Chemistry of Sweet Peptides - American Chemical Society

5 Chemistry of Sweet Peptides YASUO ARIYOSHI

Downloaded by IOWA STATE UNIV on February 28, 2017 | http://pubs.acs.org Publication Date: December 14, 1979 | doi: 10.1021/bk-1979-0115.ch005

Central Research Laboratories, Ajinomoto Co., Inc. 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki, 210, Japan

It is known that sweet-tasting compounds are quite common and their chemical structures vary widely. In order to establish a structure-taste relationship, a large number of compounds have been tested, and several molecular theories of sweet taste have been proposed by different groups. At present, the phenomenon of sweet taste seems best explained by the tripartite functioning of the postulated AH, Β (proton donor-acceptor) system and hydro phobic site X (1, 2, 3, 4, 5). Sweet-tasting compounds possess the AH-B-X system in the molecules, and the receptor site seems to be also a trifunctional unit similar to the AH-B-X system of the sweet compounds. Sweet taste results from interaction between the receptor site and the sweet unit of the compounds. Space-filling properties are also important as well as the charge and hydro phobic properties. The hydrophile-hydrophobe balance in a molecule seems to be another important factor. After the finding of a sweet taste in L-Asp-L-Phe-OMe (aspar tame) by Mazur et al. (6), a number of a s p a r t y l d i p e p t i d e e s t e r s were synthesized by s e v e r a l groups i n order to deduce s t r u c t u r e t a s t e r e l a t i o n s h i p s , and to o b t a i n potent sweet peptides. I n the case of the peptides, the c o n f i g u r a t i o n and the conformation o f the molecule are important i n connection w i t h the s p a c e - f i l l i n g p r o p e r t i e s . The p r e f e r r e d conformations o f amino a c i d s can be shown by a p p l i c a t i o n of the extended Hiickel theory c a l c u l a t i o n . However, p r o j e c t i o n of reasonable conformations f o r d i - and t r i peptide molecules i s not e a s i l y accomplished. In the course of i n v e s t i g a t i o n s of a s p a r t y l d i p e p t i d e e s t e r s , we had to draw t h e i r chemical s t r u c t u r e s i n a u n i f i e d formula. I n an attempt to f i n d a convenient method f o r p r e d i c t i n g the sweett a s t i n g property of new peptides and, i n p a r t i c u l a r , to e l u c i d a t e more d e f i n i t e s t r u c t u r e - t a s t e r e l a t i o n s h i p s f o r a s p a r t y l d i p e p t i d e e s t e r s , we p r e v i o u s l y a p p l i e d the F i s c h e r p r o j e c t i o n technique i n drawing sweet molecules i n a u n i f i e d formula ( 4_). The sweet-tasting property of a s p a r t y l d i p e p t i d e e s t e r s has been s u c c e s s f u l l y explained on the b a s i s o f the g e n e r a l s t r u c t u r e s shown i n Figure 1 (4). A peptide w i l l t a s t e sweet when i t takes 0-8412-0526-4/79/47-115-133$05.00/0 © 1979 American Chemical Society Boudreau; Food Taste Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

FOOD TASTE CHEMISTRY


134

COOH

COOH I CH

I

ÇH

2

2

H^C-*NH H^C—NH CO

2

CO

I

I

NH

NH Figure 1. General structure for sweet peptides: Rj = small hydrophobic side chain (1 ~ 4 atoms); R = larger hydrophobic side chain (3 ~6 atoms) (4) 2

R^C-^H

H^Ç—Ri R Ri £ R (A) Sweet

R 2

(B) Not sweet

COOH Figure 2. General structure for sweet amino acids: R, is not restricted; R = H,CH ,orC H (12) 2

3

2

5

2

2

R ^C^NH r Ri 2

2

Boudreau; Food Taste Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2

5.

