StructureâActivity Relationship of Sweet Molecules - ACS Symposium

Chapter 11

Structure—Activity Relationship of Sweet Molecules 1

2

3

P. A. Temussi , F. Lelj , and T. Tancredi 1

Dipartimento di Chimica, Università di Napoli, via Mezzocannone 4, 80134 Napoli, Italy Dipartimento di Chimica, Università della Basilicata, Potenza, Italy Istituto Chimica M.I.B. del C.N.R., via Toiano 6, Arco Felice, Napoli, Italy

Downloaded by IOWA STATE UNIV on April 19, 2017 | http://pubs.acs.org Publication Date: December 31, 1991 | doi: 10.1021/bk-1991-0450.ch011

2

3

The shape of the receptor active site of the receptor for sweet molecules, previously defined on the basis of a combination of rigid and flexible molds, has been refined using the shape of naphthimidazolesulfonic acids, a class of very large and rigid tastants. The new shape is still consistent with all previously examined sweet molecules. The model is illustrated by means of the fit of numerous rigid and flexible sweet and bitter tastants. Sweetness is a stimulus imparted by a very large number of molecules of widely different chemical nature (J). The availability of many agonists and the practical relevance of sweeteners has stimulated numerous SAR studies and the development of general models of the receptor active site (2-J 2). The oldest attempts to find common features among sweet molecules might be called one-dimensional structural approaches, meaning that all emphasis was placed on the chemical constitution without any reference to their actual three dimensional shape. They tried to attribute specific sweetening power to groups of atoms like C H , CHO, N 0 , COOH etc. (termed glucophores) that ought to modify basic taste properties of compounds much the way analogous substitutions modify optical spectra of the same compounds. All classifications (J,2) based on glucophores proved inadequate since, in general, it is not possible to attribute specific properties to single groups. Besides, there are several outstanding exceptions to this generalization; in particular, there are always numerous tasteless compounds containing one or more typical glucophores, e.g. N-methyl saccharin, sodium aniline sulfate, 3nitro-4-methylaniline, etc. 3

2

0O97-6156/91/045O-O143$O6.00/0 © 1991 American Chemical Society Walters et al.; Sweeteners ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


144

SWEETENERS:

DISCOVERY, M O L E C U L A R DESIGN, A N D CHEMORECEPTION

The first structural (and electronic) feature identified in most sweet molecules is the so-called AH-B entity (3). Shallenberger and Acree pointed out that nearly all sweet molecules (even those lacking any other constitutional similarity) have a hydrogen bond donor (AH) and a hydrogen bond acceptor (B) separated by ca. 0.3 nm. Thus the sweet taste of sugars, amino acids, saccharin, chloroform, olefinic alcohols and meta-nitroanilines was attributed to their ability to form two hydrogen bonds with a complementary B-AH entity of the receptor. It must be noted that it is possible to find this AH-B entity in many other sweet compounds not quoted by Shallenberger and Acree (3), but also in many bitter and tasteless compounds. The main weakness of this theory however, is that the importance of both three-dimensional shape and volume is still overlooked. A trivial example is that molecules whose volume is larger than that of the receptor active site can never fit it even if they possess the correct electronic features (the AH-B entity and/or others). Most models of the sweet receptor active site subsequently proposed (5, 9-12) include the AH-B feature and a third (dispersion forces) site at the apex of a triangle (5): they offer satisfactory explanations of many sweet compounds but fail to explain the relationship among sweet, bitter and tasteless compounds, even when they belong to the same chemical class. A Receptor Active Site Model We have tried to identify the main features of the receptor active site for sweet molecules by combining several empirical observations (taken from the literature) with rigorous geometrical criteria. The AH-B feature of Shallenberger was taken as a sufficient unifying criterion to assume that all sweet molecules (possessing it) interact with the same receptor. Another interesting observation of Shallenberger and Acree (4) was that simple amino acids can taste sweet only if their asymmetric α-carbon has a D configuration. Shallenberger and Acree attributed this behavior to the existence, in the receptor active site, of a steric barrier placed 0.3 nm below the plane of the AH-B feature, that prevents accommodation of L-amino acids with side chains bulkier than that of alanine. These hypotheses give the clue for a possible mapping of the receptor active site. The key to a quantitative mapping was found through the peculiar relationship between sweet and bitter molecules. Simple amino acids and peptides apparently change their taste quantitatively from sweet to bitter when the chirality of the α-carbon adjacent to the AH-B entity (i.e. the amino and carboxyl groups respectively) is changed (4,5,J3,J4). This behavior, if taken at face value, would seem to point to the existence of two mirrorimage receptor active sites, i.e., to two mirror-image receptor proteins. This is clearly a paradox if, as is necessary, the two

Walters et al.; Sweeteners ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


11.

