Three-Dimensional Model for the Sweet Taste Receptor - American

Kare, M.R., Eds.; Academic: New York, 1981; pp 175-203. 2. Price, S.; DeSimone, J.A. Chem. Senses Flavour 1977, 2, 427. 3. Shimazaki, K.; Sato, M.; Na...
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Chapter 16

Three-Dimensional Model for the Sweet Taste Receptor Development and Use J . Chris Culberson and D. Eric Walters

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The NutraSweet Company, Mt. Prospect, I L 60056

A model has been developed to aid in understanding the structure-taste relationships of dipeptides, guanidines, urea-dipeptides and related compounds. This model was built using conformational analysis and electrostatic potential calculations on a few highly potent representative compounds. It is consistent with known structure activity relationships in these series of compounds, and has been used to (1) design new potent analogues of known sweeteners; (2) identify structural and electronic features leading to bitterness; (3) design inhibitors of sweetness which may be useful in classifying sweeteners on the basis of receptor type. It is currently being used to design novel sweeteners. The t r a n s d u c t i o n of sweet taste i s believed to b e initiated b y receptor protein(s) located o n t h e surface o f the taste cell. T o date no receptor has been isolated, although m a n y attempts have been m a d e (1-3). G i v e n t h e l a c k o f a n i s o l a t e d r e c e p t o r p r o t e i n , m o d e l s of t h e sweet taste r e c e p t o r m u s t b e i n f e r r e d f r o m t h e a v a i l a b l e s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s (SAR). Models of t h e sweet taste receptor have g r o w n i n their complexity over time a s n e w sweeteners have been discovered. Shallenberger a n d Acree were t h e first to t r y to e x p l a i n sweetness i n terms of a model. Their model contains two r e c o g n i t i o n u n i t s , a h y d r o g e n b o n d d o n o r (AH) a n d a h y d r o g e n b o n d a c c e p t o r (B) l o c a t e d f r o m 2 . 5 t o 4 . 0 a n g s t r o m s a p a r t (4). T h i s model allows one to rationalize t h e sweet taste of most sweet c o m p o u n d s , b u t a large n u m b e r of non-sweet c o m p o u n d s w o u l d b e predicted to b e sweet b y this model. Shallenberger a n d coworkers l a t e r a d d e d a " s t e r i c b a r r i e r " t o t h e i r m o d e l (5) t o a c c o u n t f o r t h e sweetness of D b u t n o t L amino acids. E v e n w i t h this additional constraint, t h e m o d e l c a n not b e u s e d to reliably predict n e w sweet

