E. T. Contis et al.

(Editors)
Food Flavors: Formation, Analysis and Packaging Influences
© 1998 Elsevier Science B.V. All rights reserved

Thirty years of the AH-B theory

T. E. Acree^, R. S. Shallenberger^, and S. Ebeling^,

^Department of Food Science & Technology, Cornell University, Geneva, NY,

14456 "University College Cork, Cork, Ireland.

Abstract

Thirty years ago two of us (Shallenberger & Acree) published a paper enti-
tled the "Molecular Theory of Sweet Taste" in Nature[l]. The model developed
in that paper for sweetness was based on a structure-activity relationship be-
tween the simplest sweet tasting compounds and their structural features of the
stimulants and has become known as the AH-B theory. The theory described
with considerable success the structural features necessary for sweetness but it
was not sufficient to predict sweetness. That is, not all compounds that satisfied
the theory tasted sweet nor was the theory able to predict potency level espe-
cially for the very high potency sweeteners subsequently synthesized. However,
all sweet compounds seemed to have an identifiable AH-B feature. This paper
will review the last thirty years in sweetness research and discuss the role of
the AH-B theory in its development.

1. 1963 - 1971

There are two motives that have driven structure-activity research (SAR) into
sweetness. One is the desire to predict the taste of molecules from their struc-
ture (this is primarily of commercial value), and the other is to understand taste
in terms of its chemical ecology and how it evolved (this is primarily of academic
interest). In a paper publish in 1963 Robert Shallenberger summarized the re-
lationship between sugar structure and sweet taste response in terms of the
idea of a functional group for sweetness called the "saporfic group": a pair of vic-
inal hydroxyl groups [2].
 OH

OH

A fact that did not seem to support this hypothesis was that most molecules con-
taining a saporific group were not sweet. Clearly, the taste active structure that
caused the sweet response was more complicated than just a pair of vicinal hy-
droxyIs. This was demonstrated clearly by mannose which could be prepared in
two crystalline diastereoisomeric forms a- and P-. Both a- and P-D-mannose
have three glycol groups but a- is sweet and P- is bitter. In fact all mono- and
disaccharides, sugar alcohols and similar polyols are characterized by the pres-
ence of multiple glycol groups. Therefore, some relationship between the glycol
hydroxyl groups determines sweetness and in the case of mannose bitterness.
Although one glycol group in a - D-mannose has a dihedral angle greater that
60^ assuming the preferred chair conformation, exactly how this subtile struc-
tural difference could cause such a profound difference in taste was difficult to
envision?

H OH H H
a - D - mannopyranose (sweet) P - D - mannopyranose (bitter)

The prevailing hypothesis, then as it is now, was that the taste active com-
pounds acted as a ligand that bound to a receptor protein on the surface of a
taste receptor cell causing an allosteric effect on the inside of the cell inducing a
change in ionic conductance resulting in depolarization. For the idea to work
the receptor protein - ligand complex must undergo a change in at least its ter-
tiary structure. However, these changes could include changes in the ligand as
well as the receptor protein. Therefore, the conformation of the ligand as it ap-
proaches the receptor may not be as important as the conformation it can as-
sume in the receptor - ligand complex. Predicting activity from the most stable
structure in solution may be the wrong approach. This led to the idea that the
 3

glycol group induces sweetness when it can assume a dihedral nearer to the
gauch (60°) configuration than either the eclipsed (0°) or anticlinal (180°). In
this view a dihedral angle somewhat less than gauch must be obtainable and
the energy necessary to do this would be the determinant for sweet taste.

