylbutyric acid as the model substrate. configuration of carboxylic acids: MM and AM1/PM3
Their NMR showed that the higher RS values were methods were used to calculate the geometries and
obtained with ethyl 2-hydroxy-2-(9-anthryl) acetate relative stabilities of the conformers of the 9-AHA
(9-AHA; see Figure 131) as the auxiliary, and, esters; D NMR data were used to provide experimen-
therefore, 9-AHA was chosen for further studies as tal evidence of the theoretical findings; and aromatic
a CDA. shielding effect.87
Theoretical and experimental studies showed the The studies previously described all established
suitability of this reagent for the assignment of the that the conformational composition of any ester of
82 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 135. Structure of trans-arylcyclohexanols.

9-AHA with an R-chiral carboxylic acid is as repre-

sented in Figure 132: two main forms, ap (major)
and sp (minor), are in conformational equilibrium.
The L1 substituent in the (R)-9-AHA ester is shielded
in the ap conformation, while the L2 substituent
remains unaffected. In contrast, in the sp conforma-
tion, the L2 substituent is shielded while the L1
substituent remains unaffected (see Figure 132a). In
the case of the (S)-9-AHA ester, substituent L2 is
shielded in the ap conformation and substituent L1
is shielded in the sp conformation (see Figure 132b).
Because the population of the ap conformer is higher
than that of the sp conformer, substituent L 1 is more
shielded in the (R)-9-AHA ester than in the (S)-
derivative (RS < 0) and, conversely, substituent L2 Figure 136. RS values for the esters of the carboxylic
is more shielded in the (S)-9-AHA ester than in the acids shown with trans-2-phenyl-1-cyclohexanol and 9-AHA.
(R)-ester (RS > 0). The signs of the RS values Data for 9-AHA given in boldface type.
allow the assignment of the spatial position of sub-
stituents L1/L2 by comparison of the 1H NMR spectra 134c). Identification of substituents L1/L2 and their
of both diastereoisomers (see Figure 132c). placement around the asymmetric center can then
Examination by 1H NMR spectroscopy of the (R)- be directly obtained from the signs of RS, as shown
and (S)-9-AHA esters of a large number of carboxylic in Figure 134.
acids of known absolute configuration and with a
wide variety of structural features gave full experi- 3.6.2. Arylcyclohexanols
mental support to the aforementioned theory. The Other aryl alcohols different from the open-chain
RS values shown in Figure 133 serve to illustrate 1-aryl alcohols shown in Figure 131 also have been
two important facts. First, the distribution of the examined.87b Figure 135 shows their structures.
signs of RS shown in Figure 133 is perfectly When the RS values obtained for the esters of
homogeneous along the series and is consistent with the arylcyclohexanols 135.1-135.4 and a series of
both the absolute configurations of these compounds carboxylic acids of known absolute configuration are
and the conformational composition shown in Figure examined, it was observed that the commercially
132. available (1R,2S)- and (1S,2R)-2-phenyl-1-cyclohex-
Figure 134 shows the model for the assignment of anol (135.1) gave RS values that were very similar
the configuration of an acid of unknown configuration to those obtained with 9-AHA. These data also
by comparison of the 1H NMR spectra of its (R)- and indicate the following: (a) the signs of the shifts
(S)-9-AHA esters. In this model, the ap/sp equilibri- produced by 135.1 are regularly distributed in all the
um is simplified by considering the dominant ap form compounds examined (RS is positive for protons in
(Figure 130) as the only relevant one and using this substituent L1 and negative for protons in substituent
to fix the spatial location of substituents L1 and L2. L2); (b) the signs obtained are opposite to those
According to this model, the substituent that is more obtained with 9-AHA (negative for substituent L1 and
shielded in the (R)-9-AHA ester than in the (S)-9- positive for substituent L2); (c) the absolute RS
AHA ester (RS < 0) should be assigned as L1 in values obtained with 135.1 are, in all cases, similar
Figure 134c and, conversely, the substituent that is to those produced by 9-AHA for protons near the
more shielded in the (S)-9-AHA ester than in the (R)- chiral center. These facts indicate that both 135.1
9-AHA ester (RS > 0) corresponds to L2 (Figure and 9-AHA essentially show the same ability to
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 83

Figure 137. Representation of the direction of the maximum shielding in the esters of (a) 9-AHA and (b) 135.1. Models
for the determination of the absolute configuration on R-chiral carboxylic acids by NMR of their trans-2-phenyl-1-cyclohexanol
esters (c, d and e).

separate the resonances of the enantiomeric protons of its esters almost never overlap with those of the
of the substrates (see Figure 136). substrate, whereas the presence in 135.1 of numer-
Aromatic shielding-effect calculations corroborated ous different protons is a clear limitation in this
the shieldings obtained with this reagent. From a respect.
practical standpoint, 135.1 has an advantage over To analyze the role of the aryl ring in 135.1, the
9-AHA in that it is commercially available; on the possibility of increasing the RS values by changing
other hand, the signals of 9-AHA in the NMR spectra the aromatic ring was explored and new reagents
84 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 138. SR values obtained for enantiomeric mixtures of the acids shown, using (S)-methyl mandelate as the reagent
(chemical shift of the (S)-acid-(S)-methyl mandelate minus that of the (R)-acid-(S)-methyl mandelate).

b). Thus, when the phenyl group in the arylcyclohex-

anols is replaced by a larger aromatic system, the
shielding area becomes very much larger and this
should now affect not only the substituent on the
same side (L2) but also the other substituent (L1), to
some extent; therefore, on average, the RS values
will not be as high as expected. In the case of
phenylcyclohexanols, the focus of the shielding cone
Figure 139. Simplified model for arylalkoxyacetic acid is so far away from the asymmetric center that even
esters of a chiral acid according to (a) Tyrrel88 and (b) when its effect is greatly increased, it will never
Riguera.87 influence more than one substituent in each con-
with 1-naphthyl, 2-naphthyl, and 9-anthryl rings Panels c-e in Figure 137 show a graphic summary
(135.2, 135.3, and 135.4, respectively; see Figure 135) that illustrates how the absolute configuration of an
were synthesized and tested. Unfortunately, signifi- R-chiral carboxylic acid can be deduced from the signs
cant improvements in the RS values were not found of the values obtained from its esters with the
when compared to 135.1. The fact that compounds two enantiomers of any trans-2-aryl-1-cyclohexanol.
with naphthyl and anthryl rings do not produce
higher shifts than 135.1, which has a practically 3.6.3. (S)-Methyl Mandelate
identical conformation, seems to be in conflict with Other than the auxiliary reagents described by
data obtained for AMAA esters. Nevertheless, in- Rigueras group in the preceding pages, very few
spection of the structures of the esters of the aryl- papers have been devoted to the determination by
cyclohexanols indicates that the direction of maxi- NMR of absolute configuration of carboxylic acids. In
mum shielding is focused on the area immediately one such example, however, (S)-methyl mandelate
surrounding the asymmetric C atom (Figure 137a, was tested as a chiral auxiliary with racemic and

Figure 140. (a) Structure of PGDA and PGME amides and (b) empirical model for the assignment of the absolute
configuration of carboxylic acids.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 85

Figure 141. SR values for the PGDA and PGME amides of the carboxylic acids shown.

enriched mixtures of eight acids.88 Although the

number of chemical shifts reported was very limited
(only the methyl groups labeled a and b in Figure
138), a coherent shift pattern, with a homogeneous
distribution of signs for substituents L1/L2 and useful
Figure 142. Alternative model for the assignment of the
SR values was obtained. absolute configuration of carboxylic acids with PGDA or
The results obtained on using (S)-methyl mande- PGME.
late as the reagent were interpreted assuming a
conformational equilibrium and preferred conforma-
tion (CR-H not coplanar with CdO, and L2 anti to
the CdO group; see Figure 139) completely different
from those described for the esters of 9-AHA (see
Figures 132 and 134). Although this model can
effectively be used to correlate the NMR shifts with
the structure of the substrates, it is inconsistent with
the well-documented coplanarity of CR-H with
CdO, and therefore does not represent the real Figure 143. SR values for the PGME amides of com-
pound 143.1.
conformational composition of the compounds
studied;29,75-78,87 indeed, specific experimental data Although PGDA was tested with just one carboxylic
were not advanced to support the model. Therefore, acid of known absolute configuration (141.1; see
the success of this model in the prediction of correct Figure 141), and, therefore, its use as an auxiliary
R/S configuration is purely accidental and can be reagent for acids cannot be recommended, the reli-
explained because the spatial location of substituents ability of the assignments produced using PGME was
L1/L2, relative to the aryl ring, is approximately the demonstrated with the series of carboxylic acids of
same as that if the model corresponding to 9-AHA known configuration shown in Figure 141.
(Figure 132) were considered. An XRD study and NOE experiments were pre-
sented to support the model, although other studies
3.6.4. Phenylglycine Dimethyl Amide (PGDA) and have shown that, in solution, the CdO, N-H, and
Phenylglycine Methyl Ester (PGME) C-H adopt a coplanar disposition.75-78 If this is the
Phenylglycine dimethyl amide (PGDA) and phen- case, then the model should be modified to the one
ylglycine methyl ester (PGME) were proposed by shown in Figure 142, but this change does not affect
Kusumi and co-workers89 as auxiliary reagents for either the shielding distribution or the configuration
R-chiral carboxylic acids (Figure 140a). In this pro- assigned, because the position of substituents L1/L2,
cedure, the acid of unknown configuration is con- relative to the phenyl group, is approximately the
densed with the two enantiomers of the reagent and same in both cases.
the SR values of substituents L1/L2 are measured When PGME was applied to compound 143.1
in the corresponding amides. (Figure 143), an irregular distribution of SR signs
The empirical model shown in Figure 140b was was obtained90 for several protons. The authors
proposed for the interpretation of the experimental hypothesized that a phenyl-phenyl interaction could
data: substituents Hx, Hy, Hz, which correspond to be the cause of this phenomenon, but when other
L2, are more shielded by the phenyl group in the (S)- substrates that contain phenyl groups were studied
PGDA (or (S)-PGDE) amides than in the (R)-amides, (Figure 144), the signs were as one would expect from
and the opposite case occurs for substituents Ha, Hb, the configuration of the substrates and no discrep-
Hc (L1). Thus, SR (L2) < 0 and SR (L1) > 0. ancies were found. Steric hindrance or the effect of a
86 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 144. SR values for the amides of the carboxylic acids shown with PGME (obtained from the (S)-PGME amides
of the racemic acids).

Figure 146. Empirical model for the assignment of the

absolute configuration of R-oxy-R,R-disubstituted acetic
Figure 145. acids with PGME.
nearby O atom were proposed as possible causes of
the problem.
cases, the empirical model shown in Figure 146 was
PGME has been used on several occasions to
proposed and several acyclic and cyclic R-oxy-car-
deduce the absolute configuration of natural prod-
boxylic acids of known absolute configuration, includ-
ucts. Some examples in this field include the follow-
ing some natural products, were used as substrates
ing: compound 145.1, which is a degradation product
to prove the reliability of the procedure (Figure 147).
from the marine cycloperoxide plakortin;91a the HIV-1
integrase inhibitor of microbial origin, integric acid91b The method was first applied to determine the
145.2 (Figure 145); and carboxyYTX, which is an configuration at C-18 of polyether macrolide pect-
analogue of yessotoxin, isolated from mussels.91c enotoxin-692a (148.1, Figure 148). The absolute
More recently, PGME was applied to the assign- configuration of lactone 148.2, which is a derivative
ment of the configuration of R-oxy tertiary carboxylic of tanikolide, isolated from cyanobacteria93 was also
acids92 (R-oxy-R,R-disubstituted acetic acids). In these assigned this way.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 87

Figure 147. R-Oxy-R,R-disubstituted acetic acids studied with PGME.

Figure 148. (a) Structure of Pectenotoxin-6, (b) SR values in C5D5N, (c) SR values in CDCl3, and (d) structure of
Tanikolide lactone.

Figure 149. R-Oxy-R-monosubstituted acetic acids studied

with PGME.

The applicability of PGME to R-oxy-R-monosubsti-

tuted acetic acids, using the empirical model pro-
posed for R-oxy-R,R-disubstituted acetic acids in Figure 150. values for furoic acid derivatives with
which one of the R-substituents is a hydrogen, was PGME.
also investigated.94 Unfortunately, only four ex-
amples of known absolute configuration were used derivatives shown in Figure 150, the same signs
to support the reliability of this model (Figure 149). were obtained for all the relevant protons of the
In addition, Fusetani and co-workers95 found that, PGME amides. The model proposed by Kusumi is
when PGME was used for the study of the furoic acid consistent with those results.
88 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 151. (a) Structure of (aR)-BNDO. (b) Ideal conformation of an (aR)-BNDO monoester. (c) Position of the acid
substituents in the diasteromeric esters. (d) Model for the assignment of configuration from the values.

Figure 152. (a) 1H aSaR values for the BNDO esters of the carboxylic acids shown. (b) 13C aSaR values.

3.6.5. 1,1-Binaphthalene-8,8-diol (BNDO)

A completely different type of reagent was proposed
in 1996 by Japanese workers96 for the stereochemical
assignment of carboxylic acids; this is 1,1-binaph-
thalene-8,8-diol (BNDO; see Figure 151a), which
presents axial chirality and is available in the two
enantiomeric forms.
Several acids of known absolute configuration were
esterified with (aR)-, (aS)-, and/or racemic BNDO,
and a method to predict the configuration was
devised. The idealized conformation of a BNDO
monoester is depicted in Figure 151b: The naphtha- Figure 153.
lene rings are orthogonal, the carbonyl and the
hydroxyl groups are syn, and the a proton is situated due to Ha of the (aR)-BNDO monoester should
near the center of the facing naphthalene ring. appear downfield, relative to that in the (aS)-BNDO
Because of the effect of the aromatic ring, the signal monoester. The reverse should hold true for Hb
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 89

Table 14. Values of the -Methyl and Other confirm the preponderance of the ideal conformation
Diagnostic Resonances in Diastereomeric Syn and in solution.
Anti 1-Arylethylamides from 3-Methylcarboxylic
Acids The 1H values for a very limited number (four)
of chiral acids are shown in Figure 152a. 13C
values for three of those compounds were also re-
ported (Figure 152b).

3.7. Application to -Chiral Carboxylic Acids

3.7.1. Arylethylamines
Acyclic carboxylic acids containing remote stereo-
centers constitute a class of substrates for which
methods to determine their configurations were also
pursued. In fact, -substituted acids represent a
specific structural unit that is of interest in the
context of natural products chemistry. Hoye et al.97
chose the amide bond to link the reagent to the
substrate and explored the use of 1-arylethylamines
153.1-153.4 as reagents (see Figure 153a).
These compounds were tested with the homologous
series of acids 153.5-153.8 (see Figure 153b), using
the signals of the methyl groups of the amides to
assess the effectiveness of these systems as reagents.
As expected, the anthryl derivative 153.4, followed
by the naphthyl derivative 153.2, resulted in the
largest shifts. Amines 153.1 and 153.2 are com-
mercially available in optically pure form and were
therefore adopted for further studies with a series of
3-substituted acids of known absolute configuration.
The results summarized in Table 14 show how the
authors differentiate two stereochemical possibilities
(defined as syn/anti, according to the relative orienta-
tion of the benzylic methyl group and the methyl
(Figure 151c). The chemical-shift difference was substituent at C(3)) and calculate the parameter
defined as ) aS- aR; thus, in the model (defined as the difference between the chemical shifts
depicted in Figure 153d, positive and negative values in the syn and anti diastereomers) for the -methyl
are associated with the left- and right-hand sides of and other groups.
the substrate, respectively. Isobutyric acid and sev- The data obtained show that the -methyl groups
eral derivatives were used for 1H NMR analyses to are always more deshielded in the syn series than

Figure 154. Conformational model for the determination of the sign of ) (syn) - (anti) of protons in R1 and R2.

Figure 155. ) (syn) - (anti) values for amides from -chiral acids with known relative configurations Ar ) 1-Np
data given in boldface type.
90 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 158.

Figure 159. Proposed conformation for the PGME amides

of the gambieric acids based on NMR analysis. SR
Figure 156. (a) Structure of (S)-PGME. (b) Suggested (instead of RS) values were reported; the inconsistent
most stable conformation of a PGME amide. (c) Model for value is highlighted.
the assignment of the configuration.

in the anti series, whereas protons in the other

-substituent are more shielded in the syn isomerss
with the exception of the vinylic H(4) proton in the Figure 160. Formation of N-(methoxyphenylacetyl)sul-
last entry in Table 14. foximines from sulfoxides.
A working model (Figure 154) based on the NMR
results and routinely accessible calculations (AMBER
force field, as implemented in the MacroModel soft-
ware package) has been proposed. This model is
based on two assumptions: (1) the predominant
rotamer around the bond between the benzylic C
atom and the N atom is that in which the benzylic Figure 161. Model for the determination of configuration
of N-(methoxyphenylacetyl)sulfoximines from sulfoxides.
proton is eclipsed with the carbonyl group, and (2)
one of the two nonhydrogen substituents at C(3) taken from the literature. These studies confirmed
occupies the anti position, relative to the carbonyl the applicability of this method.98
In the case of the anti diastereomer, this situation 3.7.2. PGME
results in preferential shielding of R1, whereas Phenylglycine methyl ester (PGME; see Figure
preferential shielding of R2 should be observed in the 156a), proposed by Kusumi et al.89 for the determi-
syn diastereomer. As a consequence, the values nation of the configuration of R-chiral carboxylic
for protons in R1 are positive and those in R2 are acids, has also been used with acids that have the
negative. chiral center at the -position. The eight carboxylic
The scope of this method/reagent was extended by acids of known configuration shown in Figure 157
the same authors to include carboxylic acids with were used as test compounds. The signs of the RS
substituents other than a methyl group at C(3), values of the corresponding PGME amides were
including the substrates shown in Figure 155 and six coherent with their stereochemistry and the model
other examples of known absolute configuration outlined in Figure 156c.89b

Figure 157. RS values for the amides of the -chiral carboxylic acids tested with PGME.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 91

Figure 162. N-MPA-sulfoximides used in this study.

This situation is rationalized on the assumption -chiral carboxylic acid to exist in essentially the
that steric interactions cause the PGME amide of a conformation shown in Figure 156b. However, theo-

Figure 163. Polyhydroxylated compounds studied as MTPA esters.

