symposium: sweeteners and sweetness theory
Using Models to Understand and Design Sweeteners D. Eric Walters
De~aItmentof Bioloaical chemist^. .. Finch Universitv of Health Sciences The Chicago Medical School, ~ o A hChicago, IL 66064 The subject of sweeteners has appeared i n the Journal of Chemical Education many times before ( 1 4 ) .Perhaps this is because sweeteners provide a link between chemical structure and biological activity to which everyone can easily relate. Receptors (proteins which recognize chemical signals and initiate some biological response) are often just a n abstract concept. You are not aware of each neurotransmitter-receptor interaction i n your nervous system. But when you put something sweet into your mouth, you can directly feel the stimulation of your receptors. You can experience what i s meant by a threshold concentration (the minimum concentration which causes a response). You can sense receptor saturation (at some point, increasing sweete n e r concentration no longer increases t h e perceived sweetness). You can feel exactly what adaptation is (the second sip of a soft drink is not a s sweet a s the first). For those of us who study the relationship between chemical structure and biological activity, sweet taste receptors are interesting because they a r e so readily accessible-you don't have to worry about whether the compound was absorbed from the digestive system, metabolized along the wa): or exrreted horn the body belbre getting to a receptor. This circurn\~mtsmany of the complirating (kctori that may plague other structure-activity kudies.Sweeteners a r e a challenging problem because of the great structural diversity of compounds t h a t may taste sweet (see the accompanyingpaper by Ellis for some exam-
680
Journal of Chemical Education
ples). Therefore, models have been used frequently a s tools in understanding how chemical structure relates to sweet taste. Models generally are made in an effort to simplify or abstract the key features of a problem, i n order to make the problem more readily understandable. Models often provide u s a new perspective, a different way to think about our problems. Models help u s to think of experiments that will test our ideas; these experiments may indicate that a model is altogether wrong, or that i t needs more or less modification. The statistician George Box reminds us that "all models are wrong, but some are useful." Utility, not "correctness," i s the real criterion by which we judge models. In this paper, I will review the variety of ways in which models have been used to help u s understand the relationship between chemical structure and sweet taste. In many cases, these models have been useful either i n allowing us to design new sweet-tasting structures or in enabling us to estimate just how sweet a new compound is likely to be. There are many types of models to be considered. Some models have attempted to be comprehensive, that is, to incorporate every known structural type known to be sweet into one receptorlrecognition site. Other models are more limited, starting from the assumption that there may be more than one receptor type involved in sweet taste. In these cases, the authors select a series of structures which they believe are likely to all act a t a common receptor.
We also may differentiate betweenpharmacophore models and receptor site models. A pharmacophore is a collection of key structural features that are considered to be essential in receptor activation. The pharmacophore is based on the structure of the sweetener molecules. It is assumed to be complementary in its size, three-dimensional shape, and electronic charge distribution to the receptor site with which i t interacts. Because we usually cannot directly look a t the structure of the receptor site, we may infer what the receptor site i s like by looking a t the structures of compounds t h a t activate it. If we think of the sweetener molecules a s "keys" and the receptor a s a "lock," a pharmacophore model i s made u p of the features common to "keys" which work; we make reasonable guesses about what the inside of the lock i s like, based on our examination of the "keys" that fit and those that do not. Tllui, wt: ma" make mudrls thnt w e composites of thc fc:itures of actlw. compwntl.+ the pharmacophure or we nin\ make models of the site to which the active compounds bind (the receptor). Finally, we can differentiate between statistical models and structural models. I n the case of statistical models (commonlv referred to a s Quantitative Structure-Activitv ~ e l a t i o n s h i ~QSAR), s, we lbok for a statistical corre~atioh between physicalichemical properties and the measured biological activity. In structure-based models, we look for the three-dimensional shape. electronic charge distribution, and placement of hydrogen bonds that produces the desired biological response.