^^OH ^^OH ^ OH
gauch (60°) eclipsed (0°) anticlinal (180°)

Between 1963 and 1968, while a graduate student in Shallenberger's labora-

tory, Terry Acree studied the secondary and tertiary structure of the monosac-
charides in solution in an attempt to find a more successful structural descrip-
tion to predict sweetness. Although this work provided some greater detail
about sugars in solution [3-6], it did not yield any better predictors of sugar
sweetness. Meanwhile, in 1967 Shallenberger and Acree published a theory of
sweet taste chemistry that was based on simpler and more rigid structures than
the sugars. At that time, it was commonly accepted that the sweet taste mech-
anism evolved to detect biologically important primary metabolites such as sug-
ars and hydrolyzed polysaccharides at molar concentrations and that the sweet-
ness of non-sugar molecules was perhaps an artifact [7, 8]. However, it was also
assumed that these non-sugar sweeteners acted on the same sweet receptor and
t h a t their structure could be used to predict t h e structure of a "saporific"
molecules including sugars.
The simplest series of molecules t h a t have a clear sweet taste are t h e
chloromethanes. They are rigid, and the analogues chloroform, methylene chlo-
ride and chloromethane shown below are slightly sweet whereas methane and
carbon tetrachloride are not sweet.

CI CI H

CI^H>AHJ'^ H^H>AHJ'^ H^H>AH>

chloroform methylene chloride chloromethane

The structure common to these sweet analogues is the presence of a hydrogen
bis to a chlorine. Comparing this to the structure of sweet glycols with a gauch
dihedral angle we concluded that a hydrogen bondable proton located about 3 A
(0.3 nm) from an electron rich orbital also capable of forming a hydrogen bond
was required for sweetness. The proton donor was called the AH group and the
proton acceptor was called the B group: thus defining the "saporific group" as an
AH-B (3 A).
Examining the structure of a diverse group of sweet tasting molecules,
Shallenberger and Acree concluded that AH-B could be a necessary condition for
sweetness but clearly not a sufficient condition to guarantee sweetness. There
were other structural features that rendered most molecules with an AH-B at
3A non-sweet or at least dominated by some other taste property usually bit-
terness. In 1969 with the help of C. Y. Lee, Shallenberger and Acree published
a paper based on the taste of amino acids that identified the minimum require-
ments for sweetness among the chiral amino acids [9]. Starting with glycine and
alanine they pointed out that since all of the D- amino acids with side groups
larger than a methyl group or at least as large as an isobutyl group (leucine)
were non-sweet while their enantiomers ( t h e L-amino acids) tasted sweet. The
small achirial glycine with only a proton side group and the slightly larger D-
and L-alanine with only a methyl group for a side chain were all distinctly
sweet. Furthermore, glycine is functionally sweet in foods like the cooked crus-
tatae, shrimp and lobster.

D - Alanine (sweet) L - Alanine (sweet)

The simplest conclusion was that there was a steric barrier that inhibited the
binding of the D- isomer but allowed the binding of the L- isomer. Alternatively,
it could be argued that the side group of the L- isomers bound to a lipophylic
part of the receptor resulting in what was reported by Kier in 1972 as a three-
point attachment theory for the "glycophore" in which X in the figure below is a
lipophilic group [10] .
 AH

.26 nm

It seems reasonable to assume that some multiple attachment process will con-
tribute to the chirality of sweetness, but the fact that glycine is the sweetest
amino acid and it has no side chain is still puzzling. Furthermore, the observa-
tion that the enantiomers of the momosacchrides are equally sweet while the di-
astereoisomers tasted different does not speak for a simple chiral component.
For example, the two enantiomers of glucose shown below are both equally
sweet while the two anomers (diastereoisomers) of mannose taste different.

OH

HO'
H H

H OH HO
a-D-glucopyranose a-L-glucopyranose

We can summarize the taste of polyols as follows:

1. Sweet ligands are bipolar hydrogen bonding units: AH-B
2. Enantiomers of sugars are equal tasting.
3. Diastereoisomers (anomers) of sugars can have different tastes.