92 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 164. Analysis of the diastereomeric nonequivalence in the (R)-MPA esters of the enantiomers of tanshindiol (164.1).
The shielding on the methylene protons is different in each case.

retical calculations were not performed to confirm 3.9. Application to Chiral Polyhydroxylated
this hypothesis. Compounds
This reagent/model was used to establish the In many fields of chemistrysnatural products,
stereochemistry of several natural products such as organic synthesissresearchers often must determine
158.1, which is a degradation product from a diter- the absolute configuration of substrates that have
penoid isolated from an insect wax99a (see Figure 158) several chiral centers and functional groups, such as
and the gambieric acids, which are polyethers iso- polyols. Usually, when facing these situations through
lated from marine dinoflagellates.99b In the latter the use of the NMR methods, the direct application
case, the authors found it necessary to ensure that to polyalcohols of the models originally developed for
the proposed PGME model was acceptable for these monoalcohols was the solution that was used, without
compounds by performing a conformational analysis considering that such a solution could be highly
(NOE, HOHAHA). The resulting modified conforma- inappropriate. This situation was exposed in a work13
tion is shown in Figure 159. where several examples taken from the literature
illustrate the magnitude and characteristics of the
3.8. Application to Chiral Sulfoxides: MPA problem.
In those reports, attempts were made to assign the
A procedure for the determination of the absolute absolute configuration of all the asymmetric centers
configuration of chiral sulfoxides has been devel- of a polyalcohol by application of the NMR method
oped100 and requires the introduction of a chiral to the fully esterified derivatives with (R)- and (S)-
anisotropic moiety by transformation of the sul- MTPA; however, the SR data obtained generally
foxides into N-(methoxyphenylacetyl)sulfoximines. did not allow a completely unambiguous assignment
The method consists of the amination of the sulfoxide of configuration.
with O-mesitylsulfonylhydroxylamine, which pro-
ceeds with complete retention of chirality at the S
atom, followed by introduction of (R)- and (S)-MPA
at the N atom (Figure 160).
Subsequent application of the empirical model
shown in Figure 161 allows the configuration to be
deduced on the basis of the SR values (SR ) (S)
- (R)).
The utility of this model and the reliability of the
configurational assignments are based on the coher-
ence of the NMR data obtained for the MPA-derived
sulfoximides shown in Figure 162, which are derived
from eleven chiral sulfoxides of known absolute
configuration (162.5-162.15). In addition, four ra-
cemic sulfoxides were derivatized with racemic MPA
and the NMR data of the corresponding MPA-derived
sulfoximides 162.1-162.4 were shown to produce
shifts that apparently confirmed the model: the Figure 165. Polypropionate metabolites from the marine
signs are systematically arranged on both sides of mollusk Onchidium sp. (165.1 and 165.2) and 1H NMR
chemical shifts of chiral bis-MPA esters of (2R,4R)-(-)-
the MPA plane, but the absolute configuration of each pentanediol, indicating the different shielding caused by
derivative was not available for proper validation. the auxiliary reagents.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 93

Figure 166. (R)- and (S)-MPA diester of an anti-1,2-diol and expected RS signs. Arrows show the shielding produced
by the phenyl groups.
Figure 163 shows how the hexa-MTPA ester of In practice, the assignment of the configuration of
5-desacetylaltohyrtin A101 (163.1) generates val- compounds that have several functional groups with
ues around C-5, C-35, C-38, C-42, and C-47 that do the same reactivity (i.e. 2,3-pentanediol) can be
not present the necessary alternation of signs around addressed in two possible ways: either the appropri-
substituents L1/L2 and the same occurs with C-16, ate protection/deprotection protocols are used to
C-19, and C-24 of squamostatin D25b (19.5; see Figure achieve selective derivatization of each hydroxyl
19). Other examples of this type include annonaceous group or, more simply, the two alcohol groups are
acetogenins such as bullatacin12a,102a (rolliniastatin- derivatized together in a single reaction.
2, 163.2; see Figure 163), sootepensin A25a (19.4; see In the first case, the configuration at each asym-
Figure 19), 4-deoxyannomontacin102b (163.3); a don- metric C atom is examined separately, using the
naienin derivative102c (163.4); and sponge metabolites models developed for monofunctional compounds;
penaresidins23 (19.1 and 19.2; see Figure 19). however, the protection/deprotection operations will
This lack of homogeneity of the SR signs involves be time- and sample-consuming and this is a signifi-
a clear risk of misassignment and means that, to cant drawback.
some extent, the considerations known to be valid for There could be special cases where the reactivity
monoesters are not fulfilled in those bis- or tris- of the functional groups with the CDA is sufficiently
MTPA esters. The aforementioned article13 reminds different to achieve selective derivatization of one of
us that the fundamentals of the NMR method are the groups without protecting the other. One par-
related to aromatic shielding, which is a through- ticularly noteworthy example of this situation using
space phenomenon that affects protons several bonds MPA as the reagent is represented by tanshindiol
away from the hydroxylic C atom that has the (164.1; see Figure 164), which is a plant metabolite
auxiliary reagent. Therefore, the SR values ob- that possesses vicinal primary and tertiary hydroxyl
served in those polyderivatized compounds are, in groups.103 When both enantiomers of 164.1 were
fact, the result of the combined effects (shielding/ derivatized with (R)-MPA, single esters on the pri-
deshielding) of all the MTPA phenyl rings and cannot mary hydroxyl group were obtained and their NMR
be interpreted as if they were produced by just one were used for the assignment on the basis of the
phenyl group as in monofuntional compounds. models shown in Figure 164. Note that MPA is not a
94 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 167. (R)- and (S)-MPA diester of a syn-1,2-diol, and expected RS signs. Arrows show the shielding produced by
the phenyl groups.
reliable reagent for assignment of configuration of Years later, the correspondence between the abso-
primary alcohols (see section 3.2 of this review) and lute configuration of a series of diols of known
that its application in the case of tanshindiol (164.1) configuration and the NMR spectra of their bis-
is determined by the chelating ability of the tertiary AMAA derivatives was studied by the same group,
hydroxyl group. and a general methodology to determine the config-
In the second and more general approach, the two uration of 1,n-diols (1,2-diols, 1,3-diols etc.) from the
functional groups are made to react with the auxil- NMR spectra of the bis esters was presented.105a In
iary at the same time and the preparation of the bis this procedure, the diol is fully esterified with the R
derivatives is straightforward. However, as men- and the S auxiliary (MPA or 9-AMA) and the
tioned previously, the shifts observed in the NMR signs are interpreted as being the result of the
spectra of the fully derivatized compound (i.e., the combined action of the two auxiliary units.
bis-ester of the diol) cannot be interpreted using the
models for monofunctional compounds. New and This approach is based on the fact that the aro-
specific models that correlate the absolute stereo- matic shielding is a through-space phenomenon; each
chemistry of diols with the NMR spectra of their bis- reagent unit contributes to the chemical shift of a
AMAA esters have recently been described and will given proton in a different way (shielding or deshield-
be presented in the next section of this review. ing) and with a different strength, depending on the
actual structure of the diol. As a consequence, the
3.9.1. MPA and 9-AMA
The use of MPA on substrates that have several chemical shifts (and RS values) observed are the
chiral hydroxyl groups was first described by Riguera result of the additivity of the shielding/deshielding
et al.104 in a study on polypropionate metabolites from effects produced by all the auxiliary reagents present
the marine mollusk Onchidium sp. The configura- in the bis ester. This overall effect can be predicted
tions of the onchitriols 165.1 and 165.2 were estab- for each stereochemical combination if the conforma-
lished by comparison of the RS values of the bis- tional composition of the AMAA esters is known. In
MPA esters with those of selected diols of known fact, the predicted RS signs are fully in agreement
absolute configuration, such as (2R,4R)-(-)-pen- with the experimental results for many diols of
tanediol (Figure 165). known absolute configuration.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 95

Figure 168. Diagnostic RS (9-AMA, MPA) and SR (MTPA) signs for the four stereoisomers of (a) 1,2-diols, (b) 1,3-
diols and (c) 1,4-diols. R3 represents the substructure between the chiral centers in diols other than 1,2 systems.

Clearly, if the experimental values were inter- diester, which makes prediction of the sign of their
preted as if they were caused by just one reagent unit RS values impossible. For its part, the CRH and
(and neglecting the effect of the other), using the CRH are shielded only in the bis-(S)-MPA-diesters;
models for monofunctional compounds, the resulting thus, a positive RS should be expected for both
assignment may be incorrect. protons CRH and CRH. Once again, if the config-
If we consider the conformational characteristics uration of the diol were the opposite, the signs also
of the AMAA esters of secondary alcohols previously would be the opposite.
discussed, and apply them to the case of a vicinal diol, The same reasoning can be used to predict the
the schematic diagrams that are presented in Figure distribution of the RS signs in other diols different
166 result. than 1,2-diols. Figure 168 shows the distinctive RS
In Figure 166a, the shielding/deshielding zones patterns of the four stereoisomers of 1,2-, 1,3-, and
originated by the aryl group of an anti-1,2-diol 1,4-diols and constitutes a guide for the assignment
derivatized as bis-MPA ester are presented: in the of the configurations.
bis-(R)-MPA ester, the R2 group and CRH are Note that, although for monofunctional compounds,
shielded, whereas in the bis-(S)-MPA ester, only the the RS signs provide sufficient information to
R1 group and CRH are shielded. Thus, negative RS distinguish between the two enantiomers (R or S),
(R,R - S,S) values should be expected for R2, and in diols with two asymmetric centers, four stereo-
CRH, and positive RS values are expected for R1, chemical situations are possible (the syn and anti
and CRH. If the configuration of the diol were the pairs: RR, SS, RS, or SR). In fact, four different
opposite, the signs also would be opposite. combinations of shielding/deshielding effectssand,
When a syn-1,2-diol is considered (Figure 167), consequently, four different sets of RS signssare
caution must be taken into consideration, because predicted and experimentally obtained for those four
only the signals due to the protons in R positions are stereochemical arrangements: one for each stereo-
useful for assignment. Figure 167 shows that R1 and isomer. Thus, the absolute configurations of the two
R2 are both shielded in the bis-(R)- and (S)-MPA chiral centers in any of the four stereochemical
96 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

combinations of the diol can be deduced from just the

NMR of the corresponding bis-AMAA esters.
After the diol of unknown configuration has been
derivatized with the two enantiomers of the reagent
(MPA or 9-AMA), the RS values and signs of the
bis esters are calculated in the usual manner. The
next stage involves comparison of the signs of RS
of the diagnostic signals CRH and CRH with those
shown in Figure 168. If both the CRH and CRH
have the same RS sign (either both positive or both
negative), then the RS signs for R3 (but not for R1/
R2) should also be examined and all three signs
should be compared with those of Figure 168 for
In case CRH and CRH present opposite RS signs
(one positive and the other negative), the RS signs
for R1 and R2 (but not for R3) then should be
considered and all four signs should be compared
with Figure 168 for assignment.
A selection of the diols of known absolute config-
uration that have been used to demonstrate these
conclusions is shown in Figure 169 and includes 1,2-
and higher-order diols as substrates and MPA,
9-AMA (169.1-169.9) and 2-NMA102a (169.11 and
169.12) as reagents.
Extension of this methodology to the assignment
of nonsymmetrical 1,n-diols with two secondary
(chiral) hydroxyl groups, or with primary and sec-
ondary (chiral) hydroxyl groups, as well as amino
alcohols, has been conducted and shown to be
3.9.2. MTPA
If the MTPA bis esters of the diols are submitted
to the same analysis as the MPA esters, the resulting
shielding/deshielding areas around the diol frame-
work and the sign distributions are different from
those obtained with MPA and AMAA. This is due to
the diverse conformational composition of MTPA
esters; however, from a practical standpoint, the
same set of signs shown in Figure 168 for AMAA bis
esters can be applied by simply using the experimen-
tal SR values of the MTPA esters as if they were
RS. The data reported106 for compound 169.10 serve
to illustrate the application of this rule.
There are some examples in the literature of the Figure 169. Structure and RS values obtained for bis-
MPA, 9-AMA (169.1-169.9), 2-NMA (169.11-169.12), and
application of MTPA to the assignment of configu- MTPA (169.10). Data given in boldface type corresponds
ration of diols where the NMR data are interpreted to that of MPA and data given in normal typeface cor-
in a variety of ways. Interestingly, note that applica- respond to that of 9-AMA.
tion of the general model of Figure 168 to the NMR
data provided in those papers for the bis-MTPA configurations were not checked by independent
esters lead, in all cases, to the correct assignment of methods: in two cases, the work was done with
absolute configuration giving further support to the racemates and the SR values inferred from the (R)-
generality of the models developed by Rigueras MTPA derivatives).
group. According to the authors, the RS values of the
Thus, Ichikawa and co-workers107 studied the as- protons on the asymmetric carbons and their vicinal
signment of syn and anti diols by NMR of their C atoms are the only ones that can be considered for
MTPA esters; unfortunately, only a very small num- assignment purposes in the case of syn diols. The
ber of compounds (one syn (170.1) and two anti (170.2 remainder protons should be neglected. This is
and 170.3); see Figure 170) were examined (their related to the absence of the necessary homogeneity
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 97

Figure 170. (a) Diols used in this study. Models for the
determination of configuration of (b) syn- and (c) anti-1,2-
diols with MTPA. Only protons inside the box fit the rule
for syn diols.107

Figure 172. SR signs obtained from the bis-MTPA

esters of glycols. Discrepancies with the models of Figure
170 are highlighted in 172.4, 172.5, and 172.7.
Figure 171. SR values for a syn diol. The lack of
homogenity in the signs obtained for a same L substituent
are highlighted.

of RS signs in those protons in the syn diols (this

is a requirement for reliable assignments frequently
not fulfilled when MTPA is used as CDA; see Figure
171). A different model applies to anti diols (Figure
In another paper108 by the same group, the assign-
ment by NMR of the bis-MTPA esters of diols that
have aromatic groups was studied, as shown in
Figure 172 (the inadequacy of MTPA as a reagent
for alcohols that possess aromatic groups had already
been noted by Ohtani17a). This study showed that, for
the monoaromatic anti-glycols 172.1, 172.2, and
172.3, the model showed in Figure 170 was operative
but fail for the monoaromatic syn-glycol 172.4 and
the bis-aromatic syn-glycol 172.5. The fact that the
method did work with the aliphatic syn-glycols 172.6
and 172.7 suggested that - stacking could be the
cause of the disagreement between the prediction and
the experimental data.
Analysis of the NMR data, following the rules of
Figure 168, shows discrepancies in the SR signs
obtained for the protons at CR and C-R of monoaro-
matic syn-glycol 172.4 and bis-aromatic syn-glycol
172.5. This finding reinforces the idea that MTPA
should not be recommended as CDA for the assign- Figure 173. Experimental SR signs obtained for the bis-
ment of syn-glycols that posses aromatic groups nor MTPA esters of the syn- and anti-acyclic 1,3-diols shown.
for any diol of unknown relative configuration with
aromatic groups. manner proposed by Riguera et al. for 1,n-diols. The
In a more detailed and recent publication,109 a authors concluded that the method was only ap-
series of 1,3-diols (see Figure 173) was studied as bis- plicable to syn-1,3-diols, where the values of substit-
MTPA esters and their NMR spectra analyzed in the uents L1 and L2 are diagnostic (but not those of the
98 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 174. (a) Signs of the SR obtained for the bis-MTPA esters of syn- and anti-cyclohexane-1,3-diols used in this
study, and (b) predicted SR values.
central part). They also state that it should not be In the field of natural products, numerous ex-
applied to anti-1,3-diols, where substituents L1/L2 amples can be found of the assignment of configura-
cannot be considered and the central part is, in their tion by NMR of the bis- or tris-MTPA esters. Only in
opinion, not surely diagnostic. However, in all the a few cases have the authors based their assignment
cases, the SR signs obtained experimentally (Figure on the use of simpler synthetic diols of known
173) are in full agreement with those predicted by configuration as models for comparison. For example,
Rigueras model for those configurations as shown in Minale et al. determined the configuration of the side
Figure 168. chains of marine polyhydroxysteroids110a such as
In the same work, six-membered cyclic systems 175.1 and 175.2, using synthetic 2,3-dimethylpen-
were examined and four zones with distinct tane-1,2-diols and 2-methyl-3-ethylheptane-1,2-diols,
values, depending on the position around the ring, respectively, as models.110b
were proposed (see Figure 174). Five cyclohexane- The bis-MTPA derivatives of C27- and C29-alkane-
1,3-diols derived from dihydroxyvitamin D3 and with 6,8-diols (175.3; see Figure 175) were prepared in
known absolute configuration were examined as bis- another report;110c however, their NMR data were
MTPA esters. Figure 174a shows the experimental used simply to discriminate among stereoisomers (in
SR sign distributions and Figure 174b shows the fact, only the small shifts at Me-1 were considered!)
empirical models that have been used. and the absolute configuration was not proposed.
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 99

and comparison of the NMR spectra of the corre-

sponding diasteroisomeric derivatives. Nevertheless,
recently, some effort has been devoted to methods
that require the preparation of only one derivative.
The existence of such a procedure would reduce the
time and amount of sample necessary for the analysis
by a factor of 2, because only one derivative and one
derivatization step would be needed.
Two general approaches can be distinguished. The
first applies to secondary alcohols and is based on
extensive NMR data, indicating that, when a strong
auxiliary reagent such as 9-AMA (but not MPA or
MTPA) is used, comparison of the NMR spectra of
the secondary alcohol with that of one of its 9-AMA
esters (either the R or the S) allows the safe assign-
ment of the absolute configuration of the alcohol. The
procedure, which is summarized in Section 4.1.1, is
based on the comparison of the chemical shifts of
many secondary alcohols of known absolute config-
uration and the corresponding (R)- or (S)-9-AMA
ester. A related method, which makes use of glyco-
sydation shifts, is described in Section 4.1.2.
The other approach applies to secondary alcohols
Figure 175. Natural products studied as MTPA esters.
and primary amines. It consists on the controlled
4. Methods Based on a Single Derivatization modification of the conformational equilibrium of the
As we have shown, all the procedures in the MPA esters and MPA amides and is based on our
preceding pages involve the derivatization of the knowledge of the shielding/deshielding effects associ-
substrate with the two enantiomers of the reagent ated with each conformer.

Figure 176. Partial 1H NMR spectra (300 MHz) of (a) the (R)-9-anthrylmethoxyacetic acid ester of alcohol 176.1; (b)
alcohol 176.1; (c) the (S)-9-anthrylmethoxyacetic acid ester of alcohol 176.1.
100 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 177. (a) Esterification shifts (AR values) measured for the (R)-9-AMA ester of 176.1. (b) Esterification shifts
(AS values) measured for the (S)-9-AMA ester. Larger shifts are highlighted.

Figure 178. AR and AS values for (R) and (S)-9-AMA esters of alcohols 178.1-178.5. (Larger values are given in
boldface type.)

If the sp/ap conformer ratio is modified in a certain stituents L1/L2, with respect to the phenyl group of
way (i.e., an increase of the sp conformer), the NMR the auxiliary (absolute configuration known), can be
spectra should translate such a change in ratio into determined by the NMR response to the disturbance
a predictable modification in the chemical shifts in the equilibrium.
associated with the substituents (i.e., greater popula- Two different procedures have been described to
tion of the sp conformer in the (R)-MPA ester leads induce the conformational change:
to greater shielding of substituent L1 in the average (a) Modification of the equilibrium by reducing the
spectrum). In this way, the spatial position of sub- temperature of the NMR probe. This approach has
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 101

Figure 179. Diagram for the assignment of the absolute configuration of a secondary alcohol from AR or AS values.