0097-6156/91/045O-0214$06.00/0 © 1991 American Chemical Society

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

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compounds. The Shallenberger model h a s been used as a starting p o i n t for several other models. K i e r (6) a d d e d a " d i s p e r s i o n b i n d i n g " o r h y d r o p h o b i c site, 3.5 a n g s t r o m s f r o m t h e A H site a n d 5.5 a n g s t r o m s from the Β site. A s the n u m b e r of sweetener classes h a s increased, so h a s the optimal length of the h y d r o p h o b i c region. V a n d e r H e i j d e n a n d c o w o r k e r s (7) c l a i m t h e o p t i m a l A H t o h y d r o p h o b i c site length to be 10.8 a n g s t r o m s . M a z u r e t a l . (8) n o t i c e d t h e s w e e t n e s s o f a s p a r t i c a c i d b a s e d sweeteners depends o n the stereochemistry a n d size of the substituents o n the carbon atom attached to the amide nitrogen. Sweetness occurs w h e n the groups are arrayed as s h o w n i n Figure 1. Potencies are maximized w h e n the largest substituent is hydrophobic a n d polarizable. These observations led several groups to f u r t h e r investigate t h e s t r u c t u r a l r e q u i r e m e n t s of t h e large h y d r o p h o b i c g r o u p (9-11). T e m u s s i a n d coworkers selected a conformation of aspartame w h i c h t h e y b e l i e v e to b e t h e a c t i v e (sweet) o n e , u s i n g N M R a n d c o n f o r m a t i o n a l e n e r g y c a l c u l a t i o n s [12-14). Based on this conformation, they have developed a receptor site m o d e l . This m o d e l i s consistent w i t h the majority of peptide b a s e d S A R b u t w a s f o u n d to be i n c o n s i s t e n t w i t h NutraSweet i n t e r n a l S A R ( D . E . Walters a n d D . A . J o n e s , u n p u b l i s h e d observations) . T h e T e m u s s i N M R d a t a w a s r e - i n t e r p r e t e d b y v a n d e r H e i j d e n et a l . (15) to p r o d u c e a m o d e l c l o s e t o t h e o n e d e s c r i b e d b y K i e r (6) w i t h a h y d r o p h o b i c g r o u p c o n s i s t e n t w i t h t h e w o r k of B r u s s e l (9). Unfortunately, v a n der Heijden's work yields a model that is n o t c o n s i s t e n t w i t h t h e observed sweetness of a s p a r t y l a n i l i d e s (16), t r i f l u o r o a c e t y l - a s p a r t a m e (17), a n d t h e a r y l u r e a - d i p e p t i d e s ( I S ) . M a n y m o d e l s for the sweet taste receptor have been p r o p o s e d , b u t a l l of t h e m o d e l s are l i m i t e d i n t h e i r v a l u e a s tools to design n e w sweeteners. M a n y of the models are two d i m e n s i o n a l i n nature, a n d most do not a c c o u n t for the n o n - l o c a l charge distribution. In the work summarized i n this paper, we have constructed a model w h i c h is consistent with S A R of the dipeptide a n d related sweeteners. T h e model is inherently three-dimensional a n d d o e s a c c o u n t for n o n - l o c a l e l e c t r o n i c effects. I n a d d i t i o n to rationalizing k n o w n S A R , we will s h o w the u s e of this m o d e l i n a predictive a n d sweetener design mode. Development of the Model The c o n s t r u c t i o n of o u r model c a n be divided into four phases. T h e first p h a s e w a s to select appropriate c o m p o u n d s u p o n w h i c h to base the model. S e c o n d , conformational a n a l y s i s w a s done to identify low-energy conformers of these c o m p o u n d s . The next p h a s e involved s u p e r i m p o s i n g l o w energy conformers of the c h o s e n c o m p o u n d s i n s u c h a w a y a s to give a c o m m o n p a t t e r n of f u n c t i o n a l g r o u p s . T h e f i n a l step w a s to p a r t i t i o n t h e m o d e l i n t o m o l e c u l a r recognition sites.

