Tetrahedron 71 (2015) 2692e2697

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Tetrahedron
journal homepage: www.elsevier.com/locate/tet

Novozym-435 as efficient catalyst for the synthesis of benzoic and

(hetero)aromatic carboxylic acid esters
Daniela Giunta, Barbara Sechi, Maurizio Solinas *
Istituto di Chimica Biomolecolare CNReUOS SS, Trav. La Crucca 3, Sassari 07100, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Hereby we report new reaction conditions to convert benzoic acids and more general (hetero)aromatic
Received 21 November 2014 carboxylic acids into the corresponding n-heptyl esters by applying Novozym-435 as the biocatalyst in
Received in revised form 23 February 2015 cyclohexane as the solvent. Very good yields are obtained in the esterification of a plethora of substituted
Accepted 9 March 2015
acids with mild and straightforward reaction conditions. Direct esterification of the acid is favoured
Available online 14 March 2015
compared to transesterification of methyl benzoate under our reaction conditions in all cases studied.
Recycling of the immobilised enzyme is feasible although with some minor limitations.
Keywords:
Ó 2015 Published by Elsevier Ltd.
Novozym-435
Lipase
Esterification
Benzoic acids

1. Introduction biodegradable ionic liquids.5 Although, there are several synthetic

Benzoic and more general (hetero)aromatic carboxylic acid es-
ters such as A in Fig. 1 are found in a variety of important synthetic
organic molecules. Etofibrate, for example, is a drug that lowers
blood triglyceride levels.1 Chloroprocaine is a well known local
anaesthetic2 while pyrazinoic acid esters, such as B in Fig. 1, showed
a broad spectrum of antimycobacterial activity.3 Moreover, furoic
and thiophenic carboxylic acid esters such as C in Fig. 1, are potent
and selective inhibitors of human reticulocyte 15-lipoxygenase-1.4
Finally, Some N-alkylated nicotinic acid ester salts proved to be
Fig. 1. Selected examples of (hetero)aromatic carboxylic acid esters.
* Corresponding author. Tel.: þ39 079 2841219; fax: þ39 079 2841299; e-mail
address: maurizio.solinas@icb.cnr.it (M. Solinas).
http://dx.doi.org/10.1016/j.tet.2015.03.036
0040-4020/Ó 2015 Published by Elsevier Ltd.
methods for the preparation of benzoic or (hetero)aromatic esters
starting from the related acids,6 the application of biocatalysis
would be more advantageous in terms of practical laboratory scale.
Lipase catalysed esterification of carboxylic acids would lead to the
isolation of clean products by simple filtration of the reaction
mixture leaving water as the only side product. Unfortunately, es-
terification of aromatic benzoic acids with lipase is quite chal-
lenging and not broadly applied in organic synthesis on
a laboratory scale.7 Schmid and co-workers reported a study based
on a molecular dynamics simulation explaining why benzoic and
gallic acid esters cannot be obtained by Candida antarctica lipase B
(CAL-B) catalysis.8 They claim that the aromatic acyl moiety is
highly affected by steric interaction with different surrounding
residues in the enzyme pocket binding site. The presence of at least
a methylene spacer (e.g., phenyl acetic acid) is mandatory to obtain
appreciable conversion values. Nonetheless, an example of esteri-
fication of benzoic acid derivatives is reported in the literature by
Vosmann et al. They have applied CAL-B to the esterification of 3-
and 4-phenoxybenzoic acid with cis-9-octadecen-1-ol under vac-
uum and in a solventless manner, obtaining reasonable yields.9
However, this report lacks a wider substrate scope and a rather
long purification procedure is necessary to isolate the reaction
products. In contrast, Krause et al. reported the esterification of
benzoic acid and n-hexanol in a solventless system using several
lipases, including Novozym-435, although they claimed that all
tested enzymes gave negligible chemical yields.10
We have recently reported about new reaction conditions for
the esterification of benzoic acid with 1-heptanol obtaining
 D. Giunta et al. / Tetrahedron 71 (2015) 2692e2697 2693

