Why Does a Sweetener Taste Sweet? - American Chemical Society

Chapter 15

Why

Does a Sweetener Taste Sweet? A New Model

Downloaded by UNIV OF MASSACHUSETTS AMHERST on February 25, 2016 | http://pubs.acs.org Publication Date: December 31, 1991 | doi: 10.1021/bk-1991-0450.ch015

Jean-Marie Tinti and Claude Nofre Faculté de Médecine Alexis Carrel, Université Claude Bernard, Rue Guillaume Paradin, 69008, Lyon, France

A model illustrating the molecular features which allow a molecule to initiate a sweet sensation in man is described. This model is mainly deduced from structure-activity relationships between new series of intensely sweet compounds recently discovered in our Laboratory. The model assumes the existence in the sweet receptors of eight recognition sites capable of identifying eight optional interaction sites in sweeteners, designated as AH, B, G, D, Y, XH, E and E . The simultaneous binding of two of these sites can generate a sweet response, but four interactions (via sites AH, B, G and D) are necessary to induce extremely potent sweet activities. 1

2

G i v e n that various attempts to find c o m m o n stereochemical f e a t u r e s b e t w e e n s w e e t s u b s t a n c e s h a v e a l r e a d y b e e n m a d e (1-4), the proposed models have been too simple to explain t h e activity of complex molecules. T h e y a r e either n o t specific e n o u g h (including too large a n u m b e r o f c o m p o u n d s , even n o n sweet molecules) o r too specific (relevant to o n l y a few active molecules) to provide a reliable predictive value. T h e most widely accepted a n d relevant m o l e c u l a r theory i n t h e field o f sweet taste i s t h a t p u t forward i n 1963 b y Shallenberger a n d Acree (i,2) w h o proposed the existence of a n A H , Β s y s t e m i n sweeteners, Η b e i n g a n acidic h y d r o g e n linked to A , w i t h A a n d Β being two electronegative atoms separated b y 0.28-0.30 n m . I n this theory, A H w a s O H o r N H , Β a n oxygen a t o m i n groups s u c h a s C 0 H , S O 3 H , S O 2 , C O o r N O 2 , o r the nitrogen a t o m of C N , o r even a halogen atom. Shallenberger a n d Acree suggested that this ΑΗ,Β system c a n interact w i t h a c o m p l e m e n t a r y ΑΗ,Β s y s t e m i n t h e sweet receptors t h r o u g h t w o s i m u l t a n e o u s hydrogen bonds to form t h e active 2

0097-6156/91/0450-0206$06.00/0 © 1991 American Chemical Society

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


15. TIlsrn&NOFRE

207

Why Does a Sweetener Taste Sweet?

complex. I n 1 9 7 2 , K i e r (3) p r o p o s e d t h e e x i s t e n c e o f a t h i r d b i n d i n g site involving d i s p e r s i o n forces. T h i s h y d r o p h o b i c site ( n o w d e s i g n a t e d a s s i t e G) c o u l d c o o p e r a t e w i t h t h e Α Η , Β u n i t t o i n d u c e higher sweetening activities; the distances between t h i s site a n d A o r Β a t o m s w e r e e s t i m a t e d to b e a p p r o x i m a t e l y of 5.5 n m for B - G a n d 3 . 5 n m for A H - G (3,4). In 1 9 8 0 , w e d e m o n s t r a t e d t h a t C O 2 H a n d N O 2 (or C N ) g r o u p s , u n t i l t h e n c o n s i d e r e d a s t h e s a m e Β g r o u p s i n the ΑΗ,Β s y s t e m , m u s t i n fact p a r t i c i p a t e b y specific a n d d i s t i n c t i n t e r a c t i o n s w i t h t h e r e c e p t o r (5,6). W e c o n c l u d e d t h a t , i n a d d i t i o n to t h e above three i n t e r a c t i o n p o i n t s , a f o u r t h n e w b i n d i n g s i t e , n a m e d D , h a d t o b e r e t a i n e d (7). T h e significance of t h i s finding w a s demonstrated w h e n , i n 1982, we prepared a n e w very potent sweetener w h i c h was 14,000 times as sweet as sucrose ( c o m p o u n d 7, T a b l e I). T h i s c o m p o u n d w a s a h y b r i d b e t w e e n t w o other sweeteners, the 4-cyanophenylcarbamoyl-p-alanine a n d aspartame, respectively 450 a n d 2 0 0 times as sweet as sucrose (potencies g i v e n o n a w e i g h t basis) ( c o m p o u n d s 12 a n d 14 i n T a b l e I) (8). F o r the first time, t h i s h i g h sweetening activity w a s o b t a i n e d t h r o u g h the c o m b i n a t i o n of the f o u r above i n t e r a c t i o n s i t e s i n one s t r u c t u r e . In 1987, the i m p r o v e m e n t of the v a r i o u s c r i t e r i a r e q u i r e d for t h e s e f o u r b i n d i n g s i t e s l e d u s to t h e d i s c o v e r y of t h e m o s t p o t e n t s w e e t e n i n g a g e n t k n o w n to t h i s d a y . T h i s c o m p o u n d , N-[N-cyclononylamino(4-cyanophenylimino)methyl]glycine, which w e n o w c a l l s u c r o n o n i c a c i d (1), h a s a p o t e n c y o f 2 0 0 , 0 0 0 t i m e s t h a t o f s u c r o s e (9, J O ) .