TEMUSSI ET A L

Structure-Activity Relationship of Sweet Molecules

145

proteins are built of asymmetric residues of the same chirality, but can be overcome if the symmetry relationship between the two receptor proteins is of lower rank than the mirror-image relationship. In fact if one assumes that the two receptor active sites are essentially planar (or very flat) and very similar cavities, it is possible to account for the change in taste of amino acids with chirality simply by inverting the AH-B entities of the twin receptors for sweet and bitter tastes via a binary axis operation, as can be easily accomplished in nature by two single point mutations in the protein gene (6,7). Figure 1 shows the relationship between the two enantiomers of a generic amino acid of formula HOOC-CH(NH2)-CH2-R and the receptor active sites for sweet and bitter molecules, depicted here schematically as two identical boxes, whose right-hand side (shaded in the figure) represents the Shallenberger barrier. A C2 symmetry relationship relates the AH-B entities of the receptor active sites that are otherwise identical shallow cavities. Since the side of the receptor active site opposite to the Shallenberger barrier is completely open, the R moieties of the side chains of the two amino acids are almost completely outside the receptor active sites. The exact depth of the cavity representing the receptor active site can not be determined in a very detailed way, but it is possible to estimate it since it can accommodate the side chain of L-alanine, which is slightly sweet as well as the D-isomer (1,13,14). Accordingly all relevant agonist-receptor interactions are to be found only in the space comprised between the Shallenberger barrier and a parallel plane situated ca. 0.3 nm above it. On the other hand, it is not possible at the present time to estimate whether outside the open cavity the walls of the receptor are extending as a flat surface or, more likely, as the the surface of a wide funnel. This view of the receptor for sweet molecules is supported by the general observation that indeed a very large number of synthetic sweeteners are flat rigid molecules (I) and by the fact that there are both synthetic {15) and natural sweet macromolecules, i.e. the two sweet proteins monellin and thaumatin {16). In fact, with our model it is easy to envisage an interaction in which most of the sweet macromolecule is outside the receptor active site, whereas a protruding branch (a "sweet finger") is probing the receptor through the open side of the cavity. A great asset of an active site model that can be treated as a pseudo-two-dimensional model is the possibility of using, quantitatively, the wealth of data from the literature on sweet rigid molecules to map the contours of the model. This task is also greatly facilitated by the hypothesis that all sweet molecules interacting with this receptor model have the AH-B entity (or at least half of it), since it will be possible to refer all molecules to the same orientation. We exploited the fact that many synthetic sweeteners show simple trends of taste intensity with substitution. For example, saccharins substituted in position 6 of the aromatic



146

SWEETENERS:

DISCOVERY, M O L E C U L A R DESIGN, A N D CHEMORECEPTION

ring remain sweet when the hydrogen is substituted with a methyl group, an amino group, a fluorine or a chlorine; retain a sweetish taste with bromine; but abruptly lose their taste when the hydrogen is substituted with an iodine or methoxy group. This behavior was interpreted as an indication of the existence of a wall in the receptor at approximately the distance of the van der Waals radius of iodine (6). By analyzing several similar cases of substituted saccharins it was possible to identify the nearly complete shape of the receptor active site in the region of the plane surrounding the AH-B entity (6) and to delineate an upper hydrophobic region as a minimum shape of interaction by means of the structure of aspartame (17). Such a model could not be considered a complete geometrical model but rather a topological model, since only rigid molecules are fully reliable as molds and the flexibility of aspartame.is very high (IS). The final model was defined using a sweet molecule with the largest possible rigid area in the plane of AH-B, 3-anilino2-styryl-3H-naphth(l,2-d)imidazolesulfonate (henceforth called SSN), whose shape was determined by conformational studies in our laboratory (18, 19). A crucial point in comparing different sweet molecules, and hence in using an active site model, is the possibility of orienting their AH-B entities in the same way. This is not straightforward in the case of SSN since its AH-B entity is intrinsically different from those of most other sweet compounds. Strictly speaking, the anionic form of SSN has only the Β part of the entity, i.e. the -SO3" group. It can use two of the oxygens (like the oxygens of the S O 2 group of saccharin) as the Β moiety of AH-B, but the shape of AH-B is slightly different from that of the same entity in saccharin. As a general rule, however, it is fair to say that an imperfect AH-B entity can be well tolerated by the receptor active site, provided the steric fit is otherwise very good. This last requirement is certainly met by SSN since its naphthimidazolesulfonic acid moiety has a shape exactly complementary to the lower part of the active site model derived from saccharins, and fills it completely. Actually, even the upper part of SSN (i.e. the styryl moiety) is very similar to that of the original model (6), only somewhat larger. It is important to emphasize that the styryl group is completely coplanar with the naphthimidazolesulfonic acid skeleton, thus assuring a very good hydrophobic interaction with the flat surface of the receptor active site corresponding to the Shallenberger barrier. Figure 2a shows the quantitative two-dimensional contour of the new active site model, derived from a combination of the mapping with saccharins (6) and with SSN (18). The shape of the receptor active site, in the main plane of the flat cavity (xy), has been obtained optimizing the position of monoatomic apolar molecules, hereafter called S\ interacting with the mold through a simple nonbond potential described by an exp-r^ function and a 4