ARiYOSHi

Sweet Peptides

135

the formula (A), but not when i t takes the formula (Β), where R i s l a r g e r than R R i s a small hydrophobic s i d e chain with a chain l e n g t h of 1^4 atoms and R i s a l a r g e r hydrophobic s i d e chain with a chain l e n g t h of 3^6 atoms. Ri i n formula (A) serves as the hydrophobic binding s i t e (X). In the formula (A), when Ri and R are s u f f i c i e n t l y d i s s i m i l a r i n s i z e , the sweetness potency w i l l be intense, whereas when Ri and R are of s i m i l a r s i z e , the potency w i l l be weak (Table 1). The s t r u c t u r e - t a s t e r e l a t i o n s h i p s w i l l be discussed i n d e t a i l . Dipeptide e s t e r s are c l o s e l y r e l a t e d to amino a c i d s i n chemical s t r u c t u r e and p r o p e r t i e s . Hence, we s e l e c t e d amino a c i d s as the standard to which sweet peptides were r e l a t e d . The s t r u c t u r a l features of sweet-tasting amino a c i d s have been best explained by Kaneko (12) as shown i n Figure 2, i n which an amino a c i d w i l l t a s t e sweet when R i s H, CH or C H , whereas the s i z e of R i s not r e s t r i c t e d i f the amino a c i d i s s o l u b l e i n water. In the case of a s p a r t y l d i p e p t i d e e s t e r s , proton donor AH i s a f r e e α-amino group, and proton acceptor Β i s a f r e e β-carboxyl group. Therefore, the a s p a r t y l part could be r e a d i l y arranged to meet the s t r u c t u r a l requirements f o r sweet t a s t e defined by Kaneko through the F i s c h e r p r o j e c t i o n formula. The distance between the f r e e α-amino and β-carboxyl groups was considered to be w i t h i n the range defined f o r sweet molecules. In the case of the second amino a c i d such as Phe-OMe of aspartame, however, somewhat greater f l e x i b i l i t y i n drawing c o n f i g u r a t i o n s was afforded by the interchange of atoms or groups attached to the asymmetric carbon atom of the second amino a c i d . This part of amino a c i d could be replaced by a great v a r i e t y of L- or D-amino a c i d e s t e r s without l o s i n g the sweetness. This suggests that the sweet t a s t e receptor s i t e sees only the s i z e and shape of t h i s p a r t , apart from the AH-B system of L - a s p a r t i c a c i d . I t seems that the t a s t e receptor sees the second amino a c i d s as an a l k y l side chain i n the case of sweet amino a c i d s . In order to avoid confusion and to u n i f y the system, the molecular s t r u c t u r e was p r o j e c t e d so that the l a r g e s t s i d e chain attached to the asymmetric carbon atom would be at the bottom of the formula and the amino group of the peptide bond i n the upper p o s i t i o n as shown i n Figure 1. The remaining two groups such as a hydrogen atom and a smaller side chain, then l a i d i n f r o n t of the p r o j e c t i o n p l a i n . The o r i e n t a t i o n of the hydrogen atom and the smaller s i d e chain depends on the c o n f i g u r a t i o n and the s i z e of the two s i d e chains of the amino a c i d e s t e r . I t was considered that the t a s t e of the d i p e p t i d e e s t e r s changed according to the s i z e and shape of the second amino a c i d . For instance, a sweet peptide, L-Asp-L-Phe-OMe (1), corresponds to the formula (A), where R i s a methyl e s t e r group and R i s a benzyl group, whereas a nonsweet peptide, L-Asp-D-Phe-OMe (2), corresponds to the formula (B), where R i s a methyl e s t e r group and R i s a benzyl group. This evidence suggests that a peptide w i l l t a s t e sweet when i t takes the formula (A), but not when i t takes the formula 2

lt

x

2

2


2

2

3

2

5

x

x

2

x

2



1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14a. 14b. 15. 16. 17. 18a. 18b. 19. 20. 21. 22. 23. 24. 25.

6

71

n

L-Asp-L-Phe-OMe L-Asp-D-Phe-OMe ε-Ac-D-Lys L-Asp-Gly-OMe L-Asp-Gly-OEt L-Asp-Gly-0C Hn L-Asp-D-Ala-OMe L-Asp-L-Ala-OMe L-Asp-D-Abu-OMe L-Asp-L-Abu-OMe L-Asp-Gly-OPr^ L-Asp-D-Ala-OPr L-Asp-D-Abu-OPr^ L-Asp-L-Nva-OMe L-Asp-L-Nva-OMe L-Asp-L-Nva-OEt L-Asp-D-Nva-OPr L-Asp-L-Nle-OMe L-Asp-L-Nle-OEt L-Asp-L-Nle-OEt L-Asp-L-Cap-OMe L-Asp-L-Cap-OEt L-Asp-L-Ser (Ac)-OMe L-Asp-L-Ser(Bt^)- OMe L - A s p - L - S e r ( B t i ) - OEt L-Asp-L-Thr(Bt^)- OMe L-Asp-L-aThr(Bt^) -OMe

Compounds

A,Β A,Β A,Β A Β A Β A,Β A A A Β Β A A A Β A A A A A A A

A Β

Projection formula*

Table 1.