excellent yields by using the commercially available Novozym-435
and cyclohexane as the solvent.11 Due to a loss of catalytic activity
in the esterification of benzoic acid, recycling of the immobilized
lipase was not feasible for more than 4 cycles. In this paper we
show a systematic study concerning the application of Novozym-
435 for the synthesis of substituted benzoic and (hetero)aromatic
carboxylic acid esters. Moreover, we show a comparison between
esterification of benzoic acid with transesterification of methyl
benzoate. Finally, a screening of lipases and an improved recycling
experiment are reported.
2. Result and discussion
The application of lipases in the esterification of carboxylic acids
is a process known since many decades. However, with the ex-
ception of our recently discovered protocol,11 the reaction with
benzoic acid derivatives usually gives unsatisfactory results. Since
transesterification reactions may instead afford better results,12 we
decided to compare esterification of 1a with transesterification of 4
under our reaction conditions (see Fig. 2 and Experimental section
for details). Reactions were performed with 50 mg of Novozym-435
at various substrate concentrations with 1 equiv of n-heptanol. All
reactions were carried out at 60  C in 5 ml of cyclohexane. As
several commercially available lipases in our test reaction, i.e. es-
terification of benzoic acid with n-heptanol in cyclohexane (Table
1). Reactions were carried out on a 0.4 mmol scale and small
samples were analysed via GC-FID after 2, 24 and 48 h reaction time
(see Experimental section for details). As shown in Table 1, none of
the tested lipases except Novozym-435, lead to good yields of
heptyl benzoate. Less than 30% yield was obtained with lipase from
Candida rugosa similarly to what was obtained by Leszczak and
Tran-Minh applying the same catalyst in a hexane/toluene mixture
with added water (entry 1, Table 1).15 Unsupported CAL-B leads to
only traces of heptyl benzoate after 48 h reaction time (entry 2 vs 5,
Table 1). Lipase immobilised on a macroporous ion-exchange resin,
Lipozyme, gave very low yield after 48 h reaction time (entry 3,
Table 1). Being clear that only Novozym-435 was able to perform
esterification of benzoic acid within reasonable reaction time and
at mild reaction conditions we decided to explore the substrate
scope of the mentioned catalyst with several benzoic and (hetero)
aromatic carboxylic acids (see Schemes 1 and 2).
Table 1
Screening of different lipases in the esterification of 1a with n-heptanol
Entrya Enzyme Origin Yieldb (%)

depicted in Fig. 2, yield of heptyl benzoate increases linearly only 2h 24 h 48 h

for the esterification reactions reaching in all cases 100%. At low 1 Lipase Candida rugosa 0 13 27
substrate concentration (15 mM and 77 mM) quantitative conver- 2 Lipase Candida antarctica 0 2 5
sions are achieved within acceptable reaction time. Esterification of 3 Lipozyme Mucor miehei 0 5 11
4 Lipase Porcine pancreas 0 2 2
benzoic acid at 385 mM shows a very slow reaction rate, with 5 Novozym-435 Candida antarctica 11 100 100
quantitative yield achieved in about 4 days. In contrast, none of the 6 Lipase Pseudomonas fluorescens 0 2 2
transesterification reactions tested at comparable substrate con- a
All reactions were carried out on a 0.4 mmol scale with 1 equiv of n-heptanol,
centrations accomplished complete conversion. In all cases, re- 5 mL cyclohexane, 60  C.
actions follow logarithmic curves reaching plateau values so that b
Yield determined by GC-FID analysis using chlorobenzene as internal standard.
full conversion of n-heptyl benzoate is not achieved even after
several days of reaction. We speculate that these different reaction
profiles may be related to the characteristics of the organic medium
used here. Cyclohexane has a logP of 3.44 (i.e., 2754:1 organic/
aqueous) and it has no capability to form hydrogen bonds with any
solute. This may lead to variation of the water content retained in
the microenvironment at the enzyme active site affecting positively
its catalytic capability to perform esterification of benzoic acid.13
Moreover, accordingly with Liu et al. pretreatment by organic sol-
vents may increase, in some cases, the esterification activity of li-
pases by causing alteration in their secondary and tertiary Scheme 1. Esterification of (hetero)aromatic carboxylic acids with Novozym-435.
structures.14 Therefore, we next examined the catalytic activity of

Fig. 2. Esterification of benzoic acid versus transesterification of methyl benzoate under comparable reaction conditions.
2694 D. Giunta et al. / Tetrahedron 71 (2015) 2692e2697

Table 2
Novozym-435 catalysed esterification of benzoic and (hetero)aromatic carboxylic
acids

Entry Substrate Ester Yielda (%)

1 3a >99

2 3b >99
Scheme 2. Esterification of disubstituted benzoic and substituted nicotinic acids with
Novozym-435.
3 3c 63