Figure 180. Esterification shifts for the (R)- and (S)-9-AMA esters of alcohols 176.1 and 178.1-178.6, expressed as mean
values (mL1 and mL2).
found application in the assignment of the absolute preparation of only one derivative has been recently
configuration of secondary alcohols using MPA as an presented by Riguera et al.35 and consists of a
auxiliary reagent (see Section 4.1.3). comparison of the NMR spectrum of the alcohol with
(b) Modification of the equilibrium by selective that of one of its esters prepared with either the (R)-
chelation of one of the conformers. This procedure has or the (S)-9-AMA chiral auxiliary.29,30 When the two
been described for the assignment of secondary resulting spectra are compared, the signals of sub-
alcohols and primary amines using MPA as an stituents L1 and L2 can be easily distinguished,
auxiliary reagent and barium(II) salts as chelators because the protons of one of those substituents (the
(Sections 4.1.4 and 4.2, respectively). one that is under the shielding cone of the anthryl
4.1. Application to r-Chiral Secondary Alcohols ring in the derivative) move upfield in the 9-AMA
ester, with respect to the free alcohol, whereas the
4.1.1. 9-AMA Esters: Esterification Shifts protons of the other substituents (unaffected by the
A procedure for the assignment of the absolute aromatic shielding) resonate at practically the same
configuration of secondary alcohols involving the field in both the 9-AMA ester and in the free alcohol.
102 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 183. Glucosides studied by the procedure of

glucosidation shifts in 13C NMR.

Figure 181. Cyclic alcohols studied with this method.

Figure 184. Tetra-O-acetylglucosides studied by the

procedure of glucosidation shifts in 1H NMR.

Several linear secondary alcohols of known abso-

lute stereochemistry (Figure 178) have been exam-
ined in this way, and the results corroborate the
existence of the aforementioned correlation between
the spatial position of substituents L1/L2 of the
alcohol, the configuration of the auxiliary, and the
NMR shifts.
Thus, in the (R)-9-AMA esters represented in
Figure 178, the most intensely shielded protons are
all located in the right-hand substituent (L1), whereas
those that are only slightly shielded are located in
the left-hand substituent (L2) (ARL1 . ARL2):
The reverse occurs in the (S)-9-AMA esters, where
ASL1 , ASL2.
Figure 182. (a) 1H NMR spectrum of a free alcohol. (b) In this way, the configuration of a secondary
1H NMR spectrum of its (R)-9-AMA ester. (c) Inferred
alcohol such as sec-butanol (178.1) is R (as indicated
absolute configuration from the AR values.
in Figure 178) if the more intensely shielded protons
Consideration of the absolute configuration of the in the (R)-9-AMA ester are in the right-hand side of
auxiliary reagent ((R)- or (S)-9-AMA) and the spatial the projection (i.e., the ethyl substituent) and those
that are only very slightly shielded are on the left-
location of its anthryl ring allows the placement of
hand side (i.e., the methyl substituent). This ar-
substituents L1/L2 around the asymmetric C atom.
rangement results in ARL1 . ARL2. The reverse
An example illustrating the esterification shifts (ASL1 , ASL2) is observed when the enantiomeric
observed in the marine metabolite40 176.1 is shown reagent (S)-9-AMA is used and also when the con-
in Figure 176. figuration of the sec-butanol is S (opposite to that
In the (R)-9-AMA ester (Figure 176a), H(6), H(5), shown in Figure 178). A graphical resume for ap-
and H(4) are intensely shielded, with respect to the plication of this procedure is shown in the model of
same protons in the free alcohol (Figure 176b), while Figure 179.
H(9), H(10), Me(12), and Me(13) resonate at practi- The plot shown in Figure 180 represents the
cally identical chemical shifts in the ester and in the intensity of the parameter (m) produced by
alcohol (the opposite is observed in the (S)-9-AMA 9-AMA on the alcohols 176.1 and 178.1-178.5, and
ester; see Figure 176c). These esterification shifts are measured as mean values (m) of all the protons in
quantitatively expressed for each individual proton substituents L1 and L2 (mL1 and mL2).
as the difference between the chemical shift in the Application of this methodology to cyclic alcohols
free alcohol and that in the ester (AR or AS, is also possible, and compounds 181.1-181.5 (Figure
depending on which 9-AMA ester has been exam- 181) presented esterification shifts that were coher-
ined). Figure 177 shows the AR and AS values ent with their stereochemistry and the model of
obtained for compound 176.1. Figure 179. The use of the mean values mL2 and
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 103

Figure 185. (a) Correlation model for secondary alcohols as -D-glucopyranosides. (b) D - ROH values for dimethyl D-
and L-malate (in ppm, at room temperature, CDCl3).

the signals for substituent L1 are shifted to a greater

extent than those in L2 (Figure 182b).
In this way, the signals of the alcohol that are
strongly shifted upfield upon esterification with (R)-
9-AMA correspond to the protons that are located on
the same side as the anthryl group in the sp con-
former, i.e., the right-hand side of the asymmetric
center in the projection shown (substituent L1 in
Figure 182c). In turn, the protons that are slightly
shielded (as they are not affected by the aryl group)
are located on the left-hand side (substituent L2,
Figure 182c). The reverse holds for the (S)-AMA
esters and also when the configuration of the alcohol
is opposite.
Attempts to use auxiliary reagents other than
9-AMA for assignment of the configuration were
unsuccessful. Thus, when the (R)- and (S)-MPA
auxiliaries were tested with a wide range of sub-
strates, the AR and AS obtained were very small
and negative in some cases. As a consequence, the
identification of substituent L1 or L2 by association
with the shielded and nonshielded protons is not
always possible; therefore, MPA cannot be recom-
mended for this approach.
Similarly, 1- and 2-naphthylmethoxyacetic acids (1-
NMA and 2-NMA, respectively) were assayed with
linear secondary alcohols and did not give satisfac-
tory results, although a limited approach based on
certain empirical increment parameters has been
Figure 186. Alcohols of known configuration studied as
-D-glucopyranosides. 4.1.2. Glycosidation Shifts
mL1 is recommended for assignment of the config- Glycosidation Shifts in 13C NMR. A
uration of these cyclic structures. method for determining the absolute configuration
From a theoretical standpoint, the different esteri- of a secondary hydroxyl group based on characteristic
fication shifts observed for the shielded and non- glycosidation-induced shifts in 13C NMR spectroscopy
shielded substituents are explained on the basis of was reported previously in the development of NMR
the conformational composition and structure of the methods for configurational assignment.55a,b The
9-AMA esters (see Figure 46 in Section 3.1.3). In the general strategy consists of several steps that include
(R)-9-AMA ester of the alcohol of Figure 46, substitu- (a) measurement of the 13C NMR spectrum of the
ent L1 is located under the shielding cone of the secondary alcohol (in pyridine-d5), (b) synthesis of the
anthryl ring of the reagent in the sp conformer, corresponding -D- or R-D-glucopyranosides (Figure
whereas in conformer ap, it is not shielded. The 183), (c) measurement of the 13C NMR spectrum of
reverse holds for substituent L2 (shielded in the either the -D- or R-D-glucopyranoside, (d) calculation
minor conformer ap and unaffected in sp) and, as a of the glucosidation-induced shift variations for the
result, when one compares the spectrum of an alcohol anomeric carbon and for the R- and -carbons of the
in Figure 182a with that of its (R)-9-AMA derivative, aglycone using the relations (anomeric carbon, 1)
104 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 187. (a) Structure of the sp and ap conformers of an (R)-MPA ester. (b) Chemical shifts of substituents L1 and L2
in the sp and ap conformers. (c) Average chemical shifts of L1 and L2 at room temperature (T1). (d) Average chemical shifts
of L1 and L2 at low temperature (T2).

Figure 188. Distribution of shielded and deshielded areas in the (R)- and (S)-MPA esters of (-)-menthol and selected
T1T2 values.

) (alcoholic glucoside) - (methyl glucoside) and shifts were calculated as follows:

(R- and -carbons) ) (alcoholic glucoside) - -
(alcohol), and (e) the absolute configuration of the (anomeric proton, 1)
alcohol is then determined by comparison of the 13C ) (alcoholic glc-Ac4) -
NMR chemical-shift changes with the glucosidation (methyl glc-Ac4) for the sugar moiety
shifts of a series of secondary alcohols of known and
absolute configuration, classified according to their (R- and -protons)
steric hindrance and degree of substitution in the C
) (alcoholic glc-Ac4) -
atoms next to the chiral center.
(alcohol) for the aglycone unit
A caution note alerts the readers about the ap-
plication of this procedure to sterically crowded As in the 13C NMR method, comparison of the values
alcohols. obtained with those of secondary alcohols of known Tetra-O-acetyl--glucosylates in 1H configuration and different steric hindrance is neces-
NMR. The tetra-O-acetylglucosidation-induced 1H sary for assignment.
NMR shifts of a series of secondary alcohols were also The same caution note that was mentioned previ-
found highly characteristic of their absolute configura- ously applies when this procedure is intended for use
tion.55c Particularly important for the diagnostics with highly crowded alcohols.
were the protons of the aglycone attached to the C Tetra-O-benzoyl--glucosylates in 1H
atoms at the -position (see Figure 184), and accord- NMR. Another procedure based on the comparison
ingly, the tetra-O-acetylglucosidation-induced 1H NMR of the NMR spectrum of the substrate with that of a
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 105

glycosylation-induced NMR shifts. This effect is

quantified as the difference between the chemical
shifts in the D-glucopyranoside derivative and the
free alcohol ( ) D - ROH). The protons anti to
the endocyclic glucopyranoside oxygen (O-5) are
shielded due to the effect of the benzoyl group at C-2
(negative ), whereas protons syn to that O atom
show positive values, because of their proximity
to O-5, as shown in the model of Figure 185a. Figure
185b shows experimental values that fit the model
for an enantiomeric pair of alcohols.
Other alcohols of known absolute configuration
that have been used to assess the viability of this
methodology are shown in Figure 186. In some cases,
the signs are opposite to those expected (for
instance, in compounds 186.1, 186.5, 186.11, and
Figure 189. Secondary alcohols of known configuration 186.13); however, the authors state that the config-
studied through the low-temperature method. uration can still be deduced if all the other protons
are considered. However, in case of doubt, they
single derivative of an auxiliary reagent, and closely recommend that a double-derivatization procedure be
related to the previous methods, has been described used (formation of both the D- and L-benzoylgluco-
by Trujillo et al.56 for the assignment of the config- pyranosides) and the larger D - L values consid-
uration of alcohols. ered (Section rather than D - ROH.
In this procedure, the chiral alcohol is converted Attempts to assign the configuration of the alcohol
to the corresponding tetra-O-benzoyl--glucoside by using the D - ROH shift of the H atom at the
reaction with commercial 2,3,4,6-tetra-O-benzoyl-R- chiral center (C1) alone did not give successful
D-glucopyranosyl bromide in the presence of silver
trifluoromethanesulfonate and 1,1,3,3-tetramethyl-
4.1.3. Low-Temperature NMR of MPA Esters
urea. The 1H NMR spectrum of the product is then
compared with that of the free alcohol. The observed As mentioned previously, reducing the temperature
differences between the two spectra are caused by of the equilibrium should lead to an increase in the
the anisotropic effect of the benzoyl groups and the population of the most stable conformer. In the case

Figure 190. Diagram to deduce the absolute configuration of an alcohol from the experimental T1T2 signs of either its
(R)- or (S)-MPA ester.
106 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 191. Partial spectra of the (R)-MPA ester of diacetone-D-glucose at (a) 303, (b) 223, and (c) 193 K in CS2/CD2Cl2.

Figure 192. Plot of the proportional increase of RS with decreasing probe temperature T (in K) for the Me(9) group of
(-)-menthyl esters of arylmethoxyacetic acids.

of MPA esters, this is the sp conformer and, therefore, parameter is positive for substituent L1 (the group
greater shielding of the substituent located on the located on the same side as the Ph ring) and negative
same side as the Ph group in the (R)-MPA ester is for substituent L2 (the group located on the other
expected. The practical application of this approach side) and provides the configurational information.
requires the preparation of a single MPA ester (either Several alcohols of known absolute stereochemistry
the R or the S derivative) and comparison of two 1H have been used to demonstrate the general ap-
NMR spectra taken at two sufficiently different plicability of this phenomenon. Figure 188 shows the
temperatures.112 The differences in chemical shifts shielded and deshielded areas in the MPA esters of
are now measured as T1T2 (the chemical shift at the (-)-menthol. Other alcohols that have been tested are
higher temperature T1 minus that at the lower shown in Figure 189. From a practical standpoint, it
temperature T2) and obey the scheme shown in seems that temperatures lower than -70 C are not
Figure 187, where, at lower temperature, the signal necessary, and, therefore, the commonly used CDCl3
for substituent L2 moves to lower field and that of or a CS2/CD2Cl2 (4:1) mixture can be used as the
substituent L1 to higher field. The sign of this NMR solvent. The scheme shown in Figure 190
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 107

reagents.29,30 In fact, the study of the sensitivity of

chemical shifts, relative to changes in the tempera-
ture, for the esters of (-)-menthol with several
reagents (Figure 192) shows that the RS values of
MA, MPA, and 2-NMA esters increase steadily at
lower temperatures whereas those of the 9-AMA
esters remain practically constant. This is because
in the first case there is a progressive shift of the
equilibrium toward the most stable sp conformer
whereas, in the 9-AMA esters, the sp/ap ratio is at
room-temperature already shifted toward the sp and
there is little place for further displacement.

4.1.4. Complexation of MPA Esters with Barium(II)

A second procedure devised for the controlled shift
of the conformational equilibrium in AMAA deriva-
tives consists of the addition of a metal salt that is
capable of stabilizing a certain conformer by com-
plexation. Approaches for secondary alcohols113 and
primary amines are described in the literature. In
both cases, MPA was used as the auxiliary reagent
and barium(II) as the chelating agent.
Figure 193. Formation of Ba2+ complexes of the (R)-MPA The conformational equilibrium for the MPA esters
ester of a chiral secondary alcohol. The population of the of chiral alcohols is outlined in Figure 193. The
sp form increases by formation of the most stable sp-Ba2+ addition of the Ba2+ salt (i.e., Ba(ClO4)2) to the NMR
complex, so the signals for substituent L1 shift to higher
field. tube containing either the (R)- or (S)-MPA ester in
MeCN-d3 causes the spectra to change in a way that
outlines the steps that should be followed for the is practically identical to that observed in the low-
application of this methodology using either the (R)- temperature experiments, indicating that the con-
or (S)-MPA derivative. formational changes introduced are similar (i.e.,
Figure 191 shows the actual spectra of the (R)-MPA there is an increase in the population of the sp
ester of diacetone-D-glucose, taken at 303, 223, and conformer). In fact, in addition to 1H NMR evidence,
193 K, and illustrates the upfield and downfield shifts 13
C NMR and CD experiments, together with high-
of the protons situated on the shielded and non- level ab initio Hartree-Fock (HF) and density func-
shielded areas around the chiral center. tional theory (DFT) calculations, also prove that the
Curiously, when the usually more efficient reagent Ba2+ atom becomes strongly bonded to the O atoms
9-AMA was used instead of MPA, no improvement of the CdO and MeO groups, therefore leading to an
in the T1T2 values was observed and this is related increase in the stability and population of the sp form
to the different sp/ap ratio present in the two (see Figure 194).

Figure 194. HF optimized geometries for the (a) sp-Ba2+ and (b) ap-Ba2+ conformers of the (R)-MPA ester of 2-propanol.
The sp-Ba2+ geometry is more stable than that of ap-Ba2+, by 13.9 kcal/mol.
108 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Several other ions have been investigated but the

results are not as good; even the Mg2+ ion, which is
well-known for its superior ability to complex with
oxygen donors, produces lower shifts than the Ba2+
ion. The reasons for this apparent incoherence were
highlighted by ab initio studies, which showed that,
in these cases, powerful solvent-cation interactions
are operative and, thus, make these metals less
suitable, because of strong solvation by the solvent
MeCN. The importance of the Ba2+/MeCN-d3 pair is
therefore a key aspect in this method.
From the standpoint of the application of this
procedure to the assignment of absolute configura-
tion, only one derivative should be prepared (with
either the R or S auxiliary) and two NMR spectra
should be taken: one of the derivative alone and the
other after the addition of Ba2+ ions. The shifts
produced are expressed as Ba ) (MPA ester) -
(MPA ester + Ba2+) and should be positive for one
of the substituents and negative for the other. Figure
195 shows the values obtained for some representa-
tive examples of known absolute configuration that
have been used to test the coherence, reliability, and
scope of this approach.
The scheme shown in Figure 196 illustrates graphi-
cally the steps that should be followed for the
assignment of the absolute configuration of a second-
ary alcohol by this procedure.
In regard to the limitations of this method, there
is an obvious drawback in terms of those substrates
in which substituents L1/L2 contain functional groups
that are able to complex with the Ba2+ ion. In these
cases, there is no guarantee that the general confor-
mational scheme shown in Figure 193 is still opera-
tive, and, therefore, safe assignment of the substrate
cannot be assured. Examples where competitive
chelation occurs because of the presence of extra
carbonyl groups (i.e., 195.18 and 195.19 in Figure
195) have been described.113
The actual spectra of the (R)-MPA ester of di-
acetone-D-glucose before and after the addition of
Ba2+ ions are shown in Figure 197. Comparison with
the spectra for the same compound taken at different Figure 195. Selected Ba values (in ppm) obtained from
temperatures (Figure 191) illustrates the similarity the 1H NMR spectra of the (R)- and (S)-MPA esters of
structurally representative chiral esters.
in the shifts generated by both procedures because
of the increase in the population of the most stable any chelating agent, is the ap form, where the L2
sp conformers. substituent is shielded by the phenyl ring of the
reagent. Upon addition of a Ba2+ salt (e.g., Ba(ClO4)2)
4.2. Application to r-Chiral Primary Amines: to the NMR tube containing the MPA amide (MeCN-
Complexation of MPA Amides with Barium(II) d3 as the solvent), changes in the NMR chemical
shifts are observed, indicating that the equilibrium
In a similar way, the absolute configuration of is being shifted to the sp form (L1 is now the shielded
R-chiral primary amines can be deduced from the group). The changes in the chemical shifts of L1 and
NMR complexation effect produced by the addition L2 are expressed as Ba ) (MPA amide + Ba2+) -
of a Ba2+ salt on a single MPA amide, prepared from (MPA amide) and are positive for one substituent
either (R)- or (S)-MPA.77 and negative for the other. (See Figure 199.)
In this case, the summarized conformational equi- The scheme shown in Figure 200 illustrates graphi-
librium is shown in Figure 198 for an (R)-MPA cally the steps that should be followed for the
amide. The most stable conformer, in the absence of assignment of the absolute configuration of a primary
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 109

Figure 196. Diagram to deduce the absolute configuration of a chiral secondary alcohol from the Ba experimental
values of one ester (either the (R)- or the (S)-MPA derivative).

amine by this method. Figure 201 shows the changes evaluation of their enantiomeric excess, and the
produced in the spectra of the (R)-MPA amide of assignment of their absolute configuration at the
L-valine methyl ester upon the addition of Ba(ClO4)2. microscale level and in a single operation.
As discussed previously for MPA esters, amines Two different cases can be distinguished:
that have functional groups in substituents L1/L2 that (a) If the absolute configuration of each enantiomer
are able to complex with the Ba2+ ion should not be in a mixture (including racemic ones) is to be deter-
assigned by this method. 4-Oxo-R-amino acids, such mined, the mixture is derivatized with either (R)- or
as 202.1 (see Figure 202), are good examples that (S)-9-AMA, according to standard procedures. The
show the effect of such competitive chelation.114 mixture of 9-AMA esters is submitted to HPLC NMR
and comparison of the 1H NMR spectra of the two
5. New Trends HPLC peaks allows the assignment of the configu-
5.1. Hyphenated High-Pressure Liquid ration based on the shielding/deshielding effects
Chromatography (HPLC) NMRMS Techniques caused in substituents L1/L2 by the auxiliary reagent
for Automatization and Microscale Analysis of known configuration (see Figure 203 and Section
The development of hyphenated techniques for 3.1.3).
rapid separation of mixtures and spectroscopic analy- (b) If assignment of the absolute configuration of
sis of its components is a topic of great interest, a single enantiomer is the objective, then the alcohol
particularly when the amount of sample is limited is derivatized with a 3:1 mixture of (R)- and (S)-9-
and the need for automatization is high. This is the AMA. The unequal mixture of 9-AMA esters is
case for high-pressure liquid chromatography nuclear subjected to HPLC NMR and the spectra of both
magnetic resonance-mass spectroscopy (HPLC NMR- peaks are compared, taking into consideration that
MS), which has been shown to be extremely useful the major component of the mixture corresponds to
in certain pharmaceutical and medical applications. the (R)-ester and the minor component corresponds
Quite recently, Riguera et al. reported115 that the to the (S)-ester (see Figure 204).
derivatization of mixtures of enantiomeric secondary In both cases, ,1 mg of the alcohol is needed for
alcohols with 9-AMA, followed by tandem HPLC derivatization and no more than 10-15 g of the
NMR, allows the separation of the enantiomers, the mixture of 9-AMA esters, dissolved in acetonitrile-
110 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 197. Partial 1H NMR spectra (250 MHz) for the (R)-MPA ester of diacetone-D-glucose (a) before and (b) after the
addition of Ba2+.