3

3

3

3

CH2CH3 COOCH3 CH 3 CH 2 CH 2 CH 3 CH 2 CH 2 CH 2 CH 3 COOCH3 COOCH2CH3 C H 3 CH 2 CH 2 CH 2 COOCH3 COOCH2CH3 COOCH3 COOCH3 COOCH2CH3 C00CH COOCH3

Η CH

CH3CH2

CH2CH3

3

Η Η Η CH HC

CH3OOC

C00CH

Ri R 2

2

3

3

2

3

3

2

2

2

3

3

2

CH 0C0CH(CH ) CH 0C0CH(CH ) CH(CH )0C0CH(CH )2 CH(CH )OCOCH(CH )

COOCH2CH2CH3 COOCH2CH2CH3 CH 2 CH 2 CH 3 COOCH3 COOCH2CH3 COOCH2CH2CH3 CH 2 C H 2 C H 2 CH 3 CH 2 C H 2 CH 2 C H 3 COOCH2CH3 C H 2 C H 2 C H 2 CH 2 CH 2 C H 3 C H 2 CH 2 CH 2 CH 2 CH CH 3 CH2OCOCH3

3

COOCH3 COOCH COOCH2CH2CH3

COOCH3 COOCH2CH3 COOCeHxx COOCH3 COOCH3

C H 2 C eHg CH2C6H3

Taste of a s p a r t y l peptides


-

50 2-3

-10

45 45 5 0 47

-

0 14 170,125 95 4 0

-16

5-10 8 13 13 25

-

180

Taste**

6 6 4 4 7 4 8 8 9 9 4 8,9 9 9 9 9 4 9 7 7 7 7 9 9 9 9 9

Lit.


n

A A A A A A A A A A A A A,Β A A A A Β

*See F i g u r e 1 f o r p r o j e c t i o n f o r m u l a .

n

L-Asp-L-Ile-OMe L-Asp-L-Lys-OMe L-Asp-L-Lys(Ac)-OMe L-Asp-L-t-HyNle-OMe L-Asp-L-e-HyNle-OMe L-Asp-L-MPA L-Asp-L-HMPA L-Asp-D-Ser-OPr L-Asp-D-Ala-OBu« L-Asp-D-Ser-OBu L-Asp-D-Thr-OPr^ L-Asp-D-aThr-OPrn L-Asp-Gly-Gly-OMe L-Asp-D-Ala-Gly-OMe L-Asp-D-Abu-Gly-OMe L-Asp-D-Val-Gly-OMe L-Asp-L-Ama (OFn) -OMe L-Asp-D-Ama(OFn)-OMe

Projection formula* 3

3

3

COOCH3 CH3OOC

3

2

3

2

2

3

3

2

CH CH OH CH OH CH CH OH CH(CH )0H CH(CH )0H Η CH CH 2 CH 3 CH(CH )

COOCH3 COOCH3 COOCH3 COOCH3

C00CH

Ri 2

2

2

2

3

3

2

3

3

2

3

CONHCH 2 COOCH 3 CONHCH 2 COOCH CONHCH 2 COOCH CONHCH COOCH3 COO-fenchyl COO-fenchyl

COOCH2CH2CH3

2

C00CH CH CH

COOCH 2 CH 2 CH 2 CH 3

CH2C5H5 CH2C6H5 COOCH2CH2CH3 COOCH2CH2CH2CH3

2

2

CH(0H)CH CH CH CH(OH)CH CH CH

CH2CH2CH2CH2NHCOCH3

CH(CH3)CH CH3 CH 2 CH 2 CH 2 CH 2 NH 2

R

**Numbers represent the sweetness potency of the compound as a m u l t i p l e of s u c r o s e . In a d d i t i o n , 0 = t a s t e l e s s , - = b i t t e r .

26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

Compounds


6 9 9 9 9 10 10 9 7 9 9 9 7 9 7 7 11 11

_

1.2 7 18 50 1 320 50 70 150 40 0 3 1 0 22000 0

-

Lit.