However, the taste of the amino acids were summarized differently:

1. Sweet ligands are bipolar hydrogen bonding units: AH - B
2. Enantiomers of the amino acids are different tasting if
they have a side group larger than alanine.
The contradictions created by these two summaries were the subject of numer-
ous studies and structural activity relation investigations but none could resolve
them with a single ligand receptor model.
2. 1972-1991
Over the next 20 years, the apparent contradictions posed by the tastes of amino
acids and sugars eventually resulted in several descriptions of a receptor site
structure that would accommodate a variety of "saporific groups". By 1991,
these ideas reached their greatest degree of complexity in the structure simula-
tion studies in Belitz'[ll] laboratory and the multi-attachment theory of Tinti
and Nofri[12]. These workers approached the problem by creating graphic rep-
resentations of the active site on the sweet receptor in terms of the types of
functional groups that might interact and their spatial arrangements. Shown
below is the model developed by Tinti & Nofre [12] in which the various spheres
represent different functional groups that may be involved in the ligand bind-
ing.

Common to all of these receptor site models and multi-attachment theories are
two assumptions: 1) the presence of an AH-B or equivalent and 2) the assump-
tion that not all attachments are required for binding to take place. That none
of these complex models describe necessary and sufficient conditions for taste is
their weakest feature. Information about the nature of the receptor protein, the
number of transduction mechanisms involved and the relationship between
sweet and bitter taste biochemistry would certainly help. The AH-B model for
the ligand binding to the receptor provided a reasonable idea for a transduction
mechanism: the disruption of a hydrogen bond on the receptor protein on the
outside of the cell followed by an allostericUy induced change inside the cell[13].
However, the details of this part of the transduction process shown below are
too vague to guide SAR modeling and too speculative add anything to the study
of transduction biochemistry.
 Receptor site

H
Saporific ligand

There were two other facts about taste that were puzzHng. The first was the
discovery in the early 1980's of a sweetness inhibitor by Michael Lindley [14,
15]. The phenoxyalkonic acids inhibited both the amino acid based sweeteners
(aspartame) and the polyol sweeteners (sucrose) in exactly the same way with
exactly the same competitive inhibition. This would strongly suggest that the
same limiting steps were being inhibited and therefore the transduction mecha-
nisms for both types of sweeteners were perhaps the same or at least shared
some transduction components. Furthermore, that sweetness inhibition oc-
curred put into question any modeling based on the taste intensity of a series of
molecules. If sweetness intensity is a balance between inhibitory elements and
stimulant structures, perhaps on the same molecule, then interpreting sweet-
ness intensities in terms of ligand binding affinities would be erroneous. The
complex chemistry of most natural products predicts that inhibition is most
surely is an important component of many real food systems.
The second set of puzzeling facts about taste was the inability of many
sweeteners, the amino acid based sweetners for example, to produce as intense a
sweetness as sucrose. Polyols are not very potent sweeteners nor would they
need to be if their taste was simply an indication of metabolically important
concentrations. However, as shown below sucrose, fructose, etc., are one of the
most intense sweeteners where as the the extremely potent sweeteners like as-
partame are never as sweet as the polyols [16].
 Intensity
100 • High Intensity (sucrose)

80

High Potency (aspartame)

T
-4 -2 0
Log (Concentration)

This distinction between high intensity sweeteners as opposed to high potency

sweeteners would tend to indicate different transduction mechanisms.
However, as Lindely pointed out in a paper presented at the first American
Chemoreception Science symposium meeting in 1975 [17] "Assuming that there
is in fact a direct relationship between structure and taste, I think the only con-
clusion to be drawn from this [contradictory facts] is that there is something
missing." Exactly what was missing became clearer when evidence for a trans-
duction mechanism based on G-proteins found in the taste membranes of many
non-human models began to accumulate.