ο

1

O n the b a s i s of these f i n d i n g s , s t r u c t u r a l l y different s w e e t e n i n g a g e n t s w e r e r e v i e w e d (see T a b l e I f o r s o m e t y p i c a l e x a m p l e s ) , i n t h e s e a r c h for c o m m o n p a t t e r n s of s i m i l a r a t o m s o r g r o u p s , a n d a n e w t h e o r y for t h e m o l e c u l a r m e c h a n i s m s i n v o l v e d i n t h e s w e e t e n i n g a c t i v i t y ( T a b l e I) i s p r e s e n t e d . T h e existence of a receptor c o n t a i n i n g at least eight specific recognition sites is proposed. These sites correspond sterically a n d c h e m i c a l l y to eight o p t i o n a l i n t e r a c t i o n sites i n sweeteners a n d a r e d e s i g n a t e d a s s i t e s A H , B , G , D , Υ, X H , E i a n d E . T h e s p a t i a l a r r a n g e m e n t p r o p o s e d f o r t h e s e s i t e s i s g i v e n i n F i g u r e 1, a n d t h e i r average relative distances a n d their C a r t e s i a n coordinates are given i n T a b l e s II a n d III. T h e s i m u l t a n e o u s i n t e r a c t i o n of a sweetening agent w i t h a l l of t h e s e r e c e p t o r r e c o g n i t i o n sites is n o t r e q u i r e d i n o r d e r to 2



2

2

+

2

2

2

2

2

2

+

2

2

2

200 120 55 50 50 45 14 7 2 2

000 000 000 000 000 000 000 000 700 400 700 450 300 200 100 50 35 0.6 0.3

Approx. potency (sucrose=l)

CN.PhNH =C(NHc-Nonyl)Gly diCl.PhNH =C(NHCHMePh)Gly (N0 .PhNHCS)Aspartame (CN.PyrNHCS)Aspa rt ame L-Asp-NHCH (C0 Me) C0 Fn CN.PhNH+=C (NHS0 Ph)Gly (CN.PhNHCO)Aspartame CN.PhNHC(=NCN)Gly CN.PhNH =C(NH)Gly N0 . PhNHCSNH (CH ) C0 " Suosan CN. PhNHCONH (CH ) C0 " Saccharin Aspartame (6.CI.Tryptaminyl)5.tetrazole Cyclamic acid D-Tryptophan Ethylene glycol cis-1,4-c-Hexanediol

+

Abbr. Name or Formula*

3

3

+

+

+

2

2

2

2

2

3

2

2

2

2

2

2

2

2

2

4

C0"" CN ~ S0 " C0 "

3

3

NH NH + NH NH OH OH

2

2

2

C0 " C0 " C0 " C0 ~ C0 ~ C0 " C0 " C0 " C0 " C0 " C0 " C0 "

Β

NH(Ph) NH(Ph) NH (a) NH (a) NH NH(Ph) NH (a) NH(Ph) NH(Ph) NH(CH ) NH(CH ) NH(CH )

AH

Benzo Ph Indolyl c-Hexyl Indolyl

2

2

CN CN CN CN N0 N0 CN

CN CI CN CN

c-Nonyl CHMePh Bz Bz Fenchyl Ph(S0 ) Bz 2

D

G

00 CO*

CO* CO* CO* SO CO* CN (NH)