According to the pingepong mechanism, the acyl-enzyme

intermediate is first formed by reaction with a serine residue 4 3d 80
in the active site.16 Therefore, both steric and electronic prop-
erties of the substrate are likely to influence the outcome of the
esterification. All reactions were carried out at 80  C in order to 5 3e 38
overcome problems arising from the low solubility unveiled for
some aromatic acids. Moreover, it has been demonstrated that
Novozym-435 has a high thermal stability up to 100  C in certain 6 3f >99

solvents.17 For comparison, reaction time was set as 22 h for all

substrates. Benzoic and p-toluic acids are converted straightfor-
7 3g >99
wardly into the corresponding esters (Table 2, entries 1 and 2).
Increasing the steric hindrance in the p-position of the aromatic
ring by a branched or a linear alkyl chain was detrimental for
8 3h >99
catalytic activity (Table 2, entries 3 and 4). Quantitative yields
are obtained in both cases after longer reaction time. The pres-
ence of heroatoms at the p-position of the benzene ring is 9 3i <1b
generally tolerated with the exception of the p-dimethylamino
derivative, which leads to only traces of the ester 3i, even after
prolonged reaction time. 4-Methoxybenzoic acid is converted 10 3j <1b
into the corresponding heptyl ester with somehow lower
chemical yield, while p-chloro, p-trifluoromethane and p-nitro
derivatives lead quantitatively to esters 3feh within 22 h (Table
2, entries 5e9). It may be hypothesised that electron with- 11 3k 31
drawing groups at the para position lead to increased catalytic
activity of the enzyme. O-Substituted substrates with an in-
creased steric hindrance in the vicinity of the carboxylic group
(as in 1jel), are not efficiently converted into their correspond- 12 3l 4b
ing esters. Esterification of 2-methyl and 2-chloro benzoic acid
are very ineffective while in the case of the o-methoxy benzoic
acid 31% of the ester is obtained (Table 2, entries 10e12). Ac-
cordingly, less sterically demanding substrates such as the m- 13 3m >99
substituted benzoic acids are generally converted with good to
excellent yields (Table 2, entries 14e16). For the esterification of
(hetero)aromatic carboxylic acids, we expected that either the
14 3n 65
nature and the position of the heteroatom may influence the
outcome of the reaction. We speculated that the acyl-enzyme
intermediate could be influenced by hydrogen bond interaction
due to the heteroatom acceptor. For picolinic, nicotinic and iso-
15 3o >99
nicotinic acids high yields are obtained without major differ-
ences between them, at least at the reaction condition tested
(Table 2, entries 17e19). Pyrazine carboxylic acid is also con-
verted in high yield under our reaction conditions (Table 2, entry
16 3p 60
20). 5-Membered heterocycles such as 2- and 3-thiophene car-
boxylic acids are quantitatively converted into the corresponding
esters under the usual reaction conditions (Table 2, entries 21
and 22). Surprisingly, while 3-furoic acid is quantitatively con-
17 3q 91
verted into the ester 3x, 2-furoic acid leads to only 4% of the
ester 3w at the same reaction conditions (Table 2, entries 23 and
24). Ester 3w is obtained with 43% isolated yield only after very
18 3r 94
long reaction time (156 h) suggesting that the presence of the
oxygen atom ‘ortho’ to the carboxylic moiety is unfavourable for
the catalytic activity of the enzyme.
 D. Giunta et al. / Tetrahedron 71 (2015) 2692e2697 2695

Table 2 (continued ) of catalytic activity already after the 1st cycle either at 60 and 80  C
Entry Substrate Ester a
Yield (%) (see Fig. 3).11 In both experiments the catalyst was washed between
each cycle with cyclohexane before recharging another batch of
benzoic acid and alcohol (Fig. 3, method A). We decided to repeat
the recycling experiment avoiding the washing of the solid catalyst
19 3s 88
after each reaction (Fig. 3, method B). The proper amount of
Novozym-435 was placed into a small glass basket equipped with
20 3t 93 a sintered glass bottom. The glass basket was immersed into the
cyclohexane solution of benzoic acid and n-heptanol and vigorous
stirring was assured by a magnetic stirring bar. Temperature was
21 3u >99 set as 60  C and reaction time was 22 h (see Experimental section
for details). At the end of the reaction the glass basket containing
the biocatalyst was recovered from the reaction mixture and dip-
22 3v >99 ped into a new reaction solution without any further washing.
Reaction products were completely recovered from the mixture
and 100% mass balance was obtained. By this simple modification
23 3w 4b of the recycling set up, 100% yield was obtained up to 6 cycles and
the drop of catalytic activity was observed at the 7th and 8th cycles
(80% yield). Further decreasing of activity was obtained after 9 re-
24 3x >99 actions (60% yield). Such drop of activity may be due to partial
deactivation of the enzyme, which may be related to temperature
a
Isolated yield. and/or acid concentration.19
b
Determined by 1H NMR analysis of the crude reaction mixture.