3. 1991- 1997

The present theory of t a s t e transduction, recently summarized by

Lindmann[8, 18], indicates that high intensity sweeteners (polyols in particu-
lar) react with a seven-transmembrane receptor protein (SR) which is associated
with a G- protein inside the cell. The diagram below shows a schematic of the ol-
factory receptor protein found in the rat (adapted from Krieger [19]).

membrane

^COOH
It is typical of the 7-transmembrane receptor proteins usually associated with
G-proteins. In the case of olfaction the present speculation is that the cytoplas-
mic loops are associated with the G-protein inside the cell and the extra cellular
loops are involved in forming the ligand binding site. Modifications in the ter-
tiary structure of the external loops presumably provides the energy to create an
allosteric change in the cytoplasmic loops activating the G-protein. Although
the details of this part of the process are still unclear, the general idea seems
convincing since it has been repeated in so many different chemo-sensory sys-
tems [20].
In a more recent review, Naim[21] summarized some truly exciting ideas
about sweet transduction based on studies from of non-human systems. In sim-
ple terms measurements of intracellular transduction second messengers, cal-
cium ion, inosotol triphosphate (IPS) and cyclic adenosine monophosphate
(cAMP) indicates the presence of multiple mechanisms on multiple receptors.
For example, after reaction with a receptor cell saccharin caused the accumula-
tion of IPS and sucrose the accumulation of cAMP inside the cell indicating that
non-polyol sweeteners are involved in a different transduction process. The fig-
ure below shows a modification of the scheme for sweet taste transduction in the
rat circumvallate taste papillia proposed by Naim.

Si^ar
AA

AA

Sensory Nerve Hber-

The scheme shows a taste cell with two taste receptor proteins: SR, a sugar re-
ceptor protein that uses cAMP as a second messenger and NSR, a non-sugar re-
10

ceptor protein that uses IPS as a second messenger. NSR responds to saccharin,
small peptides and similar compounds. Both of these mechanisms appear to be
on the same sensory cell. Also shown in the diagram are the a, (3 and y subunits
of the G-protein that are putatively activated by the receptor protein. The in-
teresting twist to this picture is the possibility that some sweet-tasting com-
pounds labeled here as AA (called amphipathic compounds by Naim, i.e. having
both polar and non-polar properties) induce transduction by moving across the
receptor cell membrane and reacting directly with the P-y subunit. These com-
pounds are then acting like many drugs that enter cells, modify their behavior
and stimulate responses that were evolved to detect the presence of different
ligands. In the case of sweetness: caloric polyols.
The implication for SAR of sweetness created by the possibility multiple re-
ceptors with multiple mechanisms is profound. We would have to conclude that
the multiple attachment theories must represent a melange of receptor struc-
tures and this would explain the "necessary but not sufficient..." nature of their
predictive powers. We can then speculate that the following scheme for sweet
taste in which high potency sweeteners induce sweetness by disrupting the
transduction process at the G-protein while high intensity sweeteners react with
the sweet receptor protein would explain their different dose-response behavior.

Higli Fotemy " l ^ ^ Inteitaitj-

Finally, the creation of knock-out mice by Wong et al that lack a-gusducin

(presumably the a subunit of the sweet taste G-protein) inhibited both second
messenger formation at the cellular level and the taste response to bitter
(denatonium benzoate and quinine sulfate), high-intensity sweeteners (sucrose)
and high potency sweeteners (a guanidine sweetener: SC45647). This strongly
indicates that both sweet reception and bitter reception share same transduc-
tion components and that the non-sugar sweet receptor system is related to the
bitter receptor if not in fact the same as shown in the diagram below [22].
 11

Non-sugar
Non-sugar I

Exactly how the second order neurons interpret this multiple receptor - multiple
mechanism process is a little difficult to imagine. However, we should be able to
predict that if sweetness inhibitors act by inhibiting the G-protein complex they
would have the same effect on both high potency and high - intensity sweeten-
ers. Furthermore, they should also inhibit bitter compounds in a similar fash-
ion. This, however, has yet to be determined.
After thirty years, the AH-B theory remains a possible explanation for the
ligand binding chemistry that induces some sweet taste responses but it seems
to have become a minor part of what has evolved into a complicated yet elegant
story chemo-sensory response.