Y

OH OH 2

NH NH*

NH

NH* NH* NH*

XH

Interaction sites

2

;

SO CO(OMe)

CO(OMe)

CO(OMe) CO(OMe) CO(OMe)

El

2

CI

SO

CO(OFn)

E

2

* Abbreviations : Ph = phenyl substituted; CN.Ph = 4-cyanophenyl; Gly = glycine (-NHCH C0 ~); diCl.Ph = 3,5dichlorophenyl; N0 .Ph = 4-nitrophenyl; Aspartame = L-aspartyl-L-phenylalanine methyl e s t e r Asp - L-aspartyl; Bz = Benzyl group; CN.Pyr = 2-cyanopyrimidin-5-yl; Fn = fenchyl; * Peptide bond. Cpds 1 and 2 are described i n (9); 3 and 7 i n (8); 4 i n (16); 5 i n (17); 6, 8 and 9 i n (18); 10 and 12 i n (7); 11 i n (19); 13, 14, 16-18 i n (20); 15 i n (21); 19 i n (22).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Cpd No.

TABLE I . Postulated i n t e r a c t i o n s i t e s of some sweeteners. To e l i c i t sweetness, the simultaneous binding of the eight sites assumed to be involved i n the sweetener-receptor interaction i s not a prerequisite.



15. Ή Ν Ή & NOFRE

209


F i g u r e 1. S p a t i a l a r r a n g e m e n t o f t h e e i g h t p o t e n t i a l i n t e r a c t i o n s i t e s o f s w e e t e n i n g a g e n t s , w i t h (i) f o u r h i g h - a f f i n i t y s i t e s d e s i g n a t e d a s s i t e s Β f o r a n a n i o n i c g r o u p ( C 0 ~ , SO3" o r C N " ) , A H f o r a h y d r o g e n - b o n d d o n o r g r o u p ( N H , O H ), G f o r a h y d r o p h o b i c g r o u p ( h y d r o c a r b o n group) a n d D for a h y d r o g e n b o n d a c c e p t o r g r o u p ( C N , N 0 , CI) a n d (u) w i t h f o u r s e c o n d a r y s i t e s d e s i g n a t e d a s Y , X H , E i , E s i t e s , Υ, E i , E b e i n g h y d r o g e n b o n d acceptor ligands (CO, halogen atoms) a n d X H a hydrogenb o n d donor group (NH, OH). 2

2

2

2


4


210

SWEETENERS:

DISCOVERY, M O L E C U L A R

DESIGN, A N D

CHEMORECEPTION

generate a sweet response, a lower n u m b e r of sites often b e i n g s u f f i c i e n t to i n i t i a t e a s w e e t taste. T h e sweetness p o t e n c y of a c o m p o u n d w i l l d e p e n d b o t h o n the n u m b e r of active sites involved i n the molecule-receptor i n t e r a c t i o n a n d o n the effectiveness of e a c h i n d i v i d u a l i n t e r a c t i o n . T h e i n t e r a c t i o n efficiency, w h i c h i s i n fact the m a i n factor, is strictly dependent o n the space-filling p r o p e r t i e s of t h e m o l e c u l e (size a n d s h a p e ) a n d o n t h e n a t u r e , a v a i l a b i l i t y a n d o r i e n t a t i o n of the active g r o u p s i n v o l v e d i n t h e interactions. T a b l e II. A v e r a g e d i s t a n c e s * ( i n n m ) b e t w e e n t h e e i g h t i n t e r a c t i o n sites identified i n sweeteners. D

Y

XH

El

0.45

1.15

0.45

0.30

0.30

0.70

1.25

0.50

0.35

0.45

0.55

1.10

0.32

0.35

0.50

0.45

0.80

0.70

0.90

0.90

G

0.60

0.80

Β

0.28

Sites

AH

Β

G

E2

0.70

0.70

Ei

0.80

0.75

XH

0.45

Y D

* These d i s t a n c e s are the intervals s e p a r a t i n g the points c h a r a c t e r i z e d , for Β b y the average p o i n t b e t w e e n t h e n u c l e i of t h e t w o o x y g e n a t o m s of a C O 2 " g r o u p , for A H a n d X H b y t h e n u c l e u s of t h e h y d r o g e n a t o m i n a n N H or O H g r o u p , for Y, E i , E b y t h e n u c l e u s of t h e o x y g e n a t o m i n a C O g r o u p , for D b y t h e n u c l e u s of t h e n i t r o g e n a t o m i n a C N g r o u p , for G b y the c e n t e r of a cyclononyl-type hydrocarbon group represented by an a p p r o x i m a t e l y s p h e r i c a l v o l u m e w i t h a r a d i u s of a b o u t 0.4 n m . 2