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CHEMORECEPTION

C o m p o u n d s e l e c t i o n . It i s n o t c l e a r w h e t h e r t h e r e i s a s i n g l e s w e e t t a s t e r e c e p t o r o r m u l t i p l e r e c e p t o r s (19). We chose compounds w h i c h we believe m a y act at a c o m m o n site since t h e y have s i m i l a r molecular recognition units (carboxylate group, several a m i n e / a m i d e groups, a large h y d r o p h o b i c group). In addition, we chose c o m p o u n d s w i t h h i g h potencies, since we expect such c o m p o u n d s t o h a v e b e t t e r fit t o t h e r e c e p t o r s i t e . C o m p o u n d s 1-5 s h o w n i n F i g u r e 2 were selected as s t a r t i n g p o i n t s for o u r m o d e l . Compounds 1 a n d 2 are guanidine-acetic a c i d derivatives d i s c o v e r e d b y Nofre a n d T i n t i (20). C o m p o u n d 3 is a n arylurea derivative of a s p a r t a m e , a l s o d i s c o v e r e d b y Nofre a n d T i n t i (IS). T h e a s p a r t i c fenchyl ester derivative 4 is the m o s t potent of the a s p a r t i c - a m i n o m a l o n y l d i e s t e r s d e s c r i b e d b y F u j i n o (10). F i n a l l y , w e i n c l u d e d a s p a r t a m e , 5 (8), a s t h e p r o t o t y p e c o m p o u n d i n t h i s series. C o n f o r m a t i o n a l analysis. In o u r initial work, we u s e d the M M F F ( m o l e c u l a r m e c h a n i c s f o r c e field) p r o g r a m o f C h e m l a b (21). T h i s i s a m o d i f i e d v e r s i o n of A l l i n g e r ' s M M 2 p r o g r a m (22). W e g e n e r a t e d force field p a r a m e t e r s w h e r e n e c e s s a r y (urea a n d g u a n i d i n i u m g r o u p s ) u s i n g t h e m e t h o d of H o p f i n g e r a n d P e a r l s t e i n (23). Later calculations were done u s i n g Still's M a c r o M o d e l program (24), a g a i n u s i n g p a r a m e t e r s we g e n e r a t e d for u r e a a n d g u a n i d i n i u m groups. A w o r k i n g a s s u m p t i o n was made that the "active" c o n f o r m a t i o n s h o u l d be n e a r a l o c a l energy m i n i m u m (not necessarily the global m i n i m u m ) . A n o t h e r a s s u m p t i o n w a s that carboxylic acid, amine, a n d guanidine groups exist i n the ionic form in aqueous solution. S u p e r i m p o s i t i o n of conformers. The high-potency guanidine s w e e t e n e r s o f N o f r e a n d T i n t i (20) w e r e p a r t i c u l a r l y u s e f u l i n i d e n t i f y i n g t h e b e s t w a y to s u p e r i m p o s e t h e c o m p o u n d s i n o u r series. W h e r e a s dipeptides s u c h as a s p a r t a m e have h u n d r e d s of accessible low energy conformers, the p l a n a r g u a n i d i n i u m group l i m i t s the n u m b e r of conformers available. We calculated the e l e c t r o s t a t i c p o t e n t i a l (EP) f o r e a c h o f t h e s e m o l e c u l e s t o f u r t h e r aid i n the conformation selection process. The E P provides a m e a n s of u n a m b i g u o u s l y overlaying s t r u c t u r e s t h a t do n o t have identical functional groups. The E P was calculated u s i n g a point charge approximation. The charges were obtained u s i n g the I N D O / S m e t h o d (25). Low energy conformers were s u p e r i m p o s e d v i s u a l l y o n a c o m p u t e r g r a p h i c s s c r e e n to provide o p t i m u m steric overlap a n d electrostatic m a t c h i n g . Finally, a v a n der W a a l s surface over the s u p e r i m p o s e d c o m p o u n d s w a s generated, with a composite E P m a p p e d onto the surface. M o l e c u l a r r e c o g n i t i o n sites. T h e u s e of E P c a l c u l a t i o n s a l l o w s for the l o g i c a l d i v i s i o n of the m o d e l i n t o several regions. The first

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Small L-Asp-NH-

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Medium Large F i g u r e 1. S t e r e o c h e m i s t r y o f L - a s p a r t y l - d e r i v e d s w e e t e n e r s

0-Y-CV Ιί J

NH

Cr

Ν

-OOC—/

°θγ-0 -OOC -nor.

NH /

P (2) « 200,000

P (2) = 120,000

w

w

0

/

-OOC P (2) « 7,800 w

o +H

V ^ N ' 4 H -OOC^ 4 3

Ο

"COOCH

P ( 2 ) « 50,000 w

COOCH

3

à

3

H

-OOC' 8

P (2) = 180 w

Figure 2. Compounds used as templates for the model.