Figure 198. Main conformers present in the equilibrium of (R)-MPA amides in the absence and presence of a barium(II)

d3, are injected onto a standard reverse phase column experimental conditions and many examples of such
eluted with acetonitrile-d3/deuterium oxide/formic separation.
acid mixtures. The number and structural variety of the alcohols
The generality of this approach, including the that have been used to validate the procedure indi-
reliability of the NMR data obtained in acetonitrile- cate beyond doubt that this methodology is quite
d3/deuterium oxide/formic acid mixtures instead of general for secondary alcohols and that it can be used
deuteriochloroform, was demonstrated by its success- in the analytical and semipreparative scales. The
ful application to the secondary alcohols of known simplification involved in the use of the same auxil-
absolute configuration (205.1-205.18, shown in Fig- iary reagent for both the HPLC separation and the
ure 205, including saturated, unsaturated, aromatic, NMR configurational assignment, and its implemen-
acyclic, and cyclic compounds). tation in directly coupled HPLC NMR should be
The authors also mention that if actual isolation extremely useful for the automatization of many
of the 9-AMA esters of the enantiomeric alcohols is processes involving optically active compounds. HPLC
needed for preparative purposes or else, higher NMR has been recently used to purify and analyze
loading can be obtained when the HPLC separation the two MTPA esters of a very small sample of a
is performed with normal-phase HPLC and provides natural product.116
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 111

5.2. Resin-Bound Auxiliaries: The Mix and

Shake Method
In the search for new ways to simplify the configu-
rational assignment by NMR, a new procedure that
allows determining the absolute configuration in just
few minutes and without any type of separation,
workup, or manipulation being needed has been
recently presented.117 In this approach, the required
derivative/s are prepared by simply mixing a solid
matrix-bound auxiliary reagent (i.e., MPA, MTPA, or
BPG) with the chiral substrate (i.e., a primary amine
or a secondary alcohol) directly in the NMR tube and
the NMR spectra of the resulting derivatives are
obtained without any further manipulation after a
few minutes.
The key for the success of this methodology lies in
the linking of the auxiliary reagent to the solid
matrix through a mixed anhydride group, which
allows the auxiliary reagent moiety to act as an
electrophile toward the chiral substrate and permits
the solid resin to be the leaving group, liberating the
ester or amide derivative into the solution (see Figure
Resins containing either a single enantiomer or
mixtures of both enantiomers of MPA, MTPA, and
BPG, linked through anhydride groups, were pre-
pared from commercial carboxypolystyrene and used
to derivatize alcohols and amines. The reaction is
performed directly in the NMR tube, in Cl3CD, and
takes from 5 min (with primary amines) to a few
hours (with secondary alcohols), depending on the
substrate. After that time, the spectra are recorded
as usual, the resin that remains in the tube causes
no interference at all, and the assignment of absolute
configuration is conducted from the RS signs using
the corresponding configurational models (see Section
Figure 199. Selected Ba values (given in ppm) obtained 3 of this review).
from the 1H NMR spectra of the (R)- and (S)-MPA amides The resulting spectrum is sufficiently clean to allow
of structurally representative chiral amines. assignment of the absolute stereochemistry at the

Figure 200. Diagram to deduce the absolute configuration of a chiral primary amine from the experimental Ba signs,
from either the (R)- or (S)-MPA amide.
112 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 201. Partial 1H NMR spectra of the (R)-MPA amide of L-valine methyl ester in the presence (upper) and absence
(bottom) of barium perchlorate. The observed shifts are highlighted.

(those of the (R)-MPA derivative are half the size of

those for the (S)-derivative) allow the upfield/down-
field shifts (RS) to be measured.
Another successful adaptation that makes the
Figure 202. usual double derivatization (with (R)- and (S)-MPA)
and recording of two spectra unnecessary is the
microscale level (<0.5 mg of substrate). In fact, if a method based on the complexation of MPA esters
1:2 (R)/(S)-MPA resin is used, the assignment can be or amides with the Ba2+ ion (see Section 4 of this
performed in a single operation and by taking just review). This approach can also be performed directly
one spectrum because the different ratios of the NMR in the NMR tube with a resin-bound MPA (single
signals of the two diasteromeric MPA derivatives enantiomer) in CD3CN, although formation of the

Figure 203. Enantioresolution and assignment of the absolute configuration of the enantiomers of a chiral secondary
alcohol by tamden HPLC NMR of its (R)-9-AMA esters (the assignment is the opposite if (S)-9-AMA is chosen as the
Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 113

Figure 204. Assignment of the absolute configuration of a chiral secondary alcohol by derivatization with a (R)-9-AMA/
(S)-9-AMA 3:1 mixture followed by tamden HPLC NMR of the diasteromeric esters.

6. Concluding Remarks: A Critical Assessment

The interest of researchers on the use and discov-
ery of procedures for the determination of absolute
configuration of organic compounds by NMR is
evidently originated by the economy and simplicity
of the methods and the wide availability of NMR
equipments in all laboratories. These advantages
explain the very large number of papers published
on this topic that are either devoted to the proposal
of new chiral-derivatizing agents (CDAs) or mention-
ing their application to the configurational assign-
ment of a certain substrate. It is among those
working in the field of natural productssplagued
with small samples difficult to crystallizeswhere the
application of these techniques is more appreciated
and the highest number of papers is found, the same
as for those working in the synthesis of chiral
Nevertheless, the fact that, using these NMR
methods, the absolute configuration is determined in
solution and that conformational and other structural
information related to the through-space anisotropic
effect of the reagent is also obtained, wide their
applications to other fields.
There are also many publications that have pro-
posed new CDAs; however, researchers should be
aware that the reliability of the NMR assignment
depends very much on the number and structural
variety of the substrates of known absolute configu-
ration that have been used as test compounds to
validate the procedure/reagent. Consequently, of the
many reagents and procedures described in this
Figure 205. Chiral alcohols studied by this method. review, only a few can be considered to produce
reliable assignments, because, in many cases, the
derivative in this solvent takes longer time (90 min) number of test compounds is so small that no general
than in Cl3CD. behavior can be deduced from the data available.
A precedent of this methodology can be found in In the past few years, the experimental data has
the preparation of a ROMPgel support of activated been frequently complemented with theoretical work
Mosher ester (MTPA) as a means to performing the (conformational analysis, energy minimization, shield-
purification free synthesis of MTPA amides by the ing-effect calculations) and this certainly provided
Barrett group.118 supplementary support to the method/reagent in-
114 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

Figure 206. MPA, MTPA, and BPG resins used in this mix and shake methodology.

volved; however, these calculations also showed that of substrate sample is available. In a paper recently
the differences in energy between the derivatives are published, the enantioresolution of secondary alco-
frequently so small that calculations can in no way hols (either an unequal or racemic mixture of enan-
replace the use of a collection of compounds of known tiomers) and the assignment of the absolute config-
absolute configuration and representative structures uration of the enantiomers is achieved in a single
as a test compounds. operation and with just a few micrograms of sub-
In regard to the substrates investigated, most of strate by tandem HPLC NMR of the 9-AMA deriva-
the efforts have been directed to monofunctional tives. In another publication by the same group, the
compounds. Secondary alcohols and primary amines simplification of the methodology is attained using
have been the most frequently investigated, and solid matrix-bound auxiliaries that allow the deriva-
although other functional groups were also studied, tization to happen in the NMR tube and within a few
those remain the most amenable to assignment by minutes. In this way, the NMR spectra can be
NMR procedures. Quite recently, an extension of the obtained after just mixing the resin and the substrate
procedure to diols has been described, which will without other operations being necessary.
surely open the way to the assignment by NMR of In summary, NMR procedures for assignment of
other polyfunctional compounds. the absolute configuration have evolved in a few
Although many procedures that are based on the decades from the original Moshers method to a
use of NMR of nucleus different from 1H (i.e., 19F, mature field, where a menu containing a large
C) have been described and recommended on the number of CDAs is available for the study of different
basis of the simplified NMR spectra obtained, the low substrates using a variety of approaches. New excit-
sensitivity of those nuclei to the anisotropic effects ing advances will undoubtedly emerge in the future.
produced by the CDA originate very small shifts that
overcome that advantage. Nevertheless, the simpli- 7. Acknowledgment
fication of the spectra renders those reagents very
The authors are grateful to Ministerio de Ciencia
useful when the enantiomeric excess of a mixture
y Tecnologa and Xunta de Galicia for financial
(but not the configurational assignment of each
support (currently through Grant Nos. BQU2002-
enantiomer), is the objective.
01195 and PGIDT02BTF20902PR) and to their co-
Other than papers directed to widen the scope of
workers, who have contributed to many of the articles
functional groups amenable to be studied by the
described herein.
NMR procedure, in the past few years, some efforts
have been described to simplify the method and
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2002, 67, 7927. CR000665J

CHIRALITY 00:000000 (2009)

Use of (S)-BINOL as NMR Chiral Solvating Agent for the

Enantiodiscrimination of Omeprazole and its Analogs
Department of Research and Development, Esteve-Qumica S.A., Barcelona, Spain

ABSTRACT The application of (S)-1,10 -binaphthyl-2,20 -diol as NMR chiral solvating

agent (CSA) for omeprazole, and three of its analogs (lanso-, panto-, and rabe-prazole)
was investigated. The formation of diastereomeric hostguest complexes in solution
between the CSA and the racemic substrates produced sufficient NMR signal splitting
for the determination of enantiomeric excesses by 1H- or 19F-NMR spectroscopy. Using
of hydrophobic deuterated solvents was mandatory for obtaining good enantiodiscrimi-
nation, thus suggesting the importance of intermolecular hydrogen bonds in the stabili-
zation of the complexes. The method was applied to the fast quantification of the enantio-
meric purity of in-process samples of S-omeprazole. Chirality 00:000000, 2009. V C 2009

Wiley-Liss, Inc.

KEY WORDS: esomeprazole; lansoprazole; pantoprazole; rabeprazole; host-guest;

enantiorecognition; enantiomeric purity

INTRODUCTION Although several chromatographic methods for the chi-

Since the recommendations of the US Food and Drug ral analysis of omeprazole and its analogs have been pub-
Administration (FDA) and other regulatory drug agencies lished yet,1519 no information about NMR as a chiral tech-
concerning the development of chiral drugs,13 it is a com- nique for this family of drugs can be found in the litera-
mon and challenging issue in the process research of opti- ture. This is probably due to the high degradation rate of
cally pure Active Pharmaceutical Ingredients (APIs) to these compounds in water, which hinders the use of
find fast and reliable analytical methods to follow and hydrosoluble CSAs like cyclodextrins or chiral organic
quantify the enantiomeric enrichment (or the degree of acids/bases; their low affinity with chiral lanthanide
asymmetric induction) when a resolution (or an enantiose- reagents, and the lack of functional groups capable of
lective synthesis) is applied. reacting easily with Mosher reagents. As a part of our
Among the assortment of analytical techniques, and de- investigation on chemical processes for the preparation of
spite its inherent low sensitivity, NMR spectroscopy is esomeprazole, we found that the chiral host (S)-1,10 -
probably the most useful in terms of speed and minimal binaphthyl-2,20 -diol ((S)-BINOL, 2, Fig. 1), which is also
sample preparation, two key points when considering the used for the resolution of the omeprazole racemate in labo-
usually fast milestones involved in the discovery and de- ratory20 and industrial scale,21 acts as a convenient NMR
velopment of drugs. In this sense, the use of enantiomeri- CSA for the rapid and accurate quantification of low levels
cally pure chiral solvating agents (CSA),46 chiral derivatiz- of the minor enantiomer. It has been previously reported
ing agents7,8 and, more recently, chiral liquid crystal sol- that enantiopure BINOL is a good NMR chiral shift rea-
vents,9 constitute a well established toolbox for the NMR gent for sulfoxides,22 selenoxides,23 and sulfinimines,24
enantiodiscrimination of optically active molecules. and the enantiomers of BINOL and some of its derivatives
The potent proton-pump inhibitor (PPI) omeprazole have also been found to be excellent compounds for the
(5(6)-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl) chemical resolution of chiral sulfoxides.25 More in general,
methylsulfinyl)-1H-benzo[d]imidazole, 1, Fig. 1)y has been (R)-BINOL has been recently applied as NMR CSA for
until recent times one of the most used and marketed ra- some nonsulfoxide important drugs.26
cemic drugs worldwide.10,11 The stereogenic center of the In this work, we report the results of the application of
omeprazole molecule is located on the sulfur atom, and it this NMR method to the enantiodiscrimination of omepra-
has been reported that the S enantiomer (esomeprazole, zole and three structurally related analogs (lansoprazole,
(2)-(S)-1) shows higher bioavailability than the racemate
due to a difference in the rates of metabolism of both enan- Additional Supporting Information may be found in the online version of
this article.
tiomers.1214 Esomeprazole, as its magnesium salt, was *Correspondence to: Jordi Redondo, Department of Research and Devel-
approved in 2001 and is also a blockbuster anti-acid drug. opment, Esteve-Qumica S.A., Caracas street 1719, Barcelona E-08030,
Spain. E-mail:
Received for publication 1 September 2008; Accepted 17 June 2009
Omeprazole presents tautomerism in the benzimidazole group. There- DOI: 10.1002/chir.20766
fore, both tautomers must be considered when using the systematic no- Published online in Wiley InterScience
menclature. For simplicity, only one tautomer is represented in Figure 1. (
C 2009 Wiley-Liss, Inc.

Fig. 1. Molecular structures of omeprazole (1) and (S)-BINOL (2).

3, pantoprazole, 4, and rabeprazole, 5 (Fig. 2)) in different

deuterated solvents. Furthermore, some considerations
concerning the hostguest interaction between (S)-BINOL
and the S-enantiomer of 1 are presented in the light of the
NMR results.