Further we investigated the esterification reaction of some di-

substituted benzoic acids and substituted nicotinic acids with both
ethanol and n-heptanol. 2-Methoxy-4-nitrobenzoic acid leads to
low yields of esters with both alcohols, even lower yields than that
obtained with the ortho-methoxy benzoic acid (Table 3, entries 1
and 2; Table 2, entry 11). On the other hand, 3,4-dichlorobenzoic
acid is quantitatively converted into the esters 6c and 6d with
both alcohols (Table 3, entries 2 and 3). Heptyl 6-chloronicotinate
6e is quantitatively obtained by reaction with n-heptanol while
the ethyl ester is obtained with somehow lower yield (Table 3,
entries 5 and 6). 6-Methylnicotinic acid is converted with high yield
into the heptyl ester while only 62% of ethyl ester is obtained by
reaction with ethanol (Table 3, entries 7 and 8). These results seem
to indicate that, although a substantial number of benzoic acid
substrates are easily converted into the n-heptyl ester, in general
Fig. 3. Recyclability of Novozym-435 in cyclohexane.
the choice of the alcohol may also play an important role at our
reaction conditions. To further confirm that, we investigated the
3. Conclusion
reaction of benzoic acid with 2-ethylhexan-1-ol, a well known fatty
branched alcohol used in industry for the production of emollients
In the present paper we have shown new reaction conditions by,
and plasticizers. Under our reaction conditions, only 20% conver-
which it is possible to obtain very good yields in the esterification of
sion was obtained indicating the difficulties to identify some gen-
a plethora of substituted benzoic acids and, more general, (hetero)
eral and predictable rules (data not shown).
aromatic carboxylic acids with n-heptanol with Novozym-435 as
the biocatalyst and cyclohexane as the solvent. Direct esterification
Table 3 of the acid is favoured compared to transesterification of methyl
Novozym-435 catalysed esterification of disubstituted benzoic and substituted benzoate using our reaction conditions. Recycling of the immobi-
nicotinic acids
lised enzyme is feasible although with a drop of catalytic activity
Entry R1 R2 R3 X Ester Yield, % after the 6th cycle.
1 o-OCH3 p-NO2 C5H11 CH 6a 11
2 o-OCH3 p-NO2 H CH 6b 14
4. Experimental
3 m-Cl p-Cl C5H11 CH 6c >99
4 m-Cl p-Cl H CH 6d >99
5 6-Cl H C5H11 N 6e >99 4.1. General
6 6-Cl H H N 6f 92
7 6-CH3 H C5H11 N 6g 93 Substrates and reagents were commercially available and used
8 6-CH3 H H N 6h 62
without further purification. 1H and 13C NMR spectra were recorded
on a Varian Mercury (1H 400 MHz, 13C 100 MHz) with tetrame-
thylsilane (TMS) as reference. Chemical shifts are reported in ppm
Since immobilisation of enzymes is a well known strategy to and multiplicity is indicated as follow: s¼singlet; d¼doublet;