Taste**

138


(B). T h i s a l s o suggests that the AH-B concept r e p r e s e n t s o n l y a f i r s t approximation i n the case of p e p t i d e s . C e r t a i n l y , the AH-B system i s r e q u i r e d i n the molecule. However, the s t r u c t u r a l c h a r a c t e r i s t i c s of the second amino a c i d sometimes may completely mask any AH-B e f f e c t . To t e s t the above h y p o t h e s i s , we have s y n t h e s i z e d a number of p e p t i d e s w i t h o r w i t h o u t a sweet t a s t e . The C-C bonding i n R has been replaced by e t h e r , t h i o e t h e r , amide or e s t e r bond without l o s i n g sweetness. Ri i s a s m a l l s i d e c h a i n such as a methyl, e t h y l , i s o p r o p y l , or hydroxymethyl group, or an e s t e r having a s m a l l s u b s t i t u e n t . The exact chemical nature of these groups i s not c r u c i a l . The s t u d i e s on p e p t i d e s began w i t h a c o r r e l a t i o n between sweet amino a c i d s and p e p t i d e s . Since the p r o j e c t i o n formula of L-Asp-Gly-OMe (4) i s s i m i l a r i n s i z e and shape to that of ε-Ac-DLys (3) which i s sweet, we p r e d i c t e d t h a t L-Asp-Gly-OMe would t a s t e sweet i n s p i t e of the b i t t e r t a s t e i n the l i t e r a t u r e . Therefore, we synthesized the peptide and t a s t e d i t . As expected, i t was sweet and i t s sweetness potency was almost equal to that of ε-Ac-D-Lys. Thus, the d i p e p t i d e could be c o r r e l a t e d to the amino a c i d . Lengthening (5) or enlargement (6) of the a l k y l group of the e s t e r d i d not a f f e c t i t s sweetness potency (Table 1 ) . However, when a methyl group was introduced so as to protrude on the r i g h t of the p r o j e c t i o n formula of L-Asp-Gly-OMe, the r e s u l t a n t L-Asp-D-Ala-OMe (7) (8) was sweeter than L-Asp-Gly-OMe. This r e s u l t suggests that the methyl group i s i n v o l v e d i n a hydro phobic i n t e r a c t i o n a t the r e c e p t o r s i t e and causes the increased sweetness potency. On the other hand, when a methyl group was introduced so as to protrude on the l e f t of the p r o j e c t i o n formula of L-Asp-Gly-OMe, the r e s u l t a n t L-Asp-L-Ala-OMe (8) (8) was not sweet but b i t t e r . Loss of sweetness suggests that i n t e r a c t i o n w i t h the receptor s i t e may be blocked by the methyl group. This a l s o supports the idea t h a t a d i p e p t i d e w i l l not t a s t e sweet when i t takes the formula (Β), i n which R protrudes on the l e f t . I n t r o d u c t i o n of an e t h y l group i n s t e a d of the methyl group so as to protrude on the r i g h t of the p r o j e c t i o n formula of L-Asp-GlyOMe gave L-Asp-D-Abu-OMe ( 9 ) , which was 16 times sweeter than sucrose. I n t r o d u c t i o n of an e t h y l group on the o p p o s i t e s i d e gave L-Asp-L-Abu-OMe (10), which was devoid of sweetness. The low l e v e l of sweetness of ( 9 ) , as compared w i t h ( 7 ) , may show that the p o p u l a t i o n of the sweet formula (A) may s i g n i f i c a n t l y decrease because the two groups do not d i f f e r g r e a t l y i n s i z e . This idea gained f u r t h e r support when a methyl group was introduced on the r i g h t of the p r o j e c t i o n formula of L-Asp-GlyOPrtt (11) to g i v e L-Asp-D-Ala-OPr^ (12), which was 125 times sweeter than sucrose. L-Asp-D-Ala-OPr^ was about 9 times sweeter than L-Asp-Gly-OPrtt. I n the molecule of L-Asp-D-Ala-OPt^, the s i z e s of CH (Ri) and C00CH CH CH (R ) are s u f f i c i e n t l y d i s s i m i l a r . An e t h y l group was introduced i n s t e a d of the methyl group to give L-Asp-D-Abu-OPr (13), which was 95 times sweeter than sucrose and was l e s s sweeter than L-Asp-D-Ala-OPr^. Lengthening the a l k y l


2

x

3

2

2

3

2

n


5.