T a b l e II s h o w s t h a t s e v e r a l c o u p l e s o f h y d r o g e n d o n o r a n d hydrogen acceptor groups separated b y approximately 0.30 n m are present i n this m o d e l (AH-Y, A H - B , X H - Y , X H - E i , X H - E ) . S u c h a feature was identified i n sweeteners by Shallenberger a n d Acree a n d w a s the b a s i s of t h e i r t h e o r y w i t h the p o s t u l a t e of a u n i q u e ΑΗ,Β s y s t e m (i). T a b l e II s h o w s a l s o t h a t s i t e s Β ( C 0 ' ) a n d D ( N 0 / C N ) are a p p r o x i m a t e l y e q u i d i s t a n t (0.9 n m ) f r o m the hydrophobic component G. T h i s particularity m a y explain the s t r u c t u r a l a n a l o g y i n i t i a l l y a t t r i b u t e d to t h e s e two g r o u p s (1-3), before w e d e m o n s t r a t e d i n 1981 t h e i r difference for s w e e t a c t i v i t y (5-7). 2

2

2


15. ΤΙΝΉ & NOFRE


T a b l e III.


211

C a r t e s i a n c o o r d i n a t e s of t h e eight i n t e r a c t i o n s i t e s identified i n sweeteners.

AH

2.11

0.39

Β

0.00

0.00

0.00

G

3.55

5.94

-4.01

D

8.86

0.00

-0.00

2.00

4.11

-0.49

-1.01

3.86

-1.83

Y XH

-1.79

Ει

-1.78

7.04

-0.22

E2

-0.83

6.20

-3.03

T a b l e I s h o w s the c h e m i c a l h o m o g e n e i t y of the l i g a n d s r e t a i n e d a s A H , B , G , D . . . sites i n s w e e t e n e r s a n d allows u s to c o n s i d e r t h e p r o b a b l e n a t u r e of the p h y s i c a l i n t e r a c t i o n s i n v o l v e d i n the receptor recognition. Site A H is a hydrogen-bond donor group, a n d is represented b y N H or O H groups. Site Β is a n a n i o n i c g r o u p s u c h a s C 0 " , SO3" o r C N ~ ( t e t r a z o l y l ) g r o u p s , f o r m i n g a n i o n i c b o n d w i t h the receptor. Site G is a h y d r o p h o b i c group, s u c h as the alkyl, cycloalkyl or aryl groups, involved i n L o n d o n - v a n der W a a l s forces a n d h y d r o p h o b i c interactions. Site D is a h y d r o g e n - b o n d acceptor moiety p r i n c i p a l l y represented i n Table I b y C N , N 0 or CI. Site Y is a hydrogen-bond acceptor ligand s u c h as C O , S 0 , N 0 , C N or halogen. Site X H , s t r u c t u r a l l y s i m i l a r to site A H , i s a h y d r o g e n b o n d d o n o r g r o u p s u c h as a n N H or O H group. F i n a l l y , sites E i a n d E , often w o r k i n g s i m u l t a n e o u s l y , are h y d r o g e n - b o n d acceptor groups s u c h as C O , S 0 , intramolecularly hydrogen-bonded hydroxyl groups, or halogen atoms. 2

4

2

2

2

2

2

Sites A H , B , G a n d D are defined as high-affinity i n t e r a c t i o n sites since they are frequently present i n sweet-tasting c o m p o u n d s , a n d t h e i r s i m u l t a n e o u s p r e s e n c e l e a d s to the m o s t potent c o m p o u n d s ( c o m p o u n d s 1-4 i n T a b l e I). T h e i m p o r t a n c e o f t h e D g r o u p , w h i c h h a d b e e n i g n o r e d u n t i l o u r i n i t i a l o b s e r v a t i o n s (5-7), is n o w well established. This group is present i n almost all c o m p o u n d s h a v i n g sweet potencies greater t h a n 2,000 times that o f s u c r o s e ( c o m p o u n d s 1-11; F i g u r e 2). T h e other sites, Y, X H , E i a n d E , are generally a s s o c i a t e d w i t h one or several of the other A H , B , G or D sites a n d are not as c r u c i a l l y i m p o r t a n t to e l i c i t p o t e n t s w e e t e n i n g a c t i v i t i e s . The b i n d i n g of a m o l e c u l e c o n t a i n i n g two o r t h r e e of t h e s e l o w - a f f i n i t y i n t e r a c t i o n s i t e s , s e n s i t i v e to t h e o t h e r m o l e c u l a r c h a r a c t e r i s t i c s (size, s h a p e , e l e c t r o n i c a n d s p a c e - f i l l i n g p r o p e r t i e s ) i s e x p e c t e d to induce only low sweetness. 2