4. References

1. Shallenberger, R.S. and T.E. Acree, Molecular Theory of Sweet Taste.

Nature, 1967. 216(5114): p. 480-2.
2. Shallenberger, R.S., Hydrogen Bonding and the Varying Sweetness of the
Sugars. Journal of Food Science, 1963. 28(5): p. 584-589.
3. Acree, T.E., R.S. Shallenberger, and L.R. Mattick, Mutarotation ofD-galac-
tose. Tautomeric composition of an equilibrium solution in pyridine.
Carbohyd. Res., 1968. 6(4): p. 498-502.
4. Acree, T.E., Tautomerism of D-glucose, D-mannose, and D-galactose. 1969.
29(11).
5. Acree, T.E., et al., Thermodynamics and kinetics of D-galactose tau-
tomerism during mutarotation. Carbohyd. Res., 1969. 10(3): p. 355-60.
6. Acree, T.E., Chemistry of sugars in boric acid solutions. Advan. Chem. Ser.,
12

N o , 1973. .
7. Moncriff, R.W, The Chemical Senses. 3 ed. 1967, London: Leonard Hill.
8. Lindemann, B., Taste Reception. Physiol. Rev., 1996. 76(3): p. 719-766.
9. Shallenberger, R.S., T.E. Acree, and C.Y. Lee, Sweet Taste of D and L-
Sugars and Amino-acids and the Steric Nature of their Chemo-receptor Site.
Nature, 1969. 221(5180): p. 555-556.
10. Kier, L.B., A molecular theory of sweet taste. J. Pharm. Sci., 1972. 61: p.
1394-7.
11. Rohse, H. and H.-D. Belitz, Shape of Sweet Receptors Studied by Computer
Modeling, in Sweetners: Discovery, Molecular Design, and Chemoreception.
ACS Symposium Series, D.E. Walters, F.T. Orthoefer, andG.E. DuBois,
Editor. 1991, Am. Chem. Soc: Boston, p. 176-192.
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in Sweeteners: Discovery, Molecular Design, and Chemoreception. ACS
Symposium Series, D.E. Walters, F.T. Orthoefer, andG.E. DuBois, Editor.
1991, Am. Chem. Soc: Boston, p. 176-192.
13. Acree, T.E. A Molecular Theory of Sweet Taste - Amino Acids and Peptides.
in Joint Symposium on Carbohydrate /Protein Interactions: American
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14. Lindley, M.G.,. 1986, Europe.
15. Lindley, M.G., Phenoxyalkanoic Acid Sweeteness Inhibitors, in Sweetners:
Discovery, Molecular Design, and Chemoreception. ACS Symposium Series,
D.E. Walters, F.T. Orthoefer, andG.E. DuBois, Editor. 1991, Am. Chem.
Soc: Boston, p. 251-260.
16. DuBois, G.E., et al., Concentration-Response Relationships of Sweeteners, in
Sweeteners: Discovery, Molecular Design, and Chemoreception. ACS
Symposium Series. 1991, Am. Chem. Soc: Boston, p. 251-260.
17. Lindley, M.G. and G.G. Birch, Structural functions of taste in the sugar se-
ries. J. Sci. Food Agric, 1975. 26(1): p. 117-24.
18. Lindemann, B., Chemoreception: Tasting the sweet and the bitter. Curr.
Biol., 1996. 6(10): p. 1234-1237.
19. Krieger, J., et al., Cloning and Expression of Olfactory Receptors, in Adv. in
Biosciences, R. Apfelbach, et al., Editor. 1994, Elsevier Science Inc.: Oxford.
20. Brand, J.G. and A.M. Feigin, Biochemistry of sweet taste transduction. Food
Chem., 1996. 56(3): p. 199-207.
21. Naim, M., et al. Molecular aspects of Sweet Taste Transduction, in Contrib.
 13

Low- Non-Volatile Mater. Flavor Foods. 1996. Allured, Carol Stream, 111.
22. Wong, G.T., K.S. Gannon, and R.F. Margolskee, Transduction of hitter and
sweet taste by gustducin. Nature, 1996. 381(6585): p. 796-800.