ARiYOSHi

Sweet Peptides

139

group of the e s t e r of L-Asp-D-Abu-OMe increased the sweetness potency; L-Asp-D-Abu-OPr was 6 times sweeter than L-Asp-D-AbuOMe. This f a c t may show that the sweeter compound (13) takes predominantly the sweet formula (A), s i n c e the s i z e s of the two groups are s u f f i c i e n t l y d i s s i m i l a r . More i n t e r e s t i n g i s the case of L-Asp-L-Nva-OMe (14). Since the s i z e s of COOCH and CH CH CH are almost equal, both the sweet formula(A) and the nonsweet formula (B) could be drawn f o r the d i p e p t i d e , so that i t could be p r e d i c t e d that the peptide would be s l i g h t l y sweet. In f a c t , i t was only 4 times sweeter than sucrose. Of course, replacement of the methyl group by an e t h y l group r e s u l t e d i n a compound (L-Asp-L-Nva-OEt (15)) l a c k i n g i n sweetness as expected from i t s p r o j e c t i o n formula. Some d i peptides c o n t a i n i n g D-norvaline were a l s o sweet, when R and R matched the sweet formula (A), e.g., L-Asp-D-Nva-OPr (16) was 45 times sweeter than sucrose. Thus, i t i s p l a u s i b l e that a sweet response does not always depend on the c o n f i g u r a t i o n of the second amino a c i d but mainly depends on the s i z e and shape of t h i s amino a c i d e s t e r . L-Asp-L-Nle-OMe (17) was s t r o n g l y sweet, but L-Asp-L-Nle-OEt (18) was only s l i g h t l y sweet. These d i f f e r e n c e s could be e a s i l y p r e d i c t e d from examination of t h e i r p r o j e c t i o n formulas. Both the sweet formula (A) and the nonsweet formula (B) could be drawn f o r the l a t t e r peptide. L-Asp-L-Cap-OMe (19) was sweet, whereas the e t h y l e s t e r (20) was not sweet but b i t t e r , though we could draw the sweet formula (A) to i t . This may show that the increased hydrophobicity i n the molecule changed the property of the sweet peptide to a b i t t e r property, because i t has been known that b i t t e r - t a s t i n g compounds are composed of charge and hydrophobic p r o p e r t i e s . This a l s o suggests that the hydrophile-hydrophobe balance i n a sweet molecule i s a very important f a c t o r . An a l k y l s i d e c h a i n of the second amino a c i d could be replaced by an e s t e r group without l o s i n g the sweetness, e.g., L-Asp-L-Ser(Ac)-OMe (21), L-Asp-L-Ser(Bt^)-OMe (22) and L-Asp-LS e r ( B t i ) - O E t (23) were sweet. The replacement of the L - s e r i n e by L-threonine or by L-aZZothreonine r e s u l t e d i n b i t t e r compounds (24, 25). These r e s u l t s matched that the i n t r o d u c t i o n of a methyl group i n t o a sweet peptide, L-Asp-L-Nva-OMe, r e s u l t e d i n a b i t t e r substance (L-Asp-L-Ile-OMe (26)). The methyl group may b l o c k the i n t e r a c t i o n between the peptides and the sweet r e c e p t o r . From the above d i s c u s s i o n , we have concluded that a hydrophobic b i n d i n g s i t e i s necessary f o r a s e r i e s of potent sweet peptides. Next, we examined how a h y d r o p h i l i c group would a f f e c t the sweetness potency. The i n t r o d u c t i o n of an amino group to L-Asp-L-Nle-OMe (17) r e s u l t e d i n a b i t t e r compound (L-Asp-L-Lys-OMe (27)) and b l o c k i n g the amino group recovered the sweetness by some extent (28). The i n t r o d u c t i o n of a hydroxyl group i n t o a peptide with the L-L c o n f i g u r a t i o n (17, 31) r e s u l t e d i n a diminution i n the potency n