Fig. 2. Molecular structures of lansoprazole (3), pantoprazole (4) and

MATERIALS AND METHODS rabeprazole (5).
The PPIs 1, (S)-1, 3, 4, and 5 were obtained from our
laboratory using proprietary synthetic processes,21,27,28 PPIs. Conversely, no signal splitting at all was observed
and were characterized by 1H-, 19F-, and 13C-NMR as well when using hydrophilic solvents (CD3OD, acetonitrile-d3,
as by mass spectrometry. (S)-BINOL (2) was purchased acetone-d6, and DMSO-d6), thus suggesting the impor-
from Sigma-Aldrich. The absolute configuration of (S)-1 tance of the intermolecular H bonds in the formation of
and the optical purity of the sample were determined by the complexes, since hydrophobic solvents must help,
means of the chiral tests described in the European Phar- through a solvophobic effect, to establish such H bonds
macopoeia.29 All NMR measurements were obtained with between host and guest, whereas water-miscible solvents
a Varian Mercury spectrometer operating at 400.1 MHz must difficult them (significantly, the addition of only one
for 1H and 376.4 MHz for 19F. The host-guest complexes drop of DMSO-d6 to a sample prepared in any of the
were prepared by dissolving equimolecular quantities of 2 hydrophobic deuterated solvents completely removes the
and the PPI in the deuterated solvents at ambient tempera- enantiodiscrimination effect).
ture (the concentrations were around 40 mM). The assign- Table 1 shows that in the presence of one equivalent of
ment of the 1H-NMR spectra of compounds 1-5 was (S)-BINOL using CD2Cl2 as solvent, most of resonances of
accomplished by means of standard 1D and 2D NMR tech- omeprazole are split at room temperature as a conse-
niques, namely 2D COSY (1 scan per FID; 200 incre- quence of the coexistence of two diastereomeric host-
ments) and selective 1H {1H} NOE experiments (mixing guest complexes in solution. It is noteworthy that the im-
time: 800 ms, 256 scans). portant nonequivalence of H13 (DDd: 0.084 ppm) that per-
mits the easy and reliable quantification of the enantio-
RESULTS AND DISCUSSION meric purity (Fig. 3). The complexation also produced a
Enantiodiscrimination Detected by 1H-and 19F-NMR change in the chemical shift of the methylene H8AB pro-
The power of enantiodiscrimination of a NMR chiral sol- tons as well as a considerable broadening of their signals.
vent agent relies on the formation of a pair of diastereo- Similar signal splitting was found when CDCl3 or toluene-
meric complexes coexisting in solution. If the lifetime of d8 was used. Unfortunately, no information could be
such complexes is long enough on the NMR time scale, extracted from the signals of omeprazole H4 and H7 pro-
splitting could be observed for several resonances of the tons, due to the extreme broadness caused by the tautom-
molecular species, thus permitting the identification and erism of the benzimidazole group at room temperature.
quantification of the substrate single enantiomers. Hydro- Splitting was also observed for several 1H signals of
gen bonds, pp interactions, and van der Waals forces are compounds 3, 4, and 5 in the presence of (S)-BINOL. In
the common factors invoked for explaining the formation those cases, not only H13 but also the pyridine proton H12,
of hostguest complexes in solution. Moreover, the effect not present in omeprazole, showed an appreciable none-
of the solvent must be regarded as one of the important quivalence induced by the enantiodiscrimination phenom-
issues in the stabilization of the diastereomeric pairs and, enon (Fig. 4 and Table 1). In the case of compound 5, the
therefore, in the enantiodiscrimination ability of the CSA.
In this sense, it was found that, among the common deu-
Claramunt et al30 have reported that both tautomers of omeprazole can
terated solvents used in NMR spectroscopy, only the be observed by NMR at 195 K in THF-d8, with predominance of the 6-
methoxy tautomer (63%). At room temperature, however, the 1H-NMR
hydrophobic ones (namely, CDCl3, CD2Cl2, and toluene- spectrum of omeprazole consists of single signals revealing the weighted
d8) afforded good enantiodiscrimination of the studied average of the two tautomeric situations.
Chirality DOI 10.1002/chir

TABLE 1. Chemical shift nonequivalence (DDd, ppm) for protons of compounds 1, 3, 4, and 5 in presence of (S)-BINOL in
different deuterated solvents at 228C (signals from protons H4 and H7 not reported due to their broadness by effect of the
benzimidazole tautomerism)

Compound Host/guest ratio H6 H5 H8A H8B H12 H13 H15 H16 H14 H17 H18

1 (CD2Cl2) 1:1 0.000 0.042 0.018 0.084 0.033 0.005 0.017 0.004
1 (CDCl3) 1:1 0.000 0.000 0.014 0.055 0.017 0.000 0.007 0.000
1 (tol-d8) 1.5:1 bd 0.007 0.000 0.040 0.020 0.014 0.022 0.004
3 (CD2Cl2) 1:1 bd bd 0.008 0.031 0.027 0.054 0.000 0.009
3 (CDCl3) 1:1 bd bd 0.000 0.025 0.016 0.050 0.000 0.005
3 (tol-d8) 4:1 bd bd 0.032 0.000 0.000 0.016 bd 0.006
4 (CD2Cl2) 1:1 bd 0.011 0.019 0.028 0.072 0.003 0.005 0.000
4 (CDCl3) 1:1 0.022 0.014 0.020 0.025 0.075 0.000 0.008 0.000
4 (tol-d8) 1.5:1 bd 0.000 0.000 0.015 0.054 0.006 0.009 0.000
5 (CD2Cl2) 1:1 bd bd 0.000 0.021 0.035 0.060 0.007 0.000 0.012 0.000 0.000
5 (CDCl3) 1:1 bd bd 0.015 0.005 0.028 0.060 0.005 0.000 0.008 0.000 0.000
5 (tol-d8) 1:1 bd bd 0.000 0.007 0.015 0.015 bd 0.000 0.015 0.000 0.000

In all cases, H8A refers to as the downfield H8 proton and H8B to the upfield H8, broad signal.

lack of splitting in the signals from the side chain protons In addition to the induced proton nonequivalence, the
H16, H17, and H18 suggests that this part of the molecule is formation of the host-guest complex produced changes in
far away from the binding sites involved in the enantiore- the chemical shifts of the involved molecular species. In
cognition process (in fact, only H15, which are the closest
chain protons to the pyridine ring, manifest some degree
of splitting).
The effect of the enantiorecognition was also evident in
the 19F-NMR spectrum of the CHF2 group of compound 4,
whose original doublet (JF-H: 74 Hz) slightly split in the
presence of (S)-BINOL (Fig. 5). Interestingly, no extra
splitting was observed in the 19F-NMR signal of the tri-
fluoromethyl group of compound 3 (a triplet with JF-H: 7.5
Hz), probably due to the high mobility of the OCH2CF3
chain and its relatively far distance from the sites of inter-
action with (S)-BINOL.
Clearly, among the solvents studied, toluene-d8 was the
less favorable due to the relatively low solubility of com-
pounds 3 and 4 (for those compounds, the host/guest
molar ratios achieved in toluene solution were 5.4:1 and
1.3:1, respectively, as determined by integration of the cor-
responding 1H signals).

Fig. 3. Partial 1H-NMR spectrum of A: omeprazole in CDCl3; B: an

equimolecular mixture of omeprazole and (S)-BINOL in CDCl3. The Fig. 4. Partial 1H-NMR (CD2Cl2) spectra of A: 3/(S)-BINOL 1:1; B: 4/
resonances of H13 omeprazole enantiomers are indicated by arrows. Aster- (S)BINOL 1:1; C: 5/(S)-BINOL 1:1. The H12 and H13 doublets from each
isks indicate (S)-BINOL signals. enantiomer are indicated by ~ and l, respectively.
Chirality DOI 10.1002/chir

TABLE 2. 1H chemical shift increments (Dd) induced on

compounds 1, 3, 4, and 5 by complexation with one
equivalent of (S)-BINOL, in CDCl3 at 228C

Compound Assignment Dd (ppm)

1 H13 (R) 20.105

H13 (S) 20.147
H8A 20.063
H8B 20.061
H15 (R) 20.025
H15 (S) 20.037
3 H13 (d) 20.142
H13 (u) 20.192
H12 (d) 20.075
H12 (u) 20.095
H8A 20.103
H8B (d) 20.081
Fig. 5. 19F-NMR (CD2Cl2) of compound 4 in absence (A) and pres- H8B (u) 20.105
ence (B) of 1 equivalent of (S)-BINOL. H15 20.032
H14 (d) 20.031
H14 (u) 20.036
this sense, it was found that, among the resonances of 4 H13 (d) 20.118
H13 (u) 20.194
omeprazole, those of the pyridine H13 and the H8AB pro-
H12 (d) 20.047
tons displayed the more notable upfield shifts (low d val- H12 (u) 20.072
ues) in the presence of the CSA, thus suggesting an im- H8A (d) 20.101
portant interaction of this part of the molecule with the ar- H8A (u) 20.114
omatic groups of (S)-BINOL. A similar behavior was found H8B (d) 20.082
for the corresponding protons of compounds 3, 4, and 5 H8B (u) 20.101
(important upfield shifts for H13 and H8AB protons, me- 5 H13 (d) 20.137
dium shifts for H12 protons, slight shifts for methyl and H13 (u) 20.196
other aliphatic protons, see Table 2). On the other hand, H4 1 H7 20.080
most of the (S)-BINOL protons also showed upfield shifts H12 (d) 20.048
H12 (u) 20.077
when forming the complex, an effect that could be attrib-
H8A (d) 20.116
uted to magnetic anisotropy from the heteroaromatic H8A (u) 20.132
groups of the guest molecules and/or to conformational H8B (d) 20.118
changes of the (S)-BINOL molecule around its chiral axis H8B (u) 20.123
induced by the molecular recognition phenomenon.
As expected, when a molar excess of (S)-BINOL was For simplicity, increments below 20.025 not reported.
Abbreviations: (d), downfield signal; (u), upfield signal.
applied to the substrate, a shift of the complexation equi-
librium toward the associated species occurred and, conse-
quently, an increase of the signal separation was observed.
So, when the host:guest molar ratio reached 5:1 in CDCl3,
almost all the visible proton signals of omeprazole were
split. In this sample, the CH2 resonance can be described
as a pair of semioverlapped AB system signals (JA-B:
13.5 Hz), one for each diastereomeric complex (Fig. 6). A
clear pattern of four doublets was also observed for com-
pounds 3, 4, and 5, even at host/guest ratios around 1:1
(Fig. 7, as an example).

Solution Stoichiometry of the Complex

( S)-BINOL/Esomeprazole
Regarding the structural aspects of the complexes, it is
important to know the stoichiometry of the supramolecular
hostguest species in solution. One of the usual ways to
get this information is constructing a Jobs plot.31 This
method, also known as the Continuous Variation
Method,32 consists of plotting the increment in the chemi-
cal shift (Dd) of the protons of one of the components ver-
sus the mole fraction of such component. Provided no Fig. 6. Partial 1H-NMR (CDCl3) of (S)-BINOL/omeprazole, molar ratio
other secondary processes are present (for example, 5:1. The inset shows the split signal from omeprazole H8A, H8B protons.
Chirality DOI 10.1002/chir

Fig. 7. Partial 1H-NMR (CD2Cl2) spectrum of (S)-BINOL/pantoprazole

1:1 complex showing the split system of H8AB. The symbol ~ indicates
signals from one enantiomer and l from the other.

molecular aggregation due to excessive concentration of

the solutes), the maximum of the obtained parabolic curve
indicates the average solution stoichiometry of the com- Fig. 8. Continuous variation plots of H8A, H8B, and H13 protons of eso-
plexes. In our case, nine samples with a host 1 guest con- meprazole ((S) 2 1) complexed with (S)-BINOL.
stant concentration (43.4 mM) were prepared in CDCl3,
and variable amounts of esomeprazole (99% ee) were
added. The chemical shift changes induced for several cross correlation peaks were observed between host and
protons of esomeprazole (H8A, H8B, H13) were followed at guest protons, probably due to the weakness of the supra-
258C, and in all cases similar curves were obtained show- molecular association. The only reliable intermolecular
ing maxima at an approximate mole fraction of 0.5, consist- NOE contact was found between H30 of (S)-BINOL and the
ent with a 1:1 solution stoichiometry for the complex (S)- selectively irradiated H8AB esomeprazole protons, using
BINOL/esomeprazole (Fig. 8). the standard 1D NOESY sequence (Fig. 9). Although this
Some attempts were made to achieve more structural in- unique result, of course, does not permit the inference of
formation via 2D NOESY and ROESY experiments on the any preferred hostguest geometry, at least it shows the
(S)-BINOL/esomeprazole complex but, unfortunately, no transient proximity of both species and probably reflects

Fig. 9. Intermolecular NOE on H03 of (S)-BINOL after irradiation of H8AB protons of esomeprazole (intramolecular NOEs on esomeprazole H13 and
H14 protons are shown).
Chirality DOI 10.1002/chir

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5. Weisman GR. Nuclear magnetic resonance analysis using chiral sol-
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7. Yamaguchi S. Nuclear magnetic resonance analysis using chiral deriv-
atives. Asymm Synth 1983;1:125152.
8. Wenzel TJ, Wilcox JD. Chiral reagents for the determination of enan-
tiomeric excess and absolute configuration using NMR spectroscopy.
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9. Courtieu J, Lesot P, Meddour A, Merlet D, Aroulanda C. Grant DM,
Harris R., editors. Chiral liquid crystal NMR: a tool for enantiomeric
analysis. Encyclopedia of nuclear magnetic resonance, New York:
John Wiley & Sons; 2002. p 497505.
10. Lindberg P, Brandstrom A, Wallmark B, Mattsson H, Rikner L, Hoff-
man KJ. Omeprazole: the first proton pump inhibitor. Med Res Rev
11. Shi S, Klotz U. Proton pump inhibitors: an update of their clinical use
Fig. 10. Partial 1H-NMR spectrum of optically enriched omeprazole and pharmacokinetics. Eur J Clin Pharmacol 2008;64:935951.
(99.1 ee in the S-enantiomer) complexed with one equivalent of (S)- 12. Lindberg P, Keeling D, Fryklund J, Andersson T, Lundborg P, Carl-
BINOL, in CDCl3. The arrows indicate H13 proton signals from each enan- son E. Esomeprazole-enhanced bio-availability, specificity for the pro-
tiomer. ton pump and inhibition of acid secretion. Aliment Pharmacol Ther
13. Andersson T, Weidolf L. Stereoselective disposition of proton pump
the important role of the intermolecular H bonds in the inhibitors. Clin Drug Investig 2008;28:263279.
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Determination of the Enantiomeric Excess High-performance liquid chromatography enantioseparation of proton
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was checked by applying (S)-BINOL to samples of esome- 16. Jenkins AL, Hedgepeth WA. Analysis of chiral pharmaceuticals using
prazole ((S)-1) containing different amounts of its R-enan- HPLC with CD detection. Chirality 2005;17(Suppl):S24S29.
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with sufficient number of scans (typically 64) to achieve of chiral sulfoxides on polysaccharide phases by HPLC. Chirality
good signal-to-noise levels. Under such conditions, a rea-
18. Berzas-Nevado JJ, Castaneda-Penalvo G, Rodrguez-Dorado RM.
sonable limit of detection was established to be at least
Method development and validation for the separation and de-
0.5% of the minor enantiomer of omeprazole (Fig. 10), termination of omeprazole enantiomers in pharmaceutical prepara-
since it was possible to observe and integrate easily the tions by capillary electrophoresis. Anal Chim Acta 2005;533:127
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BINOL and esomeprazole 99.1% ee (enrichment deter- 19. Toribio L, Alonso C, Del Nozal MJ, Bernal JL, Jimenez JJ. Enantio-
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tion of optically active derivatives of 2-(2-pyridylmethylsulfinyl)-benz-
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chiral shift reagents, optically active 2,20 -dihydroxy-1,10 -binaphtyl and
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Chirality DOI 10.1002/chir

Chem. Educator 2004, 9, 359363 359

The Mosher Method of Determining Enantiomeric Ratios: A Microscale

NMR Experiment for Organic Chemistry

Steve Lee

School of Science and Mathematics, Roosevelt University, Chicago and Schaumburg, IL,
Received August 4, 2004. Accepted September 16, 2004.

Abstract: Moshers established technique of measuring the enantiomeric ratio of a chiral compound has been
adapted to be suitable for an undergraduate organic chemistry experiment. In this technique a chiral alcohol or
amine reacts with the Mosher acyl chloride, -methoxy--(trifluoromethyl) phenylacetyl chloride (MTPA-Cl), to
yield the corresponding diastereomeric ester or amide. The ester or amide products are analyzed by 1H and/or 19F
NMR spectroscopy, and signals for diastereotopic groups on the products are integrated to determine the ratio of
the diastereomeric products and consequently the ratio of enantiomeric substrates. This experiment has the
benefits of helping students (1) apply advanced topics in stereochemistry, such as stereoheterotopic ligands and
kinetic resolution; (2) learn a quantitative application of NMR spectroscopy, including 19F NMR; and (3) be
introduced to microscale techniques. Furthermore, even though this is a microscale NMR technique, it can be
conducted on a low-field (60- or 90-MHz) NMR spectrometer with satisfactory results.

Introduction used, and so the S enantiomer is arbitrarily shown in this

scheme. The enantiomeric substrate to be analyzed must
The most commonly used methods for determining the contain either a hydroxyl or amino functional group to permit
enantiomeric ratio of a sample of a chiral compound involve reaction with the acyl chloride. In the generic example shown
(1) chiral stationary phase HPLC or GC, (2) chiral shift in Scheme 1, (S)-1 reacts with enantiomeric alcohols (2) to
reagents with NMR spectroscopy, (3) optical rotation, or (4) yield their corresponding Mosher esters (3). The products are
derivatization into diastereomers with subsequent analysis [1]. now diastereomeric with diastereotopic groups that can thus be
Within this last method Mosher has provided a chiral distinguished and have their relative amounts measured by
derivatizing agent that has proven to become an industry NMR spectroscopy.
standard [2, 3]. This method has been updated and adapted into The racemization of the starting materials or products during
an experiment that is pedagogically beneficial for organic the reaction is not mechanistically reasonable in most cases
chemistry students as it relates to stereochemistry, 1H and 19F due to the mild reaction conditions; however, if the products
NMR spectroscopy, and microscale techniques. With this are solid at ambient temperature, differential crystallization
technique students are able to analyze a sample of a chiral could lead to an inaccurate determination of the enantiomeric
compound to determine its enantiomeric ratio and the ratio and, thus, care must be taken whenever dealing with solid
configuration of the major enantiomer. This technique can products.
either be used as its own experiment or attached to an O
asymmetric synthesis or resolution, for example, the resolution F3C RR (R,R)-3
of -methylbenzylamine [4], which is a common organic HO RR
laboratory experiment. The topics and skills for this Cl +
experiment are suitable for sophomore-level organic chemistry Ph OCH3
F3C RS (R,S)-3
courses or upper-level advanced courses. It has been used at O
Roosevelt University in our Advanced Organic Chemistry Ph OCH3
Laboratory course for several years. (S)-1 2 Mosher esters

The Mosher Method Scheme 1

An aside on the change of nomenclature of the
A pair of enantiomers is indistinguishable by NMR
configurations shown in Scheme 1 should be noted. The S
spectroscopy; however, if the two enantiomers are reacted with
configuration of 1 is shown with the relative priorities for the
a chiral compound the products can become diastereomeric
substituents on the stereogenic carbon being (1) OCH3, (2)
with diastereotopic groups that can be distinguished by NMR.
This feature is used by Mosher in his technique of determining COCl, (3) CF3, and (4) Ph, but when (S)-1 is converted to
the enantiomeric ratio of a sample of a chiral compound, and it the Mosher ester 3, the relative priorities for the substituents on
is illustrated in Scheme 1. the same carbon change to (1) OCH3, (2) CF3, (3) CO2R,
The method begins with an enantiopure sample of the chiral and (4) Ph, now with an R configuration. Thus, although the
name of the configuration changes, the absolute configuration
derivatizing agent -methoxy--(trifluoromethyl)phenylacetyl
is retained.
chloride (MTPA-Cl, 1), which is sometimes referred to as the
Mosher acyl chloride. Either the R or S enantiomer of 1 can be

2004 The Chemical Educator, S1430-4171(04)06843-X, Published on Web 11/16/2004, 10.1333/s00897040843a, 960359sl.pdf
360 Chem. Educator, Vol. 9, No. 6, 2004 Steve Lee