212

SWEETENERS:

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

CHEMORECEPTION

F i g u r e 2 . S u c r o n o n i c a c i d a c c o r d i n g to t h e m o d e l o f F i g u r e 1 (B is C 0 " , A H is N H , D is C N a n d G the c y c l o n o n y l g r o u p ; Φ represents nitrogen atoms a n d φ oxygen atoms). 2

C o n v e r s e l y , b i n d i n g of s e v e r a l h i g h - a f f i n i t y i n t e r a c t i o n s i t e s ( A H , B , G o r D) w i l l i n d u c e s i g n i f i c a n t l y h i g h s w e e t n e s s . In this case, t h e o v e r a l l c h a r a c t e r i s t i c s a t t a c h e d to e a c h b i n d i n g site (precise c h e m i c a l n a t u r e a n d relative distances) as well as the w h o l e e n v i r o n m e n t a l h i n d r a n c e of the c o m p l e m e n t a r y receptor f e a t u r e s a r e s u f f i c i e n t l y s p e c i f i c t o e x p l a i n t h e effect o f c h i r a l i t y o n s w e e t a c t i v i t y a n d to m i n i m i z e t h e e x i s t e n c e of i n a c t i v e c o m p o u n d s w h i c h fits the m o d e l . Is t h e s w e e t t a s t e a c t i v i t y m e d i a t e d t h r o u g h a s i n g l e r e c e p t o r or several distinct receptors ? The answer is still pending. Some authors have suggested that only multiple receptors c a n explain the differences existing i n c h e m i c a l s t r u c t u r e s , t h r e s h o l d sensitivities o r o t h e r p h y s i o l o g i c a l d a t a f o r v a r i o u s s w e e t e n e r s {11-13). S o m e other p h y s i o l o g i c a l d a t a are s t a n d i n g i n favor of a single type receptor, for i n s t a n c e the s a m e i n h i b i t i o n b y g y m n e m i c a c i d for d i f f e r e n t c l a s s e s o f s w e e t e n e r s [14,15). T h i s m o d e l s u g g e s t s t h e e x i s t e n c e i n h u m a n s of a s i n g l e t y p e of r e c e p t o r w i t h a t l e a s t e i g h t r e c o g n i t i o n sites a n d a b l e to i n t e r a c t w i t h s w e e t e n e r s of different s t r u c t u r a l classes. T h i s possibility does not, however, exclude the e x i s t e n c e i n m a n o r a n i m a l s of o t h e r s w e e t r e c e p t o r s c o n t a i n i n g a l o w e r n u m b e r of these r e c o g n i t i o n sites, a n d i n t e r a c t i n g w i t h a m o r e l i m i t e d n u m b e r of sweeteners. Conclusion The present m o d e l describes the m o l e c u l a r features w h i c h allow a m o l e c u l e to i n i t i a t e a s w e e t s e n s a t i o n i n m a n . It i n c l u d e s a n d clarifies the three previously postulated sites (Shallenberger a n d


15.

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& NOFRE


213

A c r e e ' s ΑΗ,Β sites a n d K i e r ' s l i p o p h i l i c site). It a s s u m e s t h e e x i s t e n c e i n t h e sweet receptors of at l e a s t eight r e c o g n i t i o n sites w i t h the c a p a c i t y to identify at least eight c o m p l e m e n t a r y a n d optional interaction sites i n sweeteners. The spatial arrangement a n d relative distances between these sites are also given.


Acknowledgments W e t h a n k T h e N u t r a S w e e t C o m p a n y , D e e r f i e l d , I l l i n o i s , for s u p p o r t of this research.

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

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RECEIVED August 27,

1990


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