3

2

2

3

1

n


2

140


(29, 30, 32). Contrary to the peptides with the L-L c o n f i g u r a t i o n , the i n t r o d u c t i o n of a hydroxyl group i n t o the L-D peptides d i d not always r e s u l t i n a diminution of t h e i r potencies, but sometimes increased t h e i r potencies. L-Asp-D-Ser-OR (R=Me, E t , P r , Pr^, Butt, Bu*- or c-hexyl) was sweeter than the corresponding peptides without a hydroxyl group, L-Asp-D-Ala-OR (9), e.g., compounds (33) and (35) were sweeter than compounds (12) and (34), r e s p e c t i v e l y . In the case of threonine-containing peptides, L-Asp-D-Thr-OR (R=Me or Pr^) was sweeter than L-Asp-D-Abu-OR which l a c k s a hydroxyl group of the D-threonine. On the contrary, when the Dthreonine was replaced by D-α Ζ Z.0 threonine, the potency diminished significantly. The IR s p e c t r a of these peptides showed that the hydroxyl a b s o r p t i o n had disappeared due to strong hydrogen bonding. I t i s considered that the apparent exceptions may be due to r i g i d i n t r a m o l e c u l a r hydrogen bonding causing l o s s of hydrop h i l i c i t y and allowing a hydrophobic group type i n t e r a c t i o n . Returning to stereoisomerism, the r e l a t i o n s h i p s between stereoisomerism and t a s t e w i l l be discussed by using stereoisomers of aspartame (L-Asp-L-Phe-OMe) as model compounds. The l a c k of sweet t a s t e i n a-L-Asp-D-Phe-OMe (6) i s r e a d i l y explained a f t e r c o n s i d e r i n g the p r o j e c t i o n formula ((2) i n Figure 3), i n which a small s i d e chain on the l e f t may cause e l i m i n a t i o n of sweetness. According to Mazur et al β-D-Asp-L-Phe-OMe (6) i s b i t t e r though i t s p r o j e c t i o n formula ((44) i n Figure 3) would suggest that i t has a sweet t a s t e . This r e s u l t can not be explained f u l l y . However, d i p e p t i d e e s t e r s c a r r y i n g a small hydrophobic group on the 5th carbon from the carbon bearing the AH(NH ) o f t e n t a s t e d b i t t e r ; e.g., L-Asp-L-Ile-OMe (26) and L-Asp-L-aThr(Bt^)-OMe (25) were b i t t e r . The l o c a t i o n of C00CH from the AH(NH ) i n the peptide (44) corresponds to that of CH i n these peptides. The l a c k of sweet t a s t e i n β-L-aspartyl d i p e p t i d e e s t e r s such as β-LAsp-Gly-OMe (7) and β-L-Asp-L-Phe-OMe (6) i s r e a d i l y explained a f t e r c o n s i d e r i n g t h e i r p r o j e c t i o n formulas ((45) i n Figure 3), i n which the second amino a c i d l i e s on the l e f t . This formula i s incompatible with that d e f i n e d f o r sweet amino a c i d s , i n which the second amino a c i d corresponds to R of Figure 2. And a l s o the peptide does not f i t the s p a t i a l b a r r i e r model f o r the receptor s i t e proposed by Shallenberger et al. (13). The l a c k of sweet t a s t e i n α-D-aspartyl d i p e p t i d e e s t e r s such as a-D-Asp-L-Phe-OMe (6) i s i n t e r p r e t e d analogously a f t e r c o n s i d e r i n g t h e i r p r o j e c t i o n formulas ((46) i n F i g u r e 3). Therefore, we have concluded that sweet-tasting a s p a r t y l d i p e p t i d e e s t e r s can be drawn as the u n i f i e d formula (A), whereas nonsweet peptides as (B) as shown i n Figure 1. There i s no asymmetric carbon atom i n aminomalonic a c i d molecule. When both of the c a r b o x y l i c a c i d s are s u b s t i t u t e d by e s t e r i f i c a t i o n with d i f f e r e n t a l c o h o l s , o p t i c a l isomers are generated. I t i s known that aminomalonic a c i d d e r i v a t i v e s r e a d i l y racemize i n s o l u t i o n under o r d i n a r y c o n d i t i o n s . L-Asp-Ama (OFn) -


n

9

2

3

2

3

2


ARiYOSHi

Sweet Peptides

COOH

COOH

CH 2

CH 2

I

I

H*-C-*NH

H^C—NH

CO

CO


2

I f o

2

I

o

H*. C-*C-0-CH

r

CHs-O-C^C-^H

3

180 α-L-L

(1)

a-L-D (2)

β-D-L (44)