two hydrogen bonds when the sweetener interacts with its receptor. Although this is a very simple model and there are now numerous known exceptions, i t served the verv important function of stimulating thoLght about the way sweeteners work. Shallenberger, Acree, and Lee (10) refined the model by adding a "steric barrier" to account for the observation that many D-amino acids are sweet, while the corresponding L-amino acids are not. Kier (11) added to the AH-B pair a~dispersionbinding site, 3.5 A from the AH group and 5.5 A from B. Van der Heijden and co-worke r s (12, 13) recalculated distances between these three sites for several structural classes of sweeteners and estimated that there may be four different receptor types, distinguished hy different distances among the three functional groups. While there are many reasons to susuect that a comureht.n*ivt single model ibr 311 known .;\rt:rrmrrs is unrenllstic Id,, we must note rhe tremendous :iucct!si which Nolre and co-workers have had in constructing and utilizing such a model. Starting from the AH-Bisteric barrier model, they mapped out a second steric barrier (151, an interaction site for nitro or cyano groups (16), and a site for binding of trifluoroacetamide groups (17). They scored a major breakthrough by comb in in^ two known sweeteners of moderate potency to form a new compound with remarkably higher potency (181, a s shown in Figure 1. (Measurement of sweetener potency involves dilution of a sweetener until a concentration is found that matches the sweetness of a given concentration [e.g., 2951 of sucrose, a s judged by a trained human panel; see reference 1 9 for details). Ultimately they derived a n eight-site model (20) that i s illustr:~red in the nccompanylng p:ipvr by k:.lis. Csing this m d d :ind t h t s ~ ~ p e r a s p n ~ t i*tructurt!, ~nat~ 'lynti and S o f w wc.1-t! ablt: to dt.iign hwanid~ne-acetic acid dcriv,~tiws.with potetac!t.~up 10 200,0011 tames that ol'sucroic!
Comprehensive Models The first comprehensive attempt to relate structure to sweetness was in a book written by Georg Cohn in 1914, entitled Die Organischen Geschmacksstoffe, which translates roughly to "The Taste of Organic Compounds" (6).In the 19th and early 20th centuries, chemists routinely described the compounds they synthesized in great detail, WAR: Statistical Models and taste was one of the common wavs to evaluate compounds (acids are sour, nitrogen-containing compounds In 1964, Hansch and Fujita (21) introduced the concept are often bitter, etc.). (Don't trv this a t home!) of QSAR (statistical correlation of physicochemical paCohn filled over 900 pages w"itb the structures of organic rameters with biological activities). Shortly afterward, compounds and their associated tastes. In addition to cataDeutsch and Hansch applied the method to a series of logu;tag thc n.rults of thouwnds of rxperim(mti. h~ made sweet nitroanilines (22). They observed a strong correlathe lirst att(vnpt to f'xtor out the eftccts of .ivrclfic futlrtion between t h e octanollwater partition coefficient ( a tional groups. ~ o h identified n hydroxyl and amino groups measure of hydrophobicity) and sweetness potency. In a as "sapophoric," and noted that they often occur i n pairs in h a n eve'better corresubsequent paper i23), ~ a k found sweet compounds. Five years later, Oertly and Myers ( 7 ) lation when aromatic substituent resonance was taken attempted to refine these observations, classifying some into account. Since then, many others have looked for stastructural features as "glucophores" and others as "auxotistical correlations between structure and sweetness in glucs." They postulated t h a t any glucophore plus any several classes of sweeteners. The table summarizes the auxogluc would produce a sweettasting compound. They noted, though, t h a t their theory was only a starting point and t h a t there were many exceptions to their rules. By f a r t h e most well-known model for explaining sweet taste Cyano-suosan.450 x sucrose is the theory of ShallenCOOCH3 herger and Acree (81, illustrated in t h e accompanying paper by Superaspartame,14.000 x sucrose Ellis (9). This model suggests t h a t a l l s w e e t - t a s t i n ~compounds contain a hydrog& bond +H,N*~ donor group (AH) and a hydro0 COOCH3 gen bond acceptor (B), separated by a distance of 2.5 to 4.0 AngAspartame, 200 x sucrose stroms. They inferred t h a t the Figure 1. Cyano-suosanand aspartame, combined along the common amino-propanoic acid segment receptor must contain a comple- (bold),produce the compound superaspaltame,with substantially higher potency. This discovey led to mentary B-AH pair t h a t forms the guandine-acetic acid compounds such as sucrononic acid with extremely high potencies.