Control Test for the Possibility of Kinetic Resolution amines because of the slower rates with sterically bulky
substrates; however to help minimize the effect of the different
In Moshers method the principle reaction might undergo a rates upon the product ratio, the reaction conditions are
kinetic resolution and so must be investigated for this designed to include an excess of the Mosher acyl chloride and
possibility in a control test. Because the reaction of the two a sufficient reaction time to ensure a complete reaction for both
enantiomeric substrates with the Mosher acyl chloride leads to diastereomeric reactions.
diastereomeric products, it must be confirmed that the ratio of
diastereomeric products accurately reflects the ratio of the The Laboratory Experiment
original enantiomers. A kinetic resolution would invalidate the
Mosher method for that particular substrate. A kinetic The chemical reagents in this experiment are all commercially
resolution involves resolving a mixture of enantiomers by available, for example, from the Sigma-Aldrich Company; however,
reacting them with a chiral, nonracemic agent and not allowing because the Mosher acyl chloride is expensive, the corresponding acid
the two reactions to go to completion. The different reaction can instead be purchased at a lower cost and converted with thionyl
chloride. The procedure involves a 50-hour reflux, and so
rates of the two enantiomers lead to one diastereomeric
unfortunately it is not conducive to most student laboratory formats.
product being formed faster than the other, thereby resolving This would have to be conducted by the instructor or laboratory
the two enantiomers. A kinetic resolution is not desired in the personnel [2].
Mosher method because this would invalidate the method for Regarding alcohol and amine substrates, four compounds have
that particular substrate; however, the effect of the different been tested and found to be suitable for this technique. Their NMR
rates is minimized by using an excess of the Mosher acyl data and configurations have been reported previously [2] and are
chloride and a sufficiently long reaction time to allow both updated here. These compounds have the added benefit of having both
reactions to proceed to completion. their racemic and enantiopure forms commercially available so that
The possibility for a kinetic resolution can be considered in samples of varying enantioenrichments can conveniently be prepared.
an example with sec-butanol (4) as shown in Scheme 2. The The compounds are sec-butanol (4), -trifluoromethylbenzyl alcohol
reactions of (S)-1 with (R)-4 and (S)-4 lead to diastereomeric (5), methyl mandelate (6), and -methylbenzylamine (7). Obviously,
countless other alcohols or amines can be tested with this technique.
products. Because a pair of enantiomers is reacting with a
chiral reagent to form diastereomeric products, it is likely that H CH3 H CF3 H CO2CH3 H CH3
the two reactions will proceed with different rates (i.e., kRR will H2N Ph
likely be different than kRS). This difference in rates could
potentially be significant enough to alter the product ratio in 4 5 6 7
which case the enantiomeric substrates would be kinetically
resolved; however, for this method to be valid, this difference The overall procedure for the experiment is logistically simple. In
in rates must not be significant enough to alter the product the first laboratory period students start their reactions by adding their
ratio. In other words, the product ratio of (R,R)-8 to (R,S)-8 reagents into a conical vial, which is left in their laboratory drawers at
needs to accurately reflect the ratio of (R)-4 to (S)-4 in order ambient temperature [5]. In the following laboratory period students
for this method to validly determine the enantiomeric ratio of 4 carry out the work-up, with no purification required, and analyze their
in the original sample. product samples by NMR spectroscopy [6]. The recommended
reaction time is 12 hours at ambient temperature, but the reaction
H O H mixture can be kept at ambient temperature for one week without a
kRR F 3C
HO O (R,R)-8 noticeable degradation of the product.
O Ph OCH3 Students can be organized into pairs or groups. One student could
F3C (R)-4 carry out the control test using a racemic sample of the substrate while
Cl + kRR kRS
the other student(s) in the group use an enantioenriched sample of the
H H same substrate. Students then compare each others NMR spectra with
(S)-1 F 3C
O (R,S)-8 the literature NMR data to fully assign their spectra, determine the
Ph OCH3 enantiomeric ratio, and determine the configuration of the major
(S)-4 enantiomer.
A sample experiment is shown in Scheme 3. In this case, (S)-1
Scheme 2 reacts with (R)-5 and (S)-5 to yield the corresponding esters (R,R)-9
and (R,S)-9. The 1H NMR spectra in Figure 1 show the signals for the
It must, therefore, be tested whether the difference in the methoxy hydrogens in 9. (These signals are split into quartets due to
rates of the two reactions (i.e., kRR and kRS) is significant long distance 1H-19F coupling.) In the control reaction, a racemic
enough to change the product ratio of (R,R)-8 to (R,S)-8. This sample of 5 was used. The 1H NMR spectrum of its product sample
possibility of kinetic resolution can be tested by running a (Figure 1a) shows that the diastereomeric products are in nearly equal
control experiment with a racemic sample of the enantiomeric amounts (51.9% to 48.1%), which indicates that the enantiomeric
substrate. If a racemic sample of the substrate reacts with the substrates are not kinetically resolved. The slight deviation from the
expected ratio of 50:50 can be explained by the use of a sample of (S)-
Mosher acyl chloride to yield a 50:50 mixture of the two
1 that was not completely enantiopure. It was labeled with 98% e.e. as
diastereomeric products, this would confirm that our method is determined by GLC according to the commercial source. From the
valid so that the ratio of diastereomeric products accurately second reaction starting with enantioenriched 5, the relative
reflects the initial ratio of enantiomeric substrates. On the other integration in its NMR spectrum (Figure 1b) indicates a
hand, if the racemic sample of the substrate reacts to yield an diastereomeric ratio of 64.2% to 35.8%, or 28.4% d.e. for (R,R)-9 and
unequal mixture of diastereomeric products, this would consequently 28.4% e.e. (R) for 5 (d.e. = diastereomeric excess = %
indicate that the substrate is not suitable for this method. For major diastereomer % minor diastereomer; e.e. = enantiomeric
this reason, the Mosher method has generally been limited to excess = % major enantiomer % minor enantiomer). This value is
sterically-unhindered, primary and secondary alcohols and close to that (25% e.e.) based upon a mass analysis of racemic and
enantiopure samples of 5 used to prepare the enantioenriched sample.

2004 The Chemical Educator, S1430-4171(04)06843-X, Published on Web 11/16/2004, 10.1333/s00897040843a, 960359sl.pdf
A Microscale NMR Experiment for Organic Chemistry Chem. Educator, Vol. 9, No. 6, 2004 361

Table 1. Summary of NMR data for the diastereomeric products from reaction with (S)-1
Diastereomeric products Configuration of substrate Chemical shifts (ppm)
OCH3 8 R 1.255 5.096 3.564 72.00
O S 1.335 5.096 3.566 72.00
Ph CF3
F 3C H O R 6.263 3.601 76.17
OCH3 S 6.342 3.479 76.40
Ph O 9
Ph CF3
H3CO2C H O R 3.769 6.094 3.697 72.20
OCH3 10 S 3.740 6.115 3.552 72.20
Ph O
Ph CF3
H 3C H O R 1.499 5.175 3.361 69.25
OCH3 S 1.539 5.175 3.403 69.44
Ph N 11
H Ph CF3
a 19
F NMR for the trifluoromethyl group in bold with CFCl3 as the internal reference set to 0 ppm.


(a) from racemic 5 (b) from enantioenriched 5

Figure 1. Expanded regions of the 1H NMR (60 MHz) spectra for (R,R)-9 and (R,S)-9 for the methoxy hydrogens.

The assignment of the signals for the methoxy hydrogens in the a microscale with typical product yields of 20 to 50 mg, it is still
diastereomeric products ( 3.601 ppm for (R,R)-9 and 3.479 ppm for suitable for a low-field NMR spectrometer. The product samples have
(R,S)-9) are based upon the chemical shifts reported by Mosher [2] also been analyzed with a higher-field 300-MHz instrument, which
and based upon the use of enantiopure (R)-5 (which was purchased provides much better separations of the signals. In this example, the
from the Sigma-Aldrich Company) to provide the major enantiomer in two signals are completely separated at 300 MHz.
the enantioenriched sample of 5. The consistency of these two A summary of the NMR data for all four diastereomeric products is
independent factors also helps to confirm the accuracy of the given in Table 1. The reactions were carried out with the S enantiomer
assignment. of 1. The table shows the chemical shifts for hydrogen or fluorine
atoms in the diastereomeric products. With only a few exceptions,
diastereotopic groups led to different chemical shifts. This data is an
F3C (R,R)-9
HO Ph O Ph update and is consistent with that reported by Mosher [2]; however, in
O Ph OCH3 one case, Mosher reported the signals for the methine hydrogens in 10
Cl +
to be identical for both diastereomers. Using a 300-MHz NMR
Ph OCH3 spectrometer, however, these signals appear separately at 6.094 and
F3C H 6.115 ppm. Complete 1H and 19F NMR data for these compounds are
(S)-1 F3C (R,S)-9
HO Ph O Ph included in the experimental section.
Ph OCH3 This technique can be adapted into a laboratory experiment in a
(S)-5 variety of ways. In a straightforward approach, students can be given
a known substrate with an unknown enantiomeric ratio that is
Scheme 3 determined by the instructor. The students would then be required to
carry out the reaction, analyze the product sample by 1H and/or 19F
The NMR spectra shown in Figure 1 are from an upgraded Anasazi
NMR, and determine the enantiomeric ratio and the major enantiomer.
EFT 60-MHz NMR spectrometer. Although the signals are not
Alternatively, the technique can be attached to another experiment
completely separated at the baseline, the spectrum is still acceptable
involving an asymmetric synthesis or resolution. For example, the
for determining the ratio of diastereomeric products for educational
resolution of -methylbenzylamine with tartaric acid is commonly
purposes. This shows that even though the technique is carried out on

2004 The Chemical Educator, S1430-4171(04)06843-X, Published on Web 11/16/2004, 10.1333/s00897040843a, 960359sl.pdf
362 Chem. Educator, Vol. 9, No. 6, 2004 Steve Lee

used in organic chemistry courses [4]. The determination of the filtered, and concentratedmaking sure that all traces of diethyl ether
enantiomeric ratio of the resolved -methylbenzylamine has been was removed [14]. Typical yields are approximately 70 to 90% with
reported to be carried out by optical rotation [4a], chiral stationary 20 to 50 mg of product. The entire residue was dissolved in 0.5 mL
phase HPLC [7], and chiral shift reagents [8]. This Mosher technique CDCl3 with TMS and filtered directly into an NMR tube. For 19F
provides another means of determining the success of the students NMR analysis, a drop of CCl3F was added as the internal standard.
resolution of -methylbenzylamine, which has not been previously Filtration was performed with a disposable glass pipette and a
reported. The technique works particularly well with - Kimwipe plug.
methylbenzylamine because the 19F NMR signals for the NMR Data. 3,3,3-Trifluoro-2-methoxy-2-phenylpropionic acid
diastereotopic trifluoromethyl groups are very far apart providing a sec-butyl ester. [(R,R)-8]: 1H NMR (CDCl3, 300 MHz, ) 7.544
pedagogically interesting NMR experiment for observing another 7.261 (m, 5H), 5.1325.056 (m, 1H), 3.564 (s, 1H), 1.5511.737 (m,
nucleus besides 1H or 13C [9]. 2H), 1.255 (d, J = 6.2 Hz, 3H), 0.939 (t, J = 7.4 Hz, 3H). 19F NMR
(CDCl3, 56.5 MHz, ) 72.0 (m, 3F). [(R,S)-8]: 1H NMR (CDCl3, 300
Summary MHz, ) 7.5447.261 (m, 5H), 5.1325.056 (m, 1H), 3.566 (s, 1H),
1.7371.551 (m, 2H), 1.335 (d, J = 6.2 Hz, 3H), 0.828 (t, J = 7.4 Hz,
The Mosher technique provides an alternative means of 3H). 19F NMR (CDCl3, 56.5 MHz, ) 72.0 (m, 3F).
determining the enantiomeric ratio of chiral alcohols and 3,3,3-Trifluoro-2-methoxy-2-phenylpropionic acid 2,2,2-
trifluoro-1-phenylethyl ester. [(R,R)-9]: 1H NMR (CDCl3, 300
amines, which can conveniently be used as a stand-alone
MHz, ) 7.5277.250 (m, 10H), 6.263 (qt, J = 6.6 Hz, 1H), 3.601 (qt,
organic experiment or attached to another experiment
J = 1.2 Hz, 3H). 19F NMR (CDCl3, 56.5 MHz, ) 72.3 (m, 3F),
involving an asymmetric synthesis or resolution. If the
76.17 (d, J = 6.6 Hz, 3F). [(R,S)-9] 1H NMR (CDCl3, 300 MHz, )
configurations of the enantiomers have been reported, students 7.5277.250 (m, 10H), 6.342 (qt, J = 6.7 Hz, 1H), 3.479 (qt, J = 1.2
can also determine the configuration of the major enantiomer. Hz, 3H). 19F NMR (CDCl3, 56.5 MHz, ) 72.3 (m, 3F), 76.40 (d, J
This technique is pedagogically beneficial as it introduces = 6.6 Hz, 3F).
microscale techniques and integrates 1H and 19F NMR 3,3,3-Trifluoro-2-methoxy-2-phenylpropionic acid methoxycar-
spectroscopy with advanced stereochemical topics that are not bonylphenylmethyl ester. [(R,R)-10]: 1H NMR (CDCl3, 300 MHz,
always sufficiently applied in organic chemistry courses. ) 7.4357.370 (m, 10H), 6.094 (s, 1H), 3.769 (s, 3H), 3.697 (qt, J =
1.2 Hz, 3H). 19F NMR (CDCl3, 56.5 MHz, ) 72.2 (m, 3F). [(R,S)-
Experimental Section 10]: 1H NMR (CDCl3, 300 MHz, ) 7.435-7.370 (m, 10H), 6.115 (s,
1H), 3.740 (s, 3H), 3.552 (qt, J = 1.1 Hz, 3H). 19F NMR (CDCl3, 56.5
General Remarks. All chemicals were purchased from the MHz, ) 72.2 (m, 3F).
Aldrich-Sigma Company. Chloroform was dried over calcium 3,3,3-Trifluoro-2-methoxy-2-phenyl-N-(1-phenylethyl)-
hydride, or its anhydrous form was purchased and used directly. propanamide. [(R,R)-11]: 1H NMR (CDCl3, 300 MHz, ) 7.533
Pyridine was dried over KOH and distilled over BaO, or its anhydrous 7.254 (m, 10H), 7.2287.014 (m, 1H), 5.2015.151 (m, 1H), 3.361
form was purchased and used directly [10]. All other chemicals were (qt, J = 1.5 Hz, 3H), 1.499 (d, J = 6.9 Hz, 3H). 19F NMR (CDCl3, 56.5
purchased and used without further purification. The sample of - MHz, ) 69.25 (m, 3F). [(R,S)-11]: 1H NMR (CDCl3, 300 MHz, )
methoxy--(trifluoromethyl)phenylacetyl chloride purchased from the 7.5337.254 (m, 10H), 7.2287.014 (m, 1H), 5.2015.151 (m, 1H),
Aldrich-Sigma Company is labeled as having 98% e.e. as determined 3.403 (qt, J = 1.5 Hz, 3H), 1.539 (d, J = 6.9 Hz, 3H). 19F NMR
by GLC [11]. 1H NMR spectra were taken on a Bruker Avance DPX (CDCl3, 56.5 MHz, ) 69.44 (m, 3F).
300-MHz (16 scans, data size = 32K) or Anasazi-upgraded Varian
360A (60 MHz, 4 scans, data size = 16K) FT-NMR spectrometer in Acknowledgments. The author would like to acknowledge
deuterochloroform (CDCl3) with tetramethylsilane (TMS) as the
the National Science Foundation (DUE 0310353), the Max
internal standard. 19F NMR spectra (56.5 MHz, 4 scans, data size =
16K) were taken on an Anasazi-upgraded Varian 360A FT-NMR Goldenberg Foundation, and Roosevelt University for funding
spectrometer in deuterochloroform (CDCl3) with the Anasazi EFT system. He would also like to thank Professor
trichlorofluoromethane (CCl3F) as the internal standard set to 0 ppm. Jeffrey Bjorklund and North Central College for the use of
Safety and Hazards. All experiments should be performed in their Bruker Avance DPX 300-MHz NMR spectrometer.
fumehoods with suitable eye and skin protection and with proper
expert supervision. Any eye and skin contact, inhalation, or ingestion Supporting Materials. A student handout, assignment, and
should be avoided with all reagents. Chloroform is a cancer suspect experimental notes for the instructor are available in a Zip file
agent. Calcium hydride is a flammable solid and moisture-sensitive.
Pyridine is flammable and an irritant. Potassium hydroxide is
corrosive. Barium oxide is corrosive and moisture-sensitive. Diethyl
ether is flammable and toxic and can form explosive peroxides. sec- References and Notes
Butanol (4) is a flammable liquid and irritant. -
1. For reviews of methods of determining enantiomeric ratios, see (a)
Trifluoromethylbenzyl alcohol (5) is an irritant. -Methylbenzylamine
Eliel, E. L.; Wilen, S. H. Stereochemistry of Organic Compounds;
(7) is corrosive and toxic. -Methoxy--(trifluoromethyl)phenylacetyl Wiley & Sons: New York, 1994; pp 214274; (b) Rothchild, R.
chloride (1) is corrosive and moisture-sensitive. Chem. Educator [Online], 1996, S1430-4171(96)03036-11(3); DOI
General Procedure for MTPA Derivatives. Into a cleaned and 10.1333/s00897960036a; (c) Rothchild, R. Enantiomer 2000, 5 (5),
dried 3-mL conical vial were added in the following order: anhydrous 457471; (d) Shah, R. D.; Nafie, L. A. Curr. Opin. Drug Discov.
pyridine (300 L), enantiopure Mosher acyl chloride (35.4 mg, 26.2 Devel. 2001, 4, 764775; (e) Finn, M. G. Chirality 2002, 14, 534
L, 0.140 mmol) [12], anhydrous chloroform [13] (300 L), and the 540; (f) Wenzel, T. J.; Wilcox, J. D. Chirality 2003, 15, 256270.
substrate (0.100 mmol). This was capped with a silicone-rubber seal, 2. Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34, 2543
stirred gently, and then left at ambient temperature in the students 2549.
drawers until the following laboratory period, usually one week. The
3. Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512519.
reaction solution was quenched with 1 mL H2O and taken up in 20
mL diethyl ether. The layers were separated, and the organic layer 4. (a) Ault, A. J. Chem. Educ. 1965, 42, 269; (b) Krause, J. G. J. Chem.
was washed with 5 mL each of 1 M HCl (aq), saturated Na2CO3 (aq), Educ. 1994, 71, 596.
and saturated NaCl (aq). The organic layer was then dried (Na2SO4),

2004 The Chemical Educator, S1430-4171(04)06843-X, Published on Web 11/16/2004, 10.1333/s00897040843a, 960359sl.pdf
A Microscale NMR Experiment for Organic Chemistry Chem. Educator, Vol. 9, No. 6, 2004 363

5. This first part of the experiment where students simply combine the the Mosher method. Mosher reports the use of this substrate with
reagents is brief; so, the instructor may wish to arrange the success [2].
laboratories so that students are finishing another experiment during 10. The chloroform and pyridine were dried by the instructor after which
the beginning of this laboratory period or use the time to introduce the drying apparatus was shown and the drying technique was
the Mosher method. explained to the students. Other instructors may wish to have
6. At Roosevelt University, the students analyze their samples by NMR laboratory personnel dry these reagents or simply purchase
on an Anasazi EFT upgraded 60-MHz spectrometer. Because they anhydrous samples.
have used the instrument in previous courses and/or laboratories, 11. Two different samples of the -methoxy--
they are able to obtain a spectrum quickly. The students use the (trifluoromethyl)phenylacetyl chloride are commercially available
spectrometer only to obtain a spectrum and then save and transfer the from the Aldrich-Sigma Company with 98% e.e. or 99+% e.e., as
spectra files via disk or email for processing (i.e., to zoom, integrate, determined by GLC.
print, etc) later on our science or university computers using the
NUTS software. This provides a rapid rotation of student use on the 12. Mosher [3] provides this additional note, which requires attention:
spectrometer while permitting students to access and analyze their The formation of a precipitate at this point indicates that the
spectra at a later time at their convenience. The author has also on pyridine was wet. A slight opalescence can be tolerated but a definite
occasion taken the class on a field trip to a nearby institution (North precipitate requires that the reaction can be abandoned and repeated
Central College) to use their 300-MHz FT-NMR. with dry reagents.
7. Krumpolc, Miroslav. J. Chem. Educ. 1991, 68, A176A178. 13. Mosher reports using CCl4 as the solvent, but due to its high costs,
CHCl3 has been used as the solvent with acceptable results.
8. Viswanathan, Tito; Toland, Alan. J. Chem. Educ. 1995, 72, 945946.
14. Even with thorough concentration on a high vacuum, diethyl ether
9. Another possible application involves the enzymatic resolution of - consistently appeared in the 1H NMR spectrum; thus, after initial
methylbenzylalcohol, which was recently reported as an organic concentration, the residue was dissolved in some CHCl3 and
chemistry experiment (Stetca, D.; Arends, I. W. C. E.; Hanefeld, U., concentrated again to ensure that all of the diethyl ether was
J. Chem. Educ. 2002, 79, 13511352.). Hanefield and coworkers removed. The NMR signals for diethyl ether appear adjacent to the
reported the determination of the enantiomeric excess by optical signals for the products.
rotation; however, the enantiomeric excess could be determined with

2004 The Chemical Educator, S1430-4171(04)06843-X, Published on Web 11/16/2004, 10.1333/s00897040843a, 960359sl.pdf


Mosher ester analysis for the determination of

absolute configuration of stereogenic (chiral) carbinol
Thomas R Hoye, Christopher S Jeffrey & Feng Shao
Department of Chemistry, University of Minnesota, 207 Pleasant Street, SE, Minneapolis, Minnesota 55455, USA. Correspondence should be addressed to T.R.H.