improve their long-term operational stability that is reflected in t¼triplet; q¼quartet; quin¼quintuplet; m¼multiplet; bs¼broad
higher catalyst productivities,18 we studied the recycling of the signal. GC-FID analysis were performed with a Agilent 6890N
biocatalyst in the esterification of benzoic acid with n-heptanol equipped with a FID (Column: Alltech Econo-CAP EC-5
under our reaction conditions. Novozym-435 showed a linear drop 30m0.25 mm i.d.0.25 mm; 80  C hold 5 min, 5  C/min to 100  C,
2696 D. Giunta et al. / Tetrahedron 71 (2015) 2692e2697
10  C/min to 250 hold 1 min). Melting points are collected using
€
a BUCHI B-540 and are uncorrected. IR spectra were recorded on
NaCl disks or KBr pellets using a Thermo Nicolet Avatar 330 FT-IR.
Elemental analyses (CHN) were performed on a PerkineElmer PE
2400 elemental analyzer.
4.2. General procedure for the esterification reaction
In a typical experiment, the benzoic acid derivative (0.4 mmol),
n-heptanol (0.4 mmol) and Novozym 435 (50 mg) in cyclohexane
(5 mL) are placed in a 10 mL glass test tube equipped with PTFE/
silicone septum on a snap-on cap. The mixture is stirred (50 rpm) at
80  C for 22 h. The crude reaction mixture is filtered through a plug
of silica gel (200e300 mesh) using dichloromethane as mobile
phase. The volatiles are removed under vacuum and the products
(unreacted acids and esters) isolated. Characterisation of esters
3aep and 6b is reported elsewhere.11
4.2.1. Heptyl picolinate (3q). Colourless oil; 1H NMR (400.1 MHz,
CDCl3): d¼8.75 (ddd, J¼1.1, 1.7, 4.7 Hz, 1H, ArH), 8.11 (dt, J¼1.1,
7.7 Hz, 1H, ArH), 7.83 (td, J¼1.7, 7.7 Hz, 1H, ArH), 7.46 (ddd, J¼1.1, 4.7,
7.7 Hz, 1H, ArH), 4.39 (t, J¼6.7 Hz, 2H, OCH2), 1.80 (quin, J¼7.0 Hz,
2H, CH2), 1.43e1.25 (br s, 8H, CH2), 0.86 (t, J¼6.9 Hz, 3H, CH3); 13C
{1H} NMR (100.6 MHz, CDCl3): d¼165.2, 149.8, 148.2, 136.9, 126.7,
125.0, 66.0, 31.6, 28.9, 28.6, 25.8, 22.5, 14.0; MS: EI (70 eV) m/z: 221
[M]þ, 106 [C5H4NCO]þ, 79 [C5H5N]þ; IR (NaCl): n 1728 cm1; Anal.
calcd for C13H19NO2: C, 70.56; H, 8.65; N, 6.33; Found: C, 70.81; H,
8.80; N, 6.71%.
4.2.2. Heptyl nicotinate (3r). Colourless oil; 1H NMR (400.1 MHz,
CDCl3): d¼9.23 (m, 1H, ArH), 8.78 (dd, J¼1.8, 4.9 Hz, 1H, ArH), 8.30
(dt, J¼1.8, 7.9 Hz, 1H, ArH), 7.40 (ddd, J¼0.7, 4.9, 7.9 Hz, 1H, ArH),
4.35 (t, J¼6.7 Hz, 2H, OCH2), 1.78 (quin, J¼6.7 Hz, 2H, CH2),
1.44e1.28 (br s, 8H, CH2), 0.89 (t, J¼6.9 Hz, 3H, CH3); 13C{1H} NMR
(100.6 MHz, CDCl3): d¼165.5, 153.4, 151.0, 137.2, 126.5, 123.4, 65.8,
31.9, 29.0, 28.8, 26.1, 22.7, 14.2; MS: EI (70 eV) m/z: 221 [M]þ, 106
[C5H4NCO]þ, 79 [C5H5N]þ; IR (NaCl): n 1727 cm1; Anal. calcd for
C13H19NO2: C, 70.56; H, 8.65; N, 6.33; Found: C, 70.41; H, 8.55; N,
6.55%.
4.2.3. Heptyl isonicotinate (3s). Colourless oil; 1H NMR (400.1 MHz,
CD3OD): d¼8.73 (dd, J¼1.7, 4.5 Hz, 2H, ArH), 7.93 (dd, J¼1.7, 4.5 Hz,