11. TEMUSSI ET AJL


sweet

bitter

Figure 1. Schematic drawing of the active site models for sweet and bitter compounds, illustrated with reference to the enantiomers of a generic amino acid. The Shallenberger barrier is represented by the shaded wall of the box. The binary axis symmetry relationship is valid only for the AH and Β groups of the receptors.

SO-

3

3-anilino-2-styryl-3//-naphth ( 1,2-d) imidazolesulfonate

American Chemical Society Library Π55 16th St, N.W. Walters etO.C al.; Sweeteners Washington, 20038

ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

147


148

SWEETENERS:

DISCOVERY, MOLECULAR DESIGN, AND CHEMORECEPTION

AH

®

0 •

ι ; c ] ο Ν

\

#

s !

ο

H

!

AH

Figure 2. Models of the receptor active sites for sweet and bitter molecules, illustrated by the fit of 3-anilino-2-styryl-3Hnaphth(l,2-d) imidafcalesulfonate (a) and 4,5,6,7-tetraiodosaccharin (b), respectively.


11.

TEMUSSI ET Al»


point charge coulomb interaction (R* = 2.111 Â, ε = 0.202 kcal/mol, q = 0. a.u.). For the apolar part of the sweet receptor active site, a total of 13 and 25 S units as a first and second shell respectively have been initially positioned around the mentioned mold molecules. For the polar part of the receptor active site, i.,e the lower part around the SO3" moiety, we have optimized (still in the xy plane) the position of a negative oxygen at (R* = 2.20 Â, ε = 0.050 kcal/mol, q = -1.0 a.u.) and of a positive nitrogen (R* = 1.82 À, ε =0.055 kcal/mol, q = +1.0 a.u.). In this second step the positions of the previously optimized positions of the *S' units were kept fixed and the saccharin molecule position optimized with respect to the "receptor active site" derived by the SSN molecule. The upper hydrophobic part, corresponding to the area previously delimited by aspartame, although more rigorous than before (6), can only represent the minimum available area, since larger compounds were not available in this region. The main features of our receptor model can be summarized as follows: (i) the receptor active site of the receptor is a shallow, flat cavity with the outer side accessible even during interaction with the agonist (ii) the lower part of the cavity contains the main "electronic features", the most important of these being the AH-B entity; this part is always essential for binding (iii) the upper part of the cavity is hydrophobic and plays an important role in the modulation of sweetness intensity. A word of caution must be said about two points before attempting to use this model for drug design. The only electronic feature we used is the AH-B feature; it is very likely that other electronic features do contribute, in particular in a dynamic way, that is by inducing slight modifications in the region around the AH-B entity. Secondly, as we mentioned previously, the model gives no clues about the shape of the walls above the flat cavity: it is likely that they are funnel-shaped and, accordingly, it may be possible that compounds with substituents extending outside the flat cavity increase the binding constant; this can in fact be one of the reasons for the extraordinary activity of some supersweet compounds derived from aspartame [vide infra). Considering that a fundamental point in the formulation of this model resides in the symmetry relationship between sweet and bitter receptors, it is easy to complete the picture with a twin model for bitter molecules (Figure 2b) that has the AH-B entity inverted by a C2 symmetry operation and a two-dimensional contour almost identical to that for sweet molecules, with the only difference that it is slightly larger, as indicated by the fact that 4,5,6,7-tetraiodosaccharin retains a slightly bitter taste (7). Figure 3 shows the comparison of the fit of 5,6-benzosaccharin (a bitter saccharin) in the two active site models. It is clear that, although possessing a perfect AH-B entity, it can only fit the bitter active site model (Figure 3b). Figure 4 illustrates another puzzling experimental observation, hitherto unexplained by any other receptor model, i.e. the fact that some sweet molecules have o


149


SWEETENERS:



150

6-Br-saccharin



AH

Figure 3. Comparison of the fit of 5,6-benzo-saccharin in the sweet (a) and bitter (b) receptor models.