Published online 4 October 2007; doi:10.1038/nprot.2007.354

2007 Nature Publishing Group

This protocol details the most commonly used nuclear magnetic resonance (NMR)-based method for deducing the configuration of
otherwise unknown stereogenic, secondary carbinol (alcohol) centers (R1R2CHOH (or the analogous amines where OH is replaced by
NH2)). This Mosher ester analysis relies on the fact that the protons in diastereomeric a-methoxy-a-trifluoromethylphenylacetic acid
(MTPA) esters (i.e., those derived from conjugation of the carbinol under interrogation with MTPA) display different arrays of
chemical shifts (ds) in their 1H NMR spectra. The protocol consists of the following: (i) preparation of each of the diastereomeric
S- and R-MTPA esters and (ii) comparative (DdSR) analysis of the 1H NMR spectral data of these two esters. By analyzing the sign of
the difference in chemical shifts for a number of analogous pairs of protons (the set of DdSR values) in the diastereomeric esters
(or amides), the absolute configuration of the original carbinol (or amino) stereocenter can be reliably deduced. A typical Mosher
ester analysis requires approximately 46 h of active effort over a 1- to 2-d period.

The absolute configuration of a non-racemic sample of a chiral HO2C CF3 HO2C CF3
molecule is a fundamental and essential property of that com- H O Ph OMe MeO Ph H O
pound. This is especially so when the compound is of biological R2 O
CF3 2R 2S R2 O
R1 R2 OH R1
relevance (e.g., a natural product or a synthetic compound under Ph OMe CIOC CF3 R1 CIOC CF3 MeO Ph

development as a possible therapeutic agent). For evidence, one 4R

Ph OMe
MeO Ph
R-MTPA ester S-MTPA ester
needs to look no further than the infamous history of the 3S 3R
differences in biological response to each of the two enantiomers
Figure 1 | Scheme for the synthesis of R- and S-Mosher esters 4 from the
(mirror image antipodes) of thalidomide1. Assignment of absolute generic carbinol 1.
configuration to compounds where that important structural detail
is not yet known is accomplished by a number of different methods. involves coupling of the hydroxyl group of the alcohol with, in two
These include (i) correlation with compounds of known config- separate but analogous experiments, each enantiomer of Moshers
uration by synthetic interconversions and comparison of optical acid (i.e., 2R and 2S). This acylation reaction is commonly
rotation by polarimetric methods, (ii) approaches based on X-ray performed using a carboxylic acid-activating agent such as N,N-
crystallographic, optical rotary dispersion, circular dichroism or dicyclohexylcarbodiimide (DCC). Alternatively, the acid chlorides
exciton chirality methods and (iii) various empirical methods 3S and 3R (MTPA-Cl), derived from 2R and 2S, respectively, can be
based on NMR spectroscopy2. Among the NMR-based methods, used directly as active acylating agents5,6. This results in the
what is now commonly referred to as Mosher ester (or amide) formation of each of the two diastereomeric Mosher esters 4R
analysis is the most frequently used. and 4S, respectively. As diastereomers have different physical and
In 1973, Dale and Mosher reported3 an NMR-based method, spectroscopic properties, Dale and Mosher recognized that their 1H
which has come to be known as Mosher ester analysis, for deducing (proton) NMR spectra would differ. They proceeded to show that
the absolute configuration of the stereogenic carbon center in by comparison of the different chemical shifts of resonances
secondary alcohols (i.e., R1R2CHOH, 1, wherein R1 a R2). They obviously corresponding to protons residing within the substitu-
used a chiral, enantiomerically pure carboxylic acid to derivatize 1, ents R1 and R2 in 4 (or the 19F atoms of the CF3 group in the MTPA
the latter having an unknown configuration. Although various moiety7), they could deduce the absolute configuration of the
acids have been studied in this role2, the discussion here will be carbinol center in the original alcohol 1.
focused on the most widely used a-methoxy-a-trifluoromethyl- In the late 1980s, the Kakisawa group810 at Tsukuba described a
phenylacetic acid (MTPA-OH, 2), commonly known as Moshers major development in the method, which has come to be known as
acid. By direct analogy, this method can also be applied to the the modified (or advanced) Mosher ester analysis. Technological
analysis of a-chiral amines (e.g., R1R2CHNH2 or R1R2CHNHR3; developments had made access to more powerful (higher field),
Mosher amide analysis3,4), but this analysis is required less com- superconducting magnetic resonance spectrometers more com-
monly, so the rest of the discussion in this protocol is confined to monplace. Among other things, this meant that a greater portion
ester derivatives of alcohol (carbinol) substrates. of the individual protons could be routinely assigned in small-
The first step in the protocol for Mosher ester analysis for the molecule compounds (e.g., those having o2,000 amu) having
determination of absolute configuration is shown in Figure 1. It moderate-to-high structural complexity. Today, it is not uncommon

NATURE PROTOCOLS | VOL.2 NO.10 | 2007 | 2451


to be able to confidently assign nearly all

proton resonances in such molecules. The MeO H O H O
thrust of the Kakisawa contribution was that O H 2 S CF R1 2 R CF
O R 3 R 3
many analogous pairs of protons residing 2
CF3 R 1
Ph OMe O H R1
R O MeO Ph
within the R1 and R2 moieties in 4R versus R
4S (i.e., many data points for a single carbi- MeO R2
nol analyte) could be analyzed, resulting in a
much higher level of confidence in the
MeO Ph Ph OMe
deduced absolute configurations for a
1 2
much broader array of substrate carbinols R R R 1

1. The self-reinforcing nature of this analysis

Diastereomer 4S Diastereomer 4R
is an integral feature of this modified Mosher
2007 Nature Publishing Group

Figure 2 | Commonly encountered representations of the conformations (rotational isomers) used for the
In addition to absolute configuration, analysis of each of the diastereomeric MTPA esters 4S and 4R. The phenyl group shielding effect is
Mosher ester derivatives can be used to indicated by the gray arrows in each representation.
gain other types of stereochemical informa-
tion. Mosher showed that one can deduce
the ratio of two enantiomers in a given sample (i.e., enantiomeric dominant conformer for 4S versus that for 4R reveals a key distin-
ratio (er [major]:[minor]), sometimes expressed as the enantio- guishing feature. Protons residing within the R2 substructure of 4S are
meric excess (%ee %major  %minor)) by measuring the relatively more shielded (and upfield in its spectrum) and, conversely,
relative intensities of analogous resonances (1H and/or 19F) in protons within the R1 substructure of 4R are relatively more shielded;
each of the diastereomers 4R and 4S5. (Complementary measure- this is the crux of the Mosher method3,7. Moreover, the reach of this
ment of this ratio is sometimes performed by gas or liquid anisotropic shielding effect extends a considerable distance within the
chromatographic analysis.) While valuable11, this variant is not molecule so that a fairly large number of (even remote) protons within
further discussed in this protocol, but it is mentioned here since each R group are differentially shielded in the two diastereomers; this is
one encounters language like Mosher ester analysis was used to the crux of the modified (or advanced) Mosher method10. A con-
determine the enantiomeric purity of this mixture in the literature. sequence of all of the above is that the signs of the DdSR (defined, by
The success of the Mosher ester method for deducing the absolute convention, to be dS  dR8,10) values for protons residing in R1 will be
configuration of a secondary alcohol relies upon the empirically positive and those in R2 will be negative. Thus, by knowing which
based (and validated) conformational picture that is shown in protons have a positive versus a negative DdSR value, one can deduce
Figure 2. Briefly, the important conformation for each diastereomer the (sub)structures of R1 versus R2, which translates directly to the
is taken to be the one in which the ester adopts the usual s-trans absolute configuration of the secondary carbinol center in 1the
arrangement about its OCO bond (analogous to the major con- original objective of the exercise.
formation for acyclic, secondary amide CN bonds), and both the Mosher ester (and amide) analysis, especially the modified version,
trifluoromethyl (CF3) substituent of the MTPA moiety and the is remarkably powerful and general. The most complementary alter-
methine proton of the secondary alcohol moiety are syn-coplanar native, predicated on the same concepts, is the use of O-methylman-
(01 dihedral angle) with the carbonyl group. Thus, all of the under- delate esters (or methoxyphenyl acetates), which was introduced by
lined atoms in the R1R2C(H)OC(O)CCF3 substructure of Trost et al.12. Although there are instances where the Mosher method
each Mosher ester are coplanar. Although this is not the only must be applied with caution (e.g., for substrates with more than one
conformation populated in the ensemble of (rapidly interconverting) carbinol and/or amine functional groups), discussion of such subtle-
rotamers available to 4, it is the one that dominates the spectroscopic ties is beyond the scope of this protocol. Interested readers are
features that distinguish the diastereomeric MTPA esters. Or, as encouraged to consult the comprehensive review by the Riguera
Mosher and coworkers stated so clearly, these conformations are group2. Finally, note that some of the developers of more recent
intended to represent a model which successfully correlates the NMR-based methods similar to the Mosher analysis have elected to
known results. These are not intended to represent the preferred use the convention DdRS dR  dS. This is opposite to the DdSR
ground state conformation of the molecules under consideration. convention used universally in Mosher analyses. One will get to the
They may in fact measure an effective average of many conforma- correct answer either way, but it is essential that users unambiguously
tions or may represent a minor conformation which, however, exerts state which way the data are being interpreted and reported.
a proportionately large differential shielding of the [R1] and [R2] The protocol detailed below consists of two main components:
groups. Admittedly the success of the correlation tends to reinforce (i) individually prepare (synthesize) each of the two diastereomeric
the belief that these do indeed represent major conformations of the MTPA esters and then (ii) comparatively analyze the 1H NMR
molecules in question, but it must still be borne in mind that the spectral data of each of the two diastereomeric MTPA esters. We
possibility exists that this is a fortuitous array which happens to serve have chosen ()-menthol (5; Fig. 3) as a representative example of
as an empirical correlation of the results7. the universe of secondary carbinols 1. Of course, the absolute
An aryl group (e.g., the phenyl substituent in each of the MTPA configuration of ()-menthol is known, but the synthesis and
esters 4) is known to impose an anisotropic, magnetic shielding effect analysis methodologies are unchanged by that fact. Given the
on protons residing above (or below) the plane of the aryl ring. This sensitivity of 1H NMR spectroscopy, it is virtually never necessary
shielding results in a more upfield chemical shift for the affected to carry out the preparation of the MTPA esters for a Mosher
(spatially proximal) proton(s) in the NMR spectrum. Inspection of the analysis on a very large scale, even when the supply of carbinol 1 is

2452 | VOL.2 NO.10 | 2007 | NATURE PROTOCOLS


large. The more common situation is that 1 is Me Me Me

precious and in short supply (e.g., in the case O 2R 2S O

of initial structural studies of a newly isolated O
( or 3S ) OH ( or 3R ) O

natural product); if necessary, the method can Ph OMe MeO Ph

Me Me Me Me Me Me
be carried out routinely on scales even below
100 mg (o1 mmol). Accordingly, we have 6R 5 6S
R-MTPA ester S-MTPA ester
provided (in Step 1) three complementary,
representative procedures for synthesis of Figure 3 | Scheme for the synthesis of R- and S-MTPA menthyl esters 6 from ()-menthol (5).
MTPA-menthyl esters 6 (Fig. 3) on different
scales and using different acylating agents.
Namely, in option A we describe the preparation of 6S from R-()- be confused/interchanged. For this reason, we recommend that the
MTPA-Cl (3R) on a 10 mg scale; in option B, 6S from R-()-MTPA- sequence of synthesis and NMR data collection and tabulation be
2007 Nature Publishing Group

Cl (3R) on a 50 mg scale and in option C, 6R from R-(+)-MTPA-OH performed for each of the diastereomeric MTPA esters serially
(2R) on a 10 mg scale. The interpretation of the data (described in rather than in parallel. Although this may seem like an obvious
Steps 27) is performed in an identical manner, regardless of how the point (as an interchange of the two samples will lead to the
pair of diastereomeric esters was prepared. assignment of precisely the wrong absolute configuration!), experi-
Throughout the protocol, it is essential that the individual ence teaches that it is an easy issue over which uncertainty can arise
samples, spectra and data for each of the two diastereomers never in the mind of the experimenter.

REAGENTS . Silica gel thin-layer chromatography (TLC) plates (SIL G/UV254, 0.25 mm
. (R)-(+)-a-Methoxy-a-trifluoromethylphenylacetic acid (2R, R-(+)-MTPA- layer thickness; Machery Nagel, M805024)
OH; e.g., Acros) . Ceric ammonium molybdate, phosphomolybdic acid, anisaldehyde
. (S)-()-a-Methoxy-a-trifluoromethylphenylacetic acid (2S, S-()-MTPA- or potassium permanganate (e.g., TCI America, Fisher Scientific or
OH; e.g., Aldrich) Aldrich)
. (S)-(+)-a-Methoxy-a-trifluoromethylphenylacetyl chloride (3S, S-(+)- EQUIPMENT
MTPA-Cl; prepared from R-(+)-MTPA-OH5,6,13, but this reagent is also . Screw-capped glass vial or other similar reaction vessel with a
commercially available; e.g., Fluka) relatively small headspace with respect to the volume of reaction
. (R)-()-a-Methoxy-a-trifluoromethylphenylacetyl chloride (3R, R-()- solvent
MTPA-Cl; prepared from S-()-MTPA-OH5,6,13, but this reagent is also . Glass microliter syringes (Hamilton) or Wiretrols (Drummond) for
commercially available; e.g., Lancaster) measuring small volumes of liquid
. ()-Menthol (5, (1R,2S,5R)-()-2-isopropyl-5-methylcyclohexanol; e.g., Aldrich) . Common glassware (round-bottomed flask, Erlenmeyer flask, disposable
. N,N-DCC (e.g., Acros) pipets, etc.)
. Pyridine (e.g., Aldrich) . Magnetic stirrer and stir bar small enough to fit the reaction vial
. 4-Dimethylaminopyridine (e.g., Aldrich) . Rotary evaporator (Buchi)
. Chloroform-d (e.g., Cambridge Isotope Lab) . Balance (Mettler)
. Hexanes, ethyl acetate, diethyl ether, dichloromethane (CH2Cl2; standard . Handheld UV lamp (UVP)
reagent-grade laboratory solvents) . Small chromatography column
. Silica gel, bulk (typically 40- to 63-mm-diameter particle size, with nominal . NMR sample tube (5 mm diameter; New Era)
60 A pore size) . 1H NMR spectrometer (Z200 MHz; Varian, Bruker)

Individually prepare (synthesize) and collect the 1H NMR spectrum for each of the two diastereomeric MTPA esters.
1| Examples of three methods are provided as follows: (A) preparation of S-MTPA-menthyl ester 6S from ()-menthol (5)
and the R-acid chloride (3R, R-()-MTPA-Cl) on a 10 mg scale; (B) preparation of S-MTPA-menthyl ester 6S from ()-menthol
(5) and the R-acid chloride (3R, R-()-MTPA-Cl) on a 50 mg scale; and (C) preparation of R-MTPA-menthyl ester 6R from
()-menthol (5), the R-Mosher acid (2R, R-(+)-MTPA-OH) and DCC on a 10 mg scale.
(A) S-MTPA-menthyl ester 6S from ()-menthol (5) and the R-acid chloride (3R, R-()-MTPA-Cl) on a 10 mg scale
(i) Fit a screw-capped 4 ml glass vial with a Teflon-coated magnetic stir bar.
(ii) Transfer ()-menthol (5, 10.0 mg, 64 mmol) and dry pyridine (16 ml, 0.20 mmol, 3.1 equiv.) to the vial.
(iii) Dissolve the contents of the vial in anhydrous CH2Cl2 or chloroform (CHCl3) (1 ml, [5] 0.064 M). If a source of one of
these anhydrous solvents is not readily available, either can be dried using the same procedure described in the following
critical step.
m CRITICAL STEP If CHCl3 is selected as the solvent, it should first be passed through a column of fresh silica gel or
alumina immediately before use to remove the ethanol (B0.75% (vol/vol)) that is present as a stabilizer in most commercial
sources. For example, insert a small cotton plug into the neck of a disposable pipet and load the pipet with fresh silica
gel (or alumina) to a height of approximately 5 cm. Pass several milliliters of CHCl3 through this column using pressure
applied from a pipet bulb.