2H, ArH), 4.37 (t, J¼6.6 Hz, 2H, OCH2), 1.79 (quin, J¼6.6 Hz, 2H, CH2),
1.47e1.26 (br s, 8H, CH2), 0.89 (t, J¼7.0 Hz, 3H, CH3); 13C{1H} NMR
(100.6 MHz, CD3OD): d¼166.1, 151.3 (2C), 139.8, 124.4 (2C), 67.1,
32.9, 30.1, 29.7, 27.1, 23.7, 14.4; MS: EI (70 eV) m/z: 221 [M]þ, 164
[M-nBu]þ, 106 [C5H4NCO]þ, 79 [C5H5N]þ; IR (NaCl): n 1728 cm1;
Anal. calcd for C13H19NO2: C, 70.56; H, 8.65; N, 6.33; Found: C,
70.54; H, 8.32; N, 6.81%.
4.2.4. Heptyl pyrazine-2-carboxylate (3t). Colourless oil; 1H NMR
(400.1 MHz, CD3OD): d¼9.25 (m, 1H, ArH), 8.83 (d, J¼2.5, 1H, ArH),
8.75 (m, 1H, ArH), 4.43 (t, J¼6.7 Hz, 2H, OCH2), 1.82 (quin, J¼6.7 Hz,
2H, CH2), 1.49e1.32 (br s, 8H, CH2), 0.91 (t, J¼6.9 Hz, 3H, CH3); 13C
{1H} NMR (100.6 MHz, CD3OD): d¼165.2, 149.2, 147.0, 145.8, 144.7,
67.4, 32.9, 30.0, 29.7, 27.0, 23.7, 14.4; MS: EI (70 eV) m/z: 222 [M]þ,
107 [C4H3N2CO]þ, 80 [C4H4N2]þ; IR (NaCl): n 1722 cm1; Anal. calcd
for C12H18N2O2: C, 64.84; H, 8.16; N, 12.60; Found: C, 65.04; H, 8.52;
N, 12.91%.
4.2.5. Heptyl thiophene-2-carboxylate (3u). Colourless oil. 1H NMR
(400.1 MHz, CDCl3): d¼7.79 (dd, J¼3.7, 1.3 Hz, 1H, ArH), 7.54 (dd,
J¼5.0, 1.3 Hz, 1H, ArH), 7.09 (dd, J¼3.7, 5.0 Hz, 1H, ArH), 4.28 (t,
J¼6.7 Hz, 2H, OCH2), 1.74 (quin, J¼6.7 Hz, 2H, CH2), 1.43e1.28 (br s,
8H, CH2), 0.89 (t, J¼6.9 Hz, 3H, CH3); 13C{1H} NMR (100.6 MHz,
CDCl3): d¼162.5, 134.3, 133.3, 132.3, 127.8, 65.4, 31.9, 29.1, 28.8, 26.0,
22.7, 14.2; MS: EI (70 eV) m/z: 226 [M]þ, 111 [C5H3SO]þ; IR (NaCl): n
1712 cm1; Anal. calcd for C12H18O2S: C, 63.68; H, 8.02; Found: C,
63.13; H, 8.31%.
4.2.6. Heptyl thiophene-3-carboxylate (3v). Colourless oil. 1H NMR
(400.1 MHz, CDCl3): d¼8.09 (dd, J¼3.1, 1.2 Hz, 1H, ArH), 7.52 (dd,
J¼5.1, 1.2 Hz, 1H, ArH), 7.30 (dd, J¼5.1, 3.1 Hz, 1H, ArH), 4.26 (t,
J¼6.7 Hz, 2H, OCH2), 1.73 (quin, J¼6.7 Hz, 2H, CH2), 1.45e1.25 (br s,
8H, CH2), 0.89 (t, J¼6.9 Hz, 3H, CH3); 13C{1H} NMR (100.6 MHz,
CDCl3): d¼163.0, 134.1, 132.6, 128.0, 126.0, 65.0, 31.9, 29.1, 28.9, 26.1,
22.7, 14.2; EI (70 eV) m/z: 226 [M]þ, 111 [C5H3SO]þ; IR (NaCl): n
1714 cm1; Anal. calcd for C12H18O2S: C, 63.68; H, 8.02; Found: C,
63.44; H, 8.56%.
4.2.7. Heptyl furan-2-carboxylate (3w). Colourless oil. 1H NMR
(400.1 MHz, CDCl3): d¼7.57 (dd, J¼0.7, 1.8 Hz, 1H, ArH), 7.17 (dd,
J¼0.7, 3.5 Hz, 1H, ArH), 6.50 (dd, J¼1.8, 3.5 Hz, 1H, ArH), 4.30 (t,
J¼6.8 Hz, 2H, OCH2), 1.74 (quin, J¼6.7 Hz, 2H, CH2), 1.42e1.27 (br s,
8H, CH2), 0.88 (t, J¼6.0 Hz, 3H, CH3); 13C{1H} NMR (100.6 MHz,
CDCl3): d¼159.0, 146.3, 145.0, 117.8, 111.9, 65.2, 31.8, 29.0, 28.8, 26.0,
22.7, 14.2; MS: EI (70 eV) m/z: 210 [M]þ, 95 [C5H3O2]þ; IR (NaCl): n
1725 cm1; Anal. calcd for C12H18O3: C, 68.55; H, 8.63; Found: C,
68.78; H, 8.66%.
4.2.8. Heptyl furan-3-carboxylate (3x). Pale yellow oil. 1H NMR
(400.1 MHz, CDCl3): d¼8.00 (dd, J¼0.7, 1.5 Hz, 1H, ArH), 7.42 (m, 1H,