COOH

I

0

COOH

R-C-CH ^ Ç - * N H 2

2

H

0

ÇH

2

R-C*-C—NH

2

H

3-L-L(or D) (45)

a-D-L(or D) (46)

R=Phe-OMe Figure 3. Projection formulas of isomers of aspartame (L-Asp-L-Phe-OMe)



142


OMe was found by Fujino et al.(11) to be 22000^33000 times sweeter than sucrose. I t i s not e x a c t l y known whether the sweett a s t i n g isomer has the L-L(or S-R) or the L-D(or S-S) c o n f i g u r a t i o n because of ready racemization. From the examination of i t s p r o j e c t i o n formula, i t could be p r e d i c t e d that the L-L(or S-R) isomer (42), i n which aminomalonic a c i d d i e s t e r takes an L ( o r R)c o n f i g u r a t i o n , would be sweet. This p r e d i c t i o n agreed with that reported i n the l i t e r a t u r e (14). In Ama-L-Phe-OMe (47) (14, 15), i t i s a l s o not known whether the sweet-tasting isomer has the L-L(or S-S) or the D-L(or R-S) c o n f i g u r a t i o n . In the case of a s p a r t y l d i p e p t i d e e s t e r s , the L-L isomer was sweet. By analogy, other researchers deduced that the L-L(or S-S) isomer ((47b) i n Figure 4) would be sweet. However, i t seemed to us that the D(or R)-configuration would be p r e f e r r e d for the aminomalonic a c i d because the D-L(or R-S) isomer ((47a) i n Figure 4) was compatible with the sweet formula and could a l s o f i t the s p a t i a l b a r r i e r model (13), whereas the L-L(or S-S) isomer could n e i t h e r f i t the receptor model nor meet the sweet formula. Further examinations of the molecular f e a t u r e s and of the model of receptor have suggested that s e v e r a l a s p a r t y l t r i p e p t i d e e s t e r s may a l s o t a s t e sweet. In confirmation of the idea, s e v e r a l t r i p e p t i d e e s t e r s have been synthesized. In the f i r s t p l a c e , LAsp-Gly-Gly-OMe (38) was synthesized as an a r b i t r a r i l y - s e l e c t e d standard of t r i p e p t i d e s , because i t was considered that t h i s peptide e s t e r had the simplest s t r u c t u r e , and c o r r e l a t i o n of other peptides to (38) was easy. The t r i p e p t i d e e s t e r was p r e d i c t e d that i t would be s l i g h t l y sweet or t a s t e l e s s because i t s p r o j e c t i o n formula was s i m i l a r i n s i z e and shape to that of L-Asp-Gly0Bu which i s 13 times sweeter than sucrose (16) and because i t i s more h y d r o p h i l i c than the d i p e p t i d e . The t r i p e p t i d e (38) was devoid of sweetness and almost t a s t e l e s s . Next, L-Asp-D-Ala-Gly-OMe (39) was synthesized i n order to evaluate the c o n t r i b u t i o n of a small s i d e c h a i n , which i s properly o r i e n t e d to e l i c i t sweetness i n the p r o j e c t i o n formula. The peptide was speculated to be sweet. As expected, i t was sweet. L-Asp-D-Abu-Gly-OMe (40) was s e l e c t e d as a next candidate i n order to determine i t s sweetness i n t e n s i t y r e l a t i v e to L-Asp-DAla-Gly-OMe (39). The sweetness i n t e n s i t y of t h i s peptide was p r e d i c t e d to be lower than that of L-Asp-D-Ala-Gly-OMe a f t e r examining t h e i r formulas. As expected, the synthesized L-Asp-DAbu-Gly-OMe was sweet, and i t s sweetness i n t e n s i t y was lower than that of L-Asp-D-Ala-Gly-OMe. F i n a l l y , L-Asp-D-Val-Gly-OMe (41) was synthesized i n order to see whether i t remained sweet. The peptide was devoid of sweetness and almost t a s t e l e s s , though D - v a l i n e - c o n t a i n i n g a s p a r t y l d i p e p t i d e e s t e r s such as L-Asp-D-Val-OPr^ (17) and L-Asp-D-ValOPr^ (8, 17), which are s i m i l a r to the t r i p e p t i d e e s t e r i n s i z e and shape and have potent sweet t a s t e . As mentioned above, the second amino a c i d of the sweet a s p a r t y l d i p e p t i d e e s t e r s could be replaced by d i p e p t i d e e s t e r s n


5.