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

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CHEMORECEPTION

region is dominated b y a n acidic group defined b y the carboxyl g r o u p s of the m o d e l c o m p o u n d s . R e g i o n two, the m a j o r N H site, i s d e f i n e d b y t h e free a m i n e of a s p a r t a m e a n d t h e a r y l - N H of t h e guanidine sweeteners. A second N H site is defined b y the dipeptide sweetener's a m i d e N H a n d the h y d r o p h o b i c N H of the guanidine sweeteners. A large h y d r o p h o b i c region d o m i n a t e s one side of the m o d e l a n d its s h a p e is quite w e l l defined. T h e m e t h y l esters of the d i p e p t i d e sweeteners define the ester r e g i o n of t h e model. T h e f i n a l r e g i o n of the m o d e l i s c h a r a c t e r i z e d b y a n electron deficient aryl ring i n the guanidine a n d arylurea-dipeptide sweeteners. We designate this region the pi-stacking region. F i g u r e 3 s h o w a s c h e m a t i c r e p r e s e n t a t i o n of the m o d e l w i t h the parts described above labeled. A p p l i c a t i o n s of t h e M o d e l W e p r o c e e d e d to t e s t t h e m o d e l to see w h e t h e r i t i s c o n s i s t e n t with k n o w n structure activity relationships i n the aspartic dipeptides, aspartic amides, aspartic anilides, arylurea-dipeptides, a n d g u a n i d i n e c l a s s e s . T h e m o d e l is c o n s i s t e n t w i t h a l l of t h e s e classes. C o m p o u n d s w h i c h have the correct p a t t e r n of E P h a v e a sweet taste. C o m p o u n d s w h i c h effectively fill the h y d r o p h o b i c r e g i o n t e n d to h a v e t h e h i g h e s t p o t e n c i e s ; c o m p o u n d s w h i c h o n l y partially occupy the hydrophobic region a n d c o m p o u n d s w h i c h e x t e n d b e y o n d t h e b o u n d a r i e s of t h i s r e g i o n t e n d to h a v e l o w potencies or non-sweet tastes. C o m p o u n d s w h i c h have a n electrond e f i c i e n t a r o m a t i c r i n g i n t h e p i - s t a c k i n g r e g i o n t e n d to h a v e enhanced potencies. F i g u r e 4 illustrates the case of a n a s p a r t y l a n i l i d e , t r i c h l o r o a c e t y l - L - a s p a r t y l - p - c y a n o p h e n y l a n i l i d e 6. The s t e r i c fit i n t o t h e m o d e l i s q u i t e g o o d . T h e e l e c t r o n i c fit i s a l s o quite good s i n c e the m o l e c u l a r r e c o g n i t i o n sites are close to c o r r e s p o n d i n g regions of the m o d e l .

HOOC

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T o t e s t t h e p r e d i c t i v e v a l u e of the m o d e l , w e m a d e p r e d i c t i o n s of t h e s w e e t n e s s of c o m p o u n d s 7 a n d 8 . B a s e d o n i t s g o o d steric a n d e l e c t r o n i c fit, w e e x p e c t e d c o m p o u n d 7 to h a v e a r e l a t i v e l y h i g h sweetness potency. O n the other h a n d , the i s o p r o p e n y l g r o u p of c o m p o u n d 8 extends w e l l b e y o n d the steric b o u n d a r y of the m o d e l a n d w a s therefore n o t expected to be v e r y p o t e n t l y sweet. C o m p o u n d 7 w a s s u b s e q u e n t l y f o u n d to h a v e a p o t e n c y of > 2 0 , 0 0 0 times s u c r o s e at a 2 % s u c r o s e sweetness level; c o m p o u n d 8 w a s

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NH-sites F i g u r e 3. T h r e e o r t h o g o n a l v i e w s of the v a n der W a a l s s u r f a c e of the receptor site model, w i t h recognition sites labeled.

f o u n d to h a v e a p o t e n c y of < 1 0 0 t i m e s s u c r o s e . We have c o n s i s t e n t l y f o u n d the s p a t i a l r e q u i r e m e n t s of the h y d r o p h o b i c r e g i o n to be r a t h e r w e l l defined.