ArH), 6.74 (m, 1H, ArH), 4.23 (t, J¼6.7 Hz, 2H, OCH2), 1.71 (quin,
J¼6.7 Hz, 2H, CH2), 1.41e1.25 (br s, 8H, CH2), 0.89 (t, J¼6.8 Hz, 3H,
CH3); 13C{1H} NMR (100.6 MHz, CDCl3): d¼163.4, 147.7, 143.7, 119.7,
109.9, 64.8, 31.9, 29.1, 28.8, 26.1, 22.7, 14.2; MS: EI (70 eV) m/z: 210
[M]þ, 95 [C5H3O2]þ; IR (NaCl): n 1726 cm1; Anal. calcd for
C12H18O3: C, 68.55; H, 8.63; Found: C, 68.14; H, 8.67%.
4.2.9. Heptyl 2-methoxy-4-nitrobenzoate (6a). Colourless oil. 1H
NMR (400.1 MHz, CDCl3): d¼7.89e7.81 (m, 3H, ArH), 4.34 (t,
J¼6.6 Hz, 2H, OCH2), 4.00 (s, 3H, OCH3), 1.74 (quin, J¼6.7 Hz, 2H,
CH2), 1.46e1.29 (br s, 8H, CH2), 0.90 (t, J¼6.8 Hz, 3H, CH3); 13C{1H}
NMR (100.6 MHz, CDCl3): d¼164.9, 159.2, 150.6, 131.9, 126.5, 114.9,
106.9, 65.9, 56.5, 31.7, 28.9, 28.6, 25.9, 22.6, 14.0; MS: EI (70 eV) m/z:
295 [M]þ, 198 [C8H8NO5]þ; IR (NaCl): n 1731 cm1; Anal. calcd for
C15H21NO5: C, 61.00; H, 7.17; N, 4.74; Found: C, 61.34; H, 7.59; N,
4.81%.
4.2.10. Ethyl 2-methoxy-4-nitrobenzoate (6b). Colourless oil; 1H
NMR (400.1 MHz, CDCl3): d¼7.89e7.82 (m, 3H, ArH), 4.40 (q,
J¼7.0 Hz, 2H, OCH2), 4.00 (s, 3H, OCH3), 1.40 (t, J¼7.0 Hz, 3H, CH3);
13 1
C{ H} NMR (100.6 MHz, CDCl3): d¼164.7, 159.2, 150.6, 131.9, 126.4,
114.9, 106.4, 61.7, 56.6, 14.2; MS: EI (70 eV) m/z: 225 [M]þ, 189
[C8H6NO5]þ; IR (NaCl): n 1733 cm1; Anal. calcd for C10H11NO5: C,
53.33; H, 4.92; N, 6.22; Found: C, 53.74; H, 4.59; N, 6.51%.
4.2.11. Heptyl 3,4-dichlorobenzoate (6c). Colourless oil. 1H NMR
(400.1 MHz, CDCl3): d¼8.12 (d, J¼2.0 Hz, 1H, ArH), 7.87 (dd, J¼8.2,
2.0 Hz, 1H, ArH), 7.53 (d, J¼8.2 Hz, 1H, ArH), 4.32 (t, J¼6.6 Hz, 2H,
OCH2), 1.77 (quin, J¼6.6 Hz, 2H, CH2), 1.45e1.30 (br s, 8H, CH2), 0.90
(t, J¼6.0 Hz, 3H, CH3); 13C{1H} NMR (100.6 MHz, CDCl3): d¼164.8,
137.4, 132.8, 131.4, 130.4, 130.3, 128.6, 65.8, 31.7, 28.9, 28.6, 25.9,
22.5, 14.0; MS: EI (70 eV) m/z: 288 [M]þ, 190 [C7H3O2Cl2]þ; IR
(NaCl): n 1708 cm1; Anal. calcd for C14H18Cl2O2: C, 58.15; H, 6.27;
Found: C, 58.24; H, 6.39%.
4.2.12. Ethyl 3,4-dichlorobenzoate (6d). White solid,
mp¼39e40  C; 1H NMR (400.1 MHz, CDCl3): d¼8.12 (d, J¼2.0 Hz,
1H, ArH), 7.87 (dd, J¼8.2, 2.0 Hz, 1H, ArH), 7.52 (d, J¼8.2 Hz, 1H,
ArH), 4.39 (q, J¼7.3 Hz, 2H, OCH2), 1.40 (t, J¼7.3 Hz, 3H, CH3); 13C
{1H} NMR (100.6 MHz, CDCl3): d¼164.7, 137.4, 132.8, 131.5, 130.4,
 D. Giunta et al. / Tetrahedron 71 (2015) 2692e2697
130.3, 128.6, 61.6, 14.2; MS: EI (70 eV) m/z: 218 [M]þ, 189
[C7H3O2Cl2]þ; IR (KBr): n 1710 cm1; Anal. calcd for C9H8Cl2O2: C,
49.35; H, 3.68; Found: C, 49.14; H, 3.49%.
4.2.13. Heptyl 6-chloronicotinate (6e). Colourless oil. 1H NMR
(400.1 MHz, CDCl3): d¼8.99 (m, 1H ArH), 8.24 (dd, J¼8.2, 2.3 Hz, 1H,
ArH), 7.42 (dd, J¼8.2, 0.8 Hz, 1H, ArH), 4.35 (t, J¼6.6 Hz, 2H, OCH2),
1.77 (quin, J¼6.7 Hz, 2H, CH2), 1.47e1.28 (br s, 8H, CH2), 0.89 (t,
J¼6.6 Hz, 3H, CH3); 13C{1H} NMR (100.6 MHz, CDCl3): d¼164.4,