NATURE PROTOCOLS | VOL.2 NO.10 | 2007 | 2453


(iv) Add the R-()-MTPA-Cl (3R, 23 ml, 0.12 mmol, 1.9 equiv.) to the mixture. This transfer can be performed using a
microliter syringe or Wiretrol.
(v) Cap the vial and stir at ambient temperature.
(vi) Monitor the progress of the reaction by TLC analysis, by eluting with a solvent composed of hexanes and ethyl acetate in
a 4:1 ratio, and visualizing the plate by UV monitoring and/or staining with an appropriate TLC stain (e.g., ceric ammo-
nium molybdate, phosphomolybdic acid, anisaldehyde or potassium permanganate). Because they are less polar, the MTPA
esters will elute on silica gel more rapidly than the precursor carbinol (i.e., with a higher retention factor (Rf) on the TLC
plate). For the case of menthol (5) to 6R, the former has an Rf of 0.46 and the latter 0.82. The choice of TLC elution
solvent will vary depending on the polarity of the starting substrate under analysis.
(vii) After the reaction is complete (typically less than 2 h), partition the reaction mixture between diethyl ether (3 ml) and
water (1 ml).
(viii) Mix the layers thoroughly.
2007 Nature Publishing Group

(ix) Separate the layers and place the ether layer into a small Erlenmeyer flask.
(x) Add 3 ml of ether to the aqueous layer that remains in the reaction vial and repeat Steps (viii) and (ix), adding this
ether layer to the original one in the Erlenmeyer flask.
(xi) Repeat Step (x).
(xii) Dry the combined ether extracts with a suitable solid drying agent (anhydrous solid Na2SO4 or MgSO4) in a 25- to 50-ml
Erlenmeyer flask. Typically, approximately 1 g of drying agent is used for the approximately 10 ml of combined organic
extracts. Swirl the suspension occasionally over a period of approximately 5 (for MgSO4) or 30 (for Na2SO4) min. In
instances where the substrate might contain acid-sensitive functionality, the use of Na2SO4 is recommended.
(xiii) Filter the ether, containing the Mosher ester, from this suspension into a round-bottomed flask (B25 ml).
(xiv) Evaporate the volatiles using a rotary evaporator under reduced pressure using a 2025 1C water bath.
(xv) Purify the product by chromatography on silica gel (any of flash14, medium pressure liquid chromatography (MPLC), HPLC,
gravity column or prep-TLC plate methods can be used), eluting with hexanes/ethyl acetate (40:1) and monitoring the
fractions using TLC (4:1, hexanes/ethyl acetate) (or another commonly available detection method like UV absorbance or
differential refractive index).
(xvi) Combine the fractions containing the purified ester product 6S and remove the solvents under reduced pressure using a
2035 1C water bath.
(xvii) If necessary or desired, remove the remaining traces of ethyl acetate and hexane elution solvents from the purified product
by attaching the sample flask to a high-vacuum source (e.g., 0.11 torr) at ambient temperature for approximately 1 h.
(xviii) Record the 1H NMR spectrum of 6S in deuterochloroform (CDCl3).
(xix) Prepare the R-MTPA-menthyl ester 6R by repeating Steps (ixvii) using S-(+)-MTPA-Cl in place of R-()-MTPA-Cl.
(xx) Record the 1H NMR spectrum of 6R in CDCl3.
(B) S-MTPA-menthyl ester 6S from ()-menthol (5) and the R-acid chloride (3R, R-()-MTPA-Cl) on a 50 lg scale
(i) Fit a screw-capped 2 ml glass vial with a Teflon-coated magnetic stir bar.
(ii) Transfer ()-menthol (5, 50 mg, 0.32 mmol) and dry pyridine (1 ml, 12.5 mmol, 39 equiv.) to the vial.
(iii) Dissolve the contents of the vial in anhydrous CDCl3 (100 ml, [5] 3.2 mM). Use of CDCl3 as the solvent allows one to
assay the reaction mixture directly by 1H NMR spectroscopy.
(iv) Add the R-()-MTPA-Cl (3R, 1 ml, 5.2 mmol, 16 equiv.) to the vial, cap the vial and stir the contents at room temperature.
(v) Perform Step 1A(vi).
(vi) After the reaction is complete (typically less than 1 h), dilute the reaction mixture with dry CDCl3 (0.6 ml).
(vii) Transfer the entire CDCl3 solution to a standard (5 mm diameter) NMR sample tube.
(viii) Record a 1H NMR spectrum.
(ix) Prepare the R-MTPA-menthyl ester 6R by repeating Steps 1B(ivii), using S-(+)-MTPA-Cl in place of R-()-MTPA-Cl.
(x) Record the 1H NMR spectrum of 6R in CDCl3.
(C) R-MTPA-menthyl ester 6R from ()-menthol (5), the R-Mosher acid (2R, R-(+)-MTPA-OH) and DCC on a 10 mg scale
(i) Fit a screw-capped 4 ml glass vial with a Teflon-coated magnetic stir bar.
(ii) Transfer ()-menthol (5, 10.0 mg, 64 mmol) and R-(+)-MTPA-OH acid (2R, 32 ml, 0.2 mmol, 3.1 equiv.) to the vial.
(iii) Dissolve the contents of the vial in anhydrous CH2Cl2 or CHCl3 (1 ml, [5] 0.064 M). If a source of one of these anhydrous
solvents is not readily available, either can be dried using the same procedure described in the critical step above,
following Step 1A(iii).
(iv) Add DCC (42 mg, 0.2 mmol, 3.1 equiv.) to the vial.
! CAUTION DCC is an irritant and can lead to sensitization. Avoid any direct contact with the skin or inhalation of particulates.
(v) Add 4-dimethylaminopyridine (25 mg, 0.20 mmol, 3.1 equiv.), tightly cap the vial and stir the contents at room
(vi) Perform Step 1A(vi).

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(vii) After the reaction is complete (typically 1224 h), filter TABLE 1 | Dd (dS  dR) data for the S- and R-MTPA-menthyl Mosher
the white precipitate through a cotton plug. This solid is esters 6S and 6R.
largely the unwanted by-product, N,N-dicyclohexylurea. DdSR (dS  dR)
(viii) Purify the product as described in Steps 1A(xivxvii). d S-ester d R-ester
(ix) Record the 1H NMR spectrum of 6R in CDCl3. (6S) (ppm) (6R) (ppm) ppm Hz (500 MHz)
(x) Prepare the S-MTPA-menthyl ester 6S by repeating
6ax 1.12 0.98 +0.14 +70
Steps (iviii), using S-()-MTPA-OH (2S) in place of 6eq 2.13 2.08 +0.05 +25
R-(+)-MTPA-OH (2R). 5Me 0.94 0.91 +0.03 +15
(xi) Record the 1H NMR spectrum of 6S in CDCl3. 5ax 1.54 1.52 +0.02 +10
1ax 4.90 4.88 +0.02 +10
Comparatively analyze the 1H NMR spectral data of each of 4ax 0.87 0.86 +0.01 +5
the two diastereomeric MTPA esters 4eq 1.70 1.70 0.00 0
2007 Nature Publishing Group

2| Unambiguously assign as many proton resonances as 3ax 1.04 1.06 0.02 10
possible in the 1H NMR spectrum for each of the 3eq 1.67 1.69 0.02 10
diastereomeric esters 4S and 4R. 2ax 1.42 1.45 0.03 15
2Mea 0.67 0.77 0.10 50
3| Determine the difference in chemical shifts (DdSR) for 2Meb 0.74 0.87 0.13 65
each of the assignable analogous pairs of protons for the 2 1.56 1.88 0.32 160
S- and R-MTPA esters according to the following convention: For the menthyl MTPA esters 6S and 6R, all proton resonances are uniquely identifiable. However, this
need not be the case, and, for many (most?) complex structures, not every proton in the compound is
Dd d(S-MTPA ester)d(R-MTPA ester) (or Dd dS SR
uniquely identifiable in its 1H NMR spectrum. In such cases, it is prudent to use only the subset of
analogous proton pairs for which unambiguous assignments are known.
dR). Gather all of the resonances of positive and, then,
negative DdSR values together. These DdSR data for the menthyl MTPA esters 6S versus 6R are presented in Table 1, listed in the
order from most positive to most negative.

4| Using the conformations shown in Figure 2 for the MTPA esters of generic secondary alcohols, decide which protons of the
esters under study are part of the R1 substituent and which part of R2. More specifically, those protons that have positive DdSR
values reside within R1, whereas those with negative values belong to R2. The structures shown in Figure 4 for the menthyl
esters 6S and 6R clearly show that the protons with positive DdSR values (i.e., those in R1) are all on one side (front) of the
plane of the MTPA moiety, whereas those with negative values are all on the opposite (back) side of that plane.
! CAUTION Occasionally, one of the proton resonances proves to be an exception to this trend. If so, it is usually for a proton
with a DdSR value that is relatively small in magnitude because this exceptional proton resides either near the plane of the
MTPA ester moiety or quite remotely. For this reason, the secondary carbinol proton itself (i.e., H1 in 6) is typically ignored in
the analysis; an important corollary is that protons having the largest DdSR values are, qualitatively, weighted very heavily in
the analysis.
5| Use the Cahn Ingold Prelog convention15 to assign the configuration of the original carbinol center as R or S. C1 of
()-menthol has the R configuration, as the substituent priority sequence is 1, OH; 2, C2; 3, C6; and 4, H.

6| Report the data. A typical chemist-friendly format for reporting the synthesis and spectral properties of a Mosher ester is
presented in Box 1. Specifically, it describes the preparation of both the S- and R-MTPA esters of ()-menthol (6S and 6R,

7| Finally, one particularly insidious aspect of the Mosher method relates to the Cahn Ingold Prelog convention nomenclature
mentioned above and represents a potential pitfall. This problem is presented as a caveat in Box 2.

50/65  TIMING
Me Steps 1A(iiv): 1520 min
H Steps 1A(v) and (vi): 13 h
Me +10 160 Me
H O H Me H H O Steps 1A(viixiv): 2040 min
Me H CF3 10 Me H CF3
O H 3 1 OMTPA O Steps 1A(xv) and (xvi): 2060 min
H 2 H
Me OMe 5
+25 Me MeO Step 1A(xvii): 530 min
4 6
Step 1A(xviii): B3.5 h
+15 H 6R Step 1A(xix): 530 min
Steps 1B(iiv): 1520 min
SR = S R
Step 1B(v): 13 h
Figure 4 | Conformations used for the analysis of the menthyl esters 6S and 6R and the resulting DdSR Steps 1B(vi) and (vii): 25 min
(dS  dR) values (in Hz) for each of the protons on the front and back sides of the menthyl moiety. Step 1B(viii): 540 min

NATURE PROTOCOLS | VOL.2 NO.10 | 2007 | 2455



Preparation of the S- and R-MTPA-menthyl esters 6S and 6R
To a stirred solution of ()-menthol (5, 10.0 mg, 64 mmol) and dry pyridine (16 ml, 0.20 mmol, 3.1 equiv.) in dry deuterochloroform (1 ml, [5]
0.064 M) at room temperature, R-()-MTPA-Cl (3R, 23 ml, 0.12 mmol, 1.9 equiv.) was added. The reaction progress was monitored by thin-layer
chromatography (TLC) on silica gel (4:1::Hex:EtOAc). After complete consumption of the menthol (B2 h), the reaction mixture was quenched
by the addition of water (B1 ml) and ether (B3 ml). The aqueous layer was extracted with two additional portions of ether (B3 ml), and the
combined organic layers were dried (Na2SO4), filtered and concentrated in vacuo. The crude product mixture was purified by silica-gel
chromatography (MPLC, eluting with hexanes/ethyl acetate (40:1, Rf 0.18)) to give the S-MTPA-menthyl ester 6S (21.5 mg, 90%) as a white
solid. 6S: 1H NMR (CDCl3) d 7.54 (m, 2H), 7.39 (m, 3H), 4.90 (ddd, 1H, J 4.5, 11.0, 11.0 Hz), 3.59 (q, 3H, J 1.0 Hz), 2.13 (dddd, 1H, J
2.0, 3.5, 4.0, 11.5 Hz), 1.70 (dddd, 1H, J 3.5, 3.5, 3.5, 12.5 Hz), 1.67 (dddd, 1H, J 3.5, 3.5, 3.5, 13 Hz), 1.56 (dsept, 1H, J 3.0, 7.0),
1.54 (ddddq, 1H, J 3.5, 3.5, 12, 12, 7.0 Hz), 1.42 (dddd, 1H, J 3.0, 3.0, 11.0, 12.5 Hz), 1.12 (ddd, 1H, J 12, 12, 12 Hz), 1.04 (dddd, 1H,
J 3.5, 12.5, 12.5, 12.5 Hz), 0.94 (d, 3H, J 7.0 Hz), 0.87 (dddd, 1H, J 4.0, 12.5, 13, 13 Hz), 0.74 (d, 3H, J 7.0 Hz), and 0.67 (d, 3H,
2007 Nature Publishing Group

J 7.0 Hz). In an entirely analogous fashion, the R-MTPA-menthyl ester 6R was prepared using S-(+)-MTPA-Cl (3S). 6R: 1H NMR (CDCl3) d 7.52
(m, 2H), 7.40 (m, 3H), 4.88 (ddd, 1H, J 4.5, 11.0, 11.0 Hz), 3.53 (q, 3H, J 1.0 Hz), 2.08 (dddd, 1H, J 2.0, 3.5, 4.0, 12 Hz), 1.88 (dsept,
1H, J 3.0, 7.0), 1.70 (dddd, 1H, J 3.5, 3.5, 3.5, 13 Hz), 1.69 (dddd, 1H, J 3, 3, 3, 13 Hz), 1.52 (ddddq, 1H, J 3.5, 3.5, 12, 12, 6.5 Hz),
1.45 (dddd, 1H, J 3.0, 3.0, 11.0, 12.5 Hz), 1.06 (dddd, 1H, J 3.5, 13, 13, 13 Hz), 0.98 (ddd, 1H, J 12, 12, 12 Hz), 0.91 (d, 3H, J 6.5
Hz), 0.87 (d, 3H, J 7.0 Hz), 0.86 (dddd, 1H, J 3.5, 12.5, 12.5, 12.5 Hz), and 0.77 (d, 3H, J 7.0 Hz).

Upon conversion of the MTPA acid (MTPA-OH) to the MTPA acid chloride (MTPA-Cl), there is a switch in the relative priority of two of the groups.
Specifically, the trifluoromethyl group (CF3) is higher in priority than the carboxyl group (COOH) in the acid, but lower than the chlorocarbonyl
group (COCl) in the acid chloride. Thus, the R enantiomer of the Mosher acid (R-(+)-MTPA-OH, 2R) gives rise to the S enantiomer of the Mosher
acid chloride (S-(+)-MTPA-Cl, 3S) (and vice versa for the S acid 2S to the R acid chloride 3R). This represents a somewhat rare instance in which
the absolute configuration of a stereocenter changes as a result of a chemical reaction, even though none of the four bonds to the stereogenic
carbon was involved in the reaction.

3 2 2 3 R1 O 2
Ph OMe Ph OMe Ph OMe
4 1 4 1 4 1

2R 3S 4R
R-(+)-MTPA-OH S-(+)-MTPA-Cl R Mosher ester
R Mosher acid S Mosher acid chloride

As was shown but not otherwise emphasized earlier in Figure 1, it follows that the R Mosher acid gives rise to the R Mosher ester, but that it is
the S Mosher acid chloride that gives rise to the R Mosher ester (since there is another change in relative priority: CF3 is lower than COCl but
higher than COOR). It is, of course, essential that every user of Mosher ester analysis considers this fact when performing the analysis.
Otherwise, again, precisely the opposite (and wrong) absolute configuration will be assigned16. Somewhat ironically, this potential problem has
been exacerbated by the fact that the acid chloride became commercially available in the early 1990s. That is, earlier workers would often
prepare the acid chloride from purchased, say, R acid, convert the derived acid chloride to the R Mosher ester and properly deduce the correct
absolute configuration, without even realizing that they were passing by way of the S acid chloride.
Finally, it is also important that users report the method by which they made their Mosher esters with absolute clarity, so that no doubt is
left in readers minds about the configuration of each MTPA ester. The experimental description in Box 1 represents one such unambiguous
presentation. If the publication venue does not lend itself to full experimental description, then an unambiguous statement like esterification
of # with (S)- and (R) MTPA-Cl occurred only at the C-8 hydroxy group to give the (R)- and (S)-MTPA esters #a and #b, respectively17 is to be
highly commended and recommended. All too often and in fact typically, authors are ambiguous on the issue of the precise origin of their R- and
S-esters. For example, simple statements like Mosher ester analysis led to the assignment of the R configurationy and R- and S-MTPA esters
were prepared from reaction of carbinol X with the MTPA acid chlorides abound and leave doubt. This does not mean that the method was
misused (i.e., that the user was unaware of the CIP switch), but it does (and should) erode the confidence that readers can have in the
conclusions reached. The only instances in which it is clear that an error was madeand we have found a number of published examples of
thisis where it is unambiguously stated that the R-Mosher ester was prepared using R-MTPA-Cl. Ironically, these cases are at least somewhat
self-correcting, since the informed reader will know to reverse whatever conclusions were drawn from these analyses.
The bottom line is that Mosher ester analysis is an extremely reliable method for determining absolute configuration, provided that users
be aware of the above pitfall and that they demonstrate that awareness by reporting the method of synthesis (origin) of each Mosher ester in
an unambiguously stated manner so that others will have the full measure of confidence in the conclusions.

2456 | VOL.2 NO.10 | 2007 | NATURE PROTOCOLS


Step 1B(ix): B3.5 h

Step 1B(x): 540 min
Steps 1C(iiv): 1520 min
Steps 1C(v) and (vi): 1224 h
Step 1C(vii): 5 min
Step 1C(viii): 3070 min
Step 1C(ix): 530 min
Step 1C(x): B24 h
Step 1C(xi): 530 min
Step 2: 1590 min
Step 3: 3060 min
Step 4: 20 min
2007 Nature Publishing Group

Step 5: 5 min
Step 6: 12 h

Troubleshooting advice regarding potential issues with the preparation, purification, and/or NMR data collection for esters 4R
and 4S can be found in Table 2.

TABLE 2 | Troubleshooting table.

Problem Possible reason Solution
Poor conversion to product Moisture in reaction mixture that consumes Use anhydrous solvents and oven-dried glassware
MTPA-Cl or DCC-activated MTPA-OH

Slow reaction Reaction solution too dilute Make sure substrate alcohol and MTPA-OH or
MTPA-Cl are present at Z0.05 M, and monitor
reaction conversion carefully by TLC analysis

Product contamination, especially for Leaching of impurities (e.g., plasticizers) Use only glass reaction vessels, pipets, etc., and
microscale from plastic labware by organic solvents Teflon rather than plastic tubing

Product contamination by excess MTPA- Need to use Z10 equiv. of MTPA-Cl because Quench the reaction mixture Me2N(CH2)3NH2 before
Cl, again, especially for microscale of limited availability of carbinol adding dilute HCl during workup3
Use stock solutions to accurately measure reagent

Overlapping resonances complicating Overall molecular complexity and/or coinci- Carry out 2D-NMR analysis (e.g., COSY or HMQC) to
NMR interpretation dental chemical shift similarity unambiguously assign overlapping resonances

Each purified diastereomeric Mosher ester (4R and 4S) will be prepared and isolated in 5080% yield from the starting
carbinol 1. 1H NMR spectroscopic data of sufficient quality for subsequent Dd analysis will be obtained for each ester. After
the prescribed DdSR analysis is performed, the absolute configuration of the carbinol 1 will have been deduced.

Published online at 5. Dale, J.A., Dull, D.L. & Mosher, H.S. a-Methoxy-a-trifluoromethylphenylacetic
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Revival as a Vital Medicine (Perseus Publishing, Cambridge, MA, 2001). Moshers acid chloride. Tetrahedron Lett. 32, 71657166 (1991).
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