Volume 72 Number 8 August 1995
681
Summary of Important QSAR Studies of Sweeteners
Structural Class
Number of Compounds
Key Parameters
Reference
Nitroanilines
9
n
22
Nitroanilines Asparlyl-dipeptides
10
ws+
33
P, f, VS
23 24
38
n,VS
25
Oximes
a n d s h a p e of t h i s p a r t of t h e molecule. Brussel and co-workers (35)made space-filling CPK models of 28 analogs and measured their dimensions. They also measured t h e volumes by immersing the models in graduated cylinders filled with liquid. They found highest potency when the length of the large s u b s t i p e n t
was between 4.8 a n d 8.8 A and its volume was t 29 A3. Fujino's o' VS 26 Asparlyl-dipeptides 21-72 -erouo. a t Takeda Chemical Industries (36) m a d e 2 1 analogs 27,28 Sulfamates and determined that the hvdron = log (octanollwaterpartition coefficient): d = Brown electronic substituent constant; P = parachor, a function of phobic group should have dimensurface tension, density. and molecular weight: f = Rekker fragmental constant. another measure of hydrophobicity sions Of 7 7 6 A, ~h~~ dis. (2q:VS = Verloop's Stetimol Substituentsize parameters (30):o =Tan polar substituentconstant: xyz = dimensions covered analogs with potencies measured from CPK models: V = substituent volume. UD to 100 times t h a t of asDartame. Van der Heijden a n d comajor parameters t h a t have Proven important in these workers (241,in their QSAR study of aspartic peptides, esstudies. We see t h a t hydrophobicity and size of substitimated the tyo-dimensional size of the hydrophobic retuents are important in almost every case. pion to be 5.9 A x 7.2 A. Iwamura's QSAR studv 1261 indicated a somewhat smaller hydrophokc region,k8 A x 3.7 Limited Pharmacophore Models A x 5.5 A. We are all like the blind men and the elephantBy limiting our attention to a set of structures that have what we are able to discern of the receptor depends on the some similarity to one another, we increase the likelihood that we are probing the nature of a single binding site. By far, the most extensive pharmacophore modeling of sweeteners h a s involved analogs of the dipeptide sweetener aspartame (Fig. 2; reference 31). This sweetener, sold under the trade name NutraSweet, was discovered acciden0 tally by J i m Schlatter a t G. D. Searle Co. in 1965 i n the course of some research on anti-ulcer drugs (32). Since that Aspartame, time, over 1000 analogs of aspartame have been syntheL-Asp-L-Phe-OCH3 sized and tested. Because i t is a peptide, aspartame is metabolized in the Figure 2. The dipeptide sweetener asparlame. body just a s any other protein would be. Like sugars and proteins, i t provides about four calories per gram. Its utility as a low-calorie sweetCC+ Ccu ener stems from the fact that i t is about 200 times a s potently sweet a s sucrose (on a weight basis). I n t h e course of exploring structure0 CH3 t a s t e relationships of a s p a r t a m e analogs, O large Mazur's group a t Searle began t o discover some of the structural requirements for highAlitame potency sweetness (33). Sweetness generally was maintained when the ~ . ~ h ~ Figure ~ 3.~(a) The l stereochemistryof ~ l ~ ~ thei small-medium-large ~ ~ substituent panem on was replaced with other ~ . acids having ~ ~ the carboni adjacent ~ to the amide ~ nitrogen in aspartic amides. (b) An example of the ~arge~ydropho~icside~chains,aslongas thees- high-potency sweetener (alitame,2,000 x sucrose) that uses this pattern. t e r group was small (e.g., methyl). Alternatively, they could use D-amino acids with small side-chains (e.g., D-alanine, methyl side chain) if they used larger ester groups. Thus, they found t h a t t h e substitution p a t t e r n a t t h e phenylalanine alpha-carbon needed to be a small group (e.g., -HI, a medium-sized group (e.g., -CH3 or -[COOCH3), and a large group (e.g., -CHz-phenyl or-COO- propyl). This simple stereochemical model (Fig. 3aj has led to the discovery of numerous analogs of aspartame with even higher potencies. One of these, alitame (Fig. 3b; reference 341, was submitted for FDA approval i n 1986. Alitame employs a Damino acid with a small side chain (D-alanine) -o and a large amide substituent (tetramethylthietane). Its potency i s about 10 times that of aspartame (2,000 times sucrose). Figure4. Schematicviewof the receptor site model derived by Temussi and coworkers How "large" should the large substituent be? (37.38).(b) Schematicview of the receptor site model derived by van der Heijden and Several groups set out to find the optimum size CO-workers(39). Nitroanilines
20
VS
25
-
a a
682
Journal of Chemical Education
a