ARiYOSHi

Sweet Peptides

143

such as D-Ala-Gly-OMe and D-Abu-Gly-OMe without l o s i n g the sweetness. However, t h e i r sweetness potencies were c o n s i d e r a b l y lower than those of a s p a r t y l d i p e p t i d e e s t e r s w i t h the s i m i l a r s i z e and shape. Replacement of the second amino a c i d of a sweet a s p a r t y l d i p e p t i d e e s t e r such as L-Asp-Gly-OBu by Gly-Gly-OMe r e s u l t e d i n l o s i n g the sweetness ((38) i n Figure 5 ) , i n s p i t e of i t s s i m i l a r i t y i n the p r o j e c t i o n formula to that of the sweet d i p e p t i d e e s t e r . These f a c t s suggest t h a t the t r i p e p t i d e e s t e r s are more h y d r o p h i l i c than the d i p e p t i d e e s t e r s and the h y d r o p h i l i c property caused the sweetness i n t e n s i t y to decrease. The conformation of the t r i p e p t i d e e s t e r s has, of course, i n f l u e n c e on the e l i c i t a t i o n of sweetness i n connection w i t h the s p a c e - f i l l i n g p r o p e r t i e s of sweet compounds. However, the conformational problem can not be discussed here because i t has not been i n v e s t i g a t e d . In the case of s m a l l - s i z e d sweeteners such as g l y c i n e (48) and a l a n i n e (49), the sweetness s e n s a t i o n occurs only by the AH-B system and the sweetness i n t e n s i t y i s low, as described p r e v i o u s l y . In the case of medium-sized sweeteners such as a s p a r t y l d i p e p t i d e e s t e r s , two types of i n t e r a c t i o n have been considered. Among the a s p a r t y l d i p e p t i d e e s t e r s without a hydrophobic b i n d i n g s i t e such as L~Asp-Gly-OPr (11), the sweetness s e n s a t i o n has occurred only by the AH-B system, l i k e g l y c i n e , and the sweetness i n t e n s i t y i s comparatively low. On the other hand, i n t r o d u c t i o n of a small hydrophobic group i n t o the sweet molecule so as to i n t e r a c t w i t h a hydrophobic s i t e of the receptor r e s u l t s i n a sweeter compound such as L-Asp-D-Ala-OPr (12). The small hydrophobic group introduced p l a y s a r o l e i n enhancing the sweetness i n t e n s i t y by forming a hydrophobic bond w i t h the r e c e p t o r s i t e . This f a c t has been s u c c e s s f u l l y explained by the theory of the AH-B-X system, i n which X i s the " d i s p e r s i o n " s i t e proposed by K i e r and has been proved e x p e r i m e n t a l l y by us to be a hydrophobic b i n d i n g s i t e ( 4 ) . Therefore, i n the case of medium-sized molecules, we have been able to conclude that formation of a hydrophobic bond causes the sweetness potency to i n c r e a s e . On the other hand, i n the case of a s p a r t y l t r i p e p t i d e e s t e r s (39, 40), i t appears that a s m a l l hydrophobic s i t e f o r hydrophobic i n t e r a c t i o n i s necessary to f i t the receptor s i t e . One problem that remains i s the mode of i n t e r a c t i o n between the sweet peptides and the receptor s i t e . Despite a great number of s t u d i e s , the mechanism of a c t i o n of sweet s t i m u l i on the receptor i s not w e l l known. Stereoisomerism can be r e s p o n s i b l e f o r d i f f e r e n c e s i n t a s t e responses, and s p a c e - f i l l i n g p r o p e r t i e s are a l s o very important. These f a c t s suggest that the receptor s i t e e x i s t s i n a three-dimensional s t r u c t u r e . In t h i s connection, the sense of sweet t a s t e i s subject to the " l o c k and key" of biological activity. The above d i s c u s s i o n s , i n c o n j u n c t i o n w i t h previous r e s u l t s , support our previous idea that the receptor s i t e f o r sweet t a s t e i s composed of the AH-B-X system and i t s most l i k e l y shape i s a "pocket" as shown i n F i g u r e 6 ( 5 ) . In t h i s model, the s p a t i a l


w

n

n


144


COOH H^C^NH

COOH 2

R-

Chemistry of Sweet Peptides - American Chemical Society

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