SWEETENERS:



152


11.

TEMUSSI ET AL.

Structure-Activity Relationship q

153

a strong bitter aftertaste. The fact that 6-bromo-saccharin can fit both receptor active sites represents a simple and satisfactory explanation of its bitter aftertaste. Most known sweet molecules, unrelated to the ones used for the mapping, do fit our model. Owing to space limitations we can only review some of them, divided into two sets: conformationally rigid and conformationally flexible tastants.


Conformationally rigid agonists One of the most interesting cases of dependence of sweetness on geometrical isomerism is that illustrated by Verkade (20) for the isomers of ethoxy-nitroanilines. Out of the ten possible isomers only one (2-ethoxy-5-nitroaniline) is sweet, while all others are tasteless. A good model for the sweet receptor active site ought to be able to discriminate among the ten isomers. It is certainly reassuring that only this isomer can fit our active site model while all others invade the wall outlined by the twodimensional contour or interfere with the AH-B entity . Figure 5 shows the fit of the sweet isomer (5d) in our active site model, together with three representative examples from the other isomers that do not fit. Equally significant is the fact that 2halogeno-5-nitroanilines are all sweet, with an increasing sweetness from F to I that parallels the fit of the halogen atom in the upper part of the model, and other 2-alkoxy-5-nitroanilines are sweet, as long as they can fit the upper part of the active site, whereas they lose all sweetness when the upper wall is invaded (20). Other interesting cases of rigid sweet molecules (I) are those of salicylic and anthranilic acids. These cases are readily explained by the simple application of the AH-B theory of Shallenberger and Acree (3), but it is impossible, on this basis, to explain why guaiacol carboxylic acid, whose chemical constitution is very similar, is bitter. Our model receptor active sites for the sweet and bitter receptors show that these are in fact trivial cases, since the first two compounds fit the sweet receptor whereas the third one can only fit the bitter receptor. Other clear-cut cases of rigid sweet molecules were already illustrated when the receptor model was only a partial geometrical model (6). Conformationally flexible agonists We have previously shown that our model active site model can be used to discriminate even among geometrical isomers of partially flexible tastants such as the three tolylureas (21). It is also easy to


SWEETENERS:


NH


2

NH \

OEt

OEt

NO

NO;

2-ethoxy-4-nitroaniline

2

2-ethoxy-3-nitroaniline OEt

OEt.

NH;

NH

Τ NO

2

3 -ethoxy-5 -nitroaniline

NO

2

2-ethoxy-5-nitroaniline


11.

TEMUSSI ET AIH


155

show that the taste of other ureas, either partially flexible (such as p-tolyl-N-methylurea or very flexible (such as suosan) can be interpreted on the basis of our model. The most interesting conformationally flexible sweet compounds however, are probably aspartame and its analogs. We have recently shown that all low-energy conformers of aspartame are characterized by similar extended backbone conformations that are compatible with only one (gauche) Asp side chain conformation (D in the notation of ref.J8) but with all three (quasi-isoenergetic) side chain conformations of the phenylalanyl moiety found in vacuo (IS), in solution (18) and in the solid state (22), i.e. F F and F , respectively. Figure 6 shows that only the first conformation (FJDJJ, Figure 6a) is consistent with our active site model, as originally proposed by the semi-quantitative receptor model (6,17,18). n


Ie

n

m

Moreover, the same conformation is the minimum energy conformation for the aspartame moiety of the N-(4-nitrophenylcarbamoyl)-L-aspartyl-L-phenylalanine methyl ester, one of the sweetest known compounds (23). This conformation was obtained from an energy minimization with respect to all internal torsional parameters for a total of 5832 conformations, using the AMBER force field (24). As starting conformations the set of most stable conformations obtained in the case of aspartame(J 7) and those obtained in the case of N-para-tolyl urea (21) were combined with the 12 most likely conformations deriving from the torsion around the C-N bond between the b carbon of Asp and the nitrogen of the urea moiety. As shown in Figure 7a, the aspartame moiety of this molecule fits perfectly into the flat cavity of the active site model, whereas the p-nitrophenylurea moiety contributes to the stabilization of the FjDn aspartame conformation with the formation of a C 7 ring, but remains completely outside the active site model, protruding through the open side of the receptor. Even more remarkable is the fact that the p-nitrophenylurea moiety can fit into the flat cavity as well. However, in the latter case, the aspartame moiety extends outside the active site model (Figure 7b). This kind of dual mode of interaction with of the active site (i.e. the entropie factor it implies) together with possible additional interactions with the walls of a funnel-shaped receptor active site (above the flat bottom cavity), may well be the reason for the exceptional sweetness potency of this molecule. It seems fair to conclude that the model receptor active sites we proposed for sweet and bitter agonists are, probably, the most general ones available at present and, in particular, the only models that can discriminate among sweet, bitter and tasteless isomers of several classes of tastants of widely different chemical constitution.