T h e i m p o r t a n c e o f t h e E P fit i s i l l u s t r a t e d b y c o m p o u n d s 9 a n d 1 0 . T h e s e c o m p o u n d s b o t h fit t h e m o d e l w e l l s t e r i c a l l y b u t have a n a r o m a t i c nitrogen a t o m near the major N H site of the receptor model. The lone pair electrons o n the r i n g nitrogen atom d r a m a t i c a l l y i n f l u e n c e the E P of the adjacent a r y l - N H g r o u p . C o m p o u n d s 9 a n d 10 are q u i t e b i t t e r , i n c o n t r a s t to t h e i r c a r b o n analogs w h i c h are intensely sweet. In general, c h a n g e s i n the E P a r o u n d the major N H site result i n bitter-tasting c o m p o u n d s .

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F i g u r e 4. Trichloroacetyl-L-aspartyl-p-cyanophenyl anilide i n the receptor model.

9

10

W e expected t h a t the r e m o v a l or m o d i f i c a t i o n of c e r t a i n r e c o g n i t i o n e l e m e n t s from a sweet c o m p o u n d w o u l d l e a d to sweet taste i n h i b i t o r s , c o m p o u n d s w h i c h c o u l d b i n d to t h e r e c e p t o r site b u t n o t trigger the sweet response. T h i s prediction w a s b o r n out i n c o m p o u n d s 11 a n d 12 . R e m o v a l o f t h e a m i n o g r o u p o f a s p a r t a m e (5) l e d t o c o m p o u n d 1 1 , s u c c i n y l - L - p h e n y l a l a n i n e m e t h y l e s t e r , w h i c h b l o c k s the sweet taste of a s p a r t a m e . C h a n g i n g t h e n a t u r e of the a c i d i c g r o u p also p r o d u c e s a c o m p o u n d t h a t effectively b l o c k s s w e e t t a s t e . C o m p o u n d 12 (26) i s a n a n a l o g o f t h e s w e e t c o m p o u n d 13 (27), i n w h i c h o n e m e t h y l e n e g r o u p h a s b e e n r e m o v e d a n d t h e carboxylate group is replaced b y a sulfonic acid.

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In a n o t h e r i n s t a n c e , the m o d e l w a s u s e d to i m p r o v e the p o t e n c y of a k n o w n s w e e t e n e r . C o m p o u n d 13, a n a n a l o g o f s u o s a n s y n t h e s i z e d b y Nofre et a l . (18), l a c k s a n y f u n c t i o n a l g r o u p s to f i l l t h e h y d r o p h o b i c g r o u p of the m o d e l . T h e a d d i t i o n of a p h e n y l g r o u p to c o m p o u n d 13 a t t h e c a r b o n b e t a to t h e c a r b o x y l a t e g r o u p y i e l d s c o m p o u n d 14 (28). B y o c c u p y i n g the h y d r o p h o b i c region of the m o d e l , a n i n c r e a s e i n p o t e n c y of one o r d e r of m a g n i t u d e w a s o b t a i n e d (see C h a p t e r 2 2 ) .

13 14 T h e m o d e l h a s a l s o b e e n u s e d to d e s i g n n e w l e a d c o m p o u n d s , w h i c h are n o t s i m p l e m o d i f i c a t i o n s of k n o w n s w e e t c o m p o u n d s . C o m p o u n d 15 w a s d e s i g n e d u s i n g the m o d e l a n d f o u n d to be moderately sweet.

Summary

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W e h a v e c o n s t r u c t e d a m o d e l of the sweet taste receptor u s i n g m o l e c u l a r d e s i g n t e c h n i q u e s . T h i s a l l o w s u s to define b o t h steric a n d e l e c t r o n i c c r i t e r i a n e c e s s a r y for s w e e t n e s s w i t h i n the dipeptide a n d g u a n i d i n e series. T h e m o d e l h a s b e e n a p p l i e d to the r a t i o n a l i z a t i o n of k n o w n S A R , the d e s i g n of h i g h e r p o t e n c y a n a l o g s

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of k n o w n sweeteners, a n d t h e d e s i g n o f n o v e l sweeteners. T h e m e t h o d of model construction outlined above c a n b e applied to other classes of sweeteners a s well, i n order to assist i n the design of n e w sweeteners.

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16. CULBERSON & WALTERS

3-D Model for the Sweet Taste223 Receptor

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