155.5, 151.1, 139.5, 125.3, 124.1, 65.8, 31.6, 28.8, 28.5, 25.8, 22.5, 13.9;
MS: EI (70 eV) m/z: 255 [M]þ, 220 [C13H18O2N]þ; IR (NaCl): n
1728 cm1; Anal. calcd for C13H18ClNO2: C, 61.05; H, 7.09; N, 5.48;
Found: C, 61.42; H, 7.35; N, 5.71%.
4.2.14. Ethyl 6-chloronicotinate (6f). Colourless oil. 1H NMR
(400.1 MHz, CDCl3): d¼9.01 (dd, J¼2.3, 0.8 Hz, 1H ArH), 8.25 (dd,
J¼8.2, 2.3 Hz, 1H, ArH), 7.41 (dd, J¼8.2, 0.8 Hz, 1H, ArH), 4.42 (q,
J¼7.3 Hz, 2H, OCH2), 1.42 (t, J¼7.3 Hz, 3H, CH3); 13C{1H} NMR
(100.6 MHz, CDCl3): d¼164.4, 155.5, 151.1, 139.5, 125.3, 124.1, 61.7,
14.2; MS: EI (70 eV) m/z: 185 [M]þ, 157 [C6H4O2NCl]þ; IR (NaCl): n
1724 cm1; Anal. calcd for C8H8ClNO2: C, 51.77; H, 4.34; N, 7.55;
Found: C, 51.58; H, 4.73; N, 7.85%.
4.2.15. Heptyl 6-methylnicotinate (6g). Colourless oil. 1H NMR
(400.1 MHz, CDCl3): d¼9.12 (d, J¼2.0 Hz, 1H, ArH), 8.19 (dd, J¼8.2,
2.0 Hz, 1H, ArH), 7.25 (d, J¼8.2 Hz, 1H, ArH), 4.34 (t, J¼6.6 Hz, 2H,
OCH2), 2.64 (s, 3H, CH3), 1.78 (quin, J¼6.6 Hz, 2H, CH2), 1.46e1.28 (br
s, 8H, CH2), 0.89 (t, J¼7.0 Hz, 3H, CH3); 13C{1H} NMR (100.6 MHz,
CDCl3): d¼165.4, 162.9, 150.3, 137.3, 123.6, 122.8, 65.3, 31.6, 28.8,
28.6, 25.9, 24.6, 22.5, 14.0; MS: EI (70 eV) m/z: 235 [M]þ, 220
[C13H18O2N]þ; (NaCl): n 1720 cm1; Anal. calcd for C14H21NO2: C,
71.46; H, 9.00; N, 5.95; Found: C, 71.54; H, 9.24; N, 6.11%.
4.2.16. Ethyl 6-methylnicotinate (6h). Colourless oil. 1H NMR
(400.1 MHz, CDCl3): d¼9.12 (d, J¼2.3 Hz, 1H ArH), 8.19 (dd, J¼8.0,
2.3 Hz, 1H, ArH), 7.25 (d, J¼8.0 Hz, 1H, ArH), 4.41 (q, J¼7.0 Hz, 2H,
OCH2), 2.64 (s, 3H, CH3), 1.41 (t, J¼7.0 Hz, 3H, CH3); 13C{1H} NMR
(100.6 MHz, CDCl3): d¼165.4, 162.9, 150.3, 137.3, 123.6, 122.9, 61.2,
24.6, 14.2; MS: EI (70 eV) m/z: 165 [M]þ, 137 [C7H7O2N]þ; IR (NaCl):
n 1722 cm1; Anal. calcd for C9H11NO2: C, 65.44; H, 6.71; N, 8.48;
Found: C, 65.72; H, 6.54; N, 8.21%.
4.3. General procedure for the lipase recycling experiment
In a typical experiment, benzoic acid (200 mg, 1.64 mmol), n-
heptanol (191, 1.64 mmol) and 20 mL cyclohexane were placed into
a glass vial. Novozym-435 (200 mg) was charged into a glass basket
equipped with a sintered glass bottom and immersed into the re-
action solution. The mixture was stirred vigorously at 60  C for 22 h.
2697
At the end of the reaction the glass basket was recovered and re-
immersed into a different glass vial containing a fresh reaction
solution and the reaction products recovered by solvent evapora-
tion. The crude reaction products were filtered on a small silica gel
column (200e300 mesh) using dichloromethane as the eluent to
separate the ester from the unreacted acid.
Acknowledgements
We gratefully acknowledge financial support from Regione
Autonoma della Sardegna (L.R. 7 agosto 2007, n. 7, grant no. CRP-
25257).
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