SWEETENERS:



156

AH

Β

Figure 5. Comparison of the fit of four of the ten isomers of nitro-ethoxy-aniline in the sweet receptor. The only sweet isomer is 2-ethoxy-5-nitroaniline (d).




TEMUSSI ET A L

AH

Β

Figure 5. Continued.


SWEETENERS:



158


TEMUSSIETJU^



11.


159

SWEETENERS: DISCOVERY, MOLECULAR DESIGN, AND CHEMORECEPTION


160

AH

Β

Figure 7. Comparison of the two possible interactions of the low-energy conformer of the N-(4-nitro-phenylcarbamoyl)-Laspartyl-L-phenylalanine methyl ester with the sweet receptor. Walters et al.; Sweeteners ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

11.

TEMUSSI ET A L


161

Acknowledgments P . A . T . w i s h e s to t h a n k Vittorio B o n g i o r n o of G l u e M o o n A r t S t u d i o (Naples, Italy) w h o , w i t h e n o r m o u s patience, m a n a g e d to c h a n g e h i s r o u g h sketches into beautiful art work.

Literature Cited


1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Moncrieff, R.W. The Chemical Senses; Hill: London, 1967. Cohn, G. Die Organischen Geschmackstoffe; Siemenroth: Berlin, 1914. Shallenberger, R.S.; Acree, T. Nature (London) 1967, 216, 480-482. Shallenberger, R.S.; Acree T.; Lee, C.Y. Nature (London) 1969, 221, 555-556. Kier, L.B. J . Pharm. Sci. 1972, 61, 1394-1397. Temussi, P.A.; Lelj, F.; Tancredi,T. J.Med. Chem. 1978, 21, 1154-1158. Tancredi, T.; Lelj, F.; Temussi, P.A. Chem. Senses and Flavor 1979, 4, 259-264. Temussi, P.A.; Lelj, F.; Tancredi,T.; Castiglione-Morelli, M.A.; Pastore,A. Int. J.Quantum Chem. 1984, 26, 889-906. van der Heijden, Α.; Brussel, L.B.P.; Peer, H.G. Food Chem. 1978, 3, 207-213. Iwamura, H. J . Med. Chem. 1981, 24, 572-578. Goodman, M.; Coddington, J.; Mierke, D.F. J.Amer. Chem. Soc. 1987, 109, 4712-4714. Walters, D.E.; Pearlstein, R.A.; Krimmel, C.P. J. Chem. Ed. 1986, 63, 869-871. Solms, J . J.Agr.Food Chem. 1969, 17, 686-688. Petrischeck, Α.; Lynen,F.; Belitz, H.D. Dtsch. Forsch. Lebensmittelchem. 1972, 5, 47-51. Zaffaroni, A. U.S. Patent 3 876 816, 1975. Van der Wel, H.; Arvidson, K. Chem. Senses Flavor 1978, 3, 291-297. Lelj, F.; Tancredi, T.; Temussi, P.A.; Toniolo, C. J. Am. Chem. Soc. 1976, 98, 6669-6674. Castiglione-Morelli, M.A.; Lelj, F.; Naider, F.; Tallon, M.; Tancredi, T.; Temussi, P.A. J.Med.Chem. 1990, 33, 514-520. Ciajolo, M.R.; Parrilli, M.; Temussi, P.A.; Tuzi, A. Acta Cryst. 1983, C39, 983-984. Verkade, P.E. Il Farmaco Ed. Sci. 1967, 23, 248-291. Ciajolo, M.R.; Lelj, F.; Tancredi, T.; Temussi, P.A.; Tuzi, A. J.Med.Chem. 1983, 26, 1060-1064. Hatada, M.; Jancarik, J.; Graves, B; Kim, S. J. Am. Chem. Soc. 1985, 107, 4279-4282. Tinti, J.-M.; Nofre, C. Fr. Demande FR 2 533 210, 1984; Chem. Abstr. 1984, 101, 152354k. Weiner, S.J.; Kollman, P. Α.; Nguyen, D.T.; Case, D. A.J.Comp. Chem. 1986, 7, 230-242.

RECEIVED August 27, 1990 Walters et al.; Sweeteners ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

StructureâActivity Relationship of Sweet Molecules - ACS Symposium

Recommend Documents

StructureâActivity Relationship of Sweet Molecules - ACS Symposium