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Activation of carboxylic acids by pyrocarbonates. Application of di-tert-butyl pyrocarbonate as condensing reagent in the synthesis of amides of protected amino acids and peptides
Tetrahedron Letters, Vol. 36, No. 39. pp. 7115-7118,1995
Pergamon
ElsevierScienceLtd Printedin GreatBritain 0040.-4039/95$9.50+0.1)0
0040-4039(95)01412-8
A C T I V A T I O N OF C A R B O X Y L I C ACIDS BY P Y R O C A R B O N A T E S . A P P L I C A T I O N OF D I - t e r t - B U T Y L P Y R O C A R B O N A T E AS C O N D E N S I N G R E A G E N T IN THE S Y N T H E S I S OF A M I D E S OF P R O T E C T E D A M I N O ACIDS AND P E P T I D E S
Vladimir
F. P o z d n e v
Institute o f Biomedical Chemistry, Russian Academy o f Medical Sciences, Moscow 119832, Russia
Abstract: Amides formation from protected amino acids and peptides was achieved in an
easy and convenient one-pot procedure using di-tert-butyl pyrocarbonate as activating agent in the presence of pyridine and ammonium hydrogencarbonate. The method gave good yields and did not induce racemization during the amidation of urethane protected amino acids.
C a r b o x y l i c acid amides is a large class of n a t u r a l and s y n t h e t i c c o m p o u n d s , i n c l u d i n g , drugs and p e p t i d e h o r m o n e s . Wide a p p l i c a t i o n o f c a r b o x a m i d e s s t i m u l a t e d an i n t e n s i v e search for m e t h o d s o f c a r b o x y l i c acids amidation. A l t h o u g h much has been done in this field o f s y n t h e t i c o r g a n i c chemistry, there is as yet no efficient method for c a r b o x y l i c acid amidation. The key p r o b l e m o f amide synthesis is the d e v e l o p m e n t of an a d e q u a t e method for carboxyl g r o u p a c t i v a t i o n . Indeed, it is the choice o f the carboxyl g r o u p a c t i v a t i n g r e a g e n t that d e t e r m i n e s a m e t h o d ' s efficiency - in p a r t i c u l a r , its c o n v e n i e n c e , o c c u r r e n c e o f side r e a c t i o n s and f o r m a t i o n of b y - p r o d u c t s . As an e x t e n s i o n o f the p r e v i o u s studies on c a r b o x y l i c acids a c t i v a t i o n by dialkyl p y r o c a r b o n a t e s 1"3, we have a t t e m p t e d to employ these c o m p o u n d s as c o n d e n s i n g r e a g e n t s in the s y n t h e s i s o f amides o f p r o t e c t e d amino acids and peptides. The work was done with c o m m e r c i a l l y a v a i l a b l e d i - t e r t - b u t y l p y r o c a r b o n a t e ( B o c 2 0 ) , a r e a g e n t widely used for the i n t r o d u c t i o n o f the B o c - p r o t e c t i n g group.
7115
7116
S e v e r a l a p p l i c a t i o n s o f B o c 2 0 are p o s s i b l e for the s y n t h e s i s o f c a r b o x y l i c acid amides. One
approach
is to
synthesize
symmetrical
anhydrides
or
active
e s t e r s 2 with
their
s u b s e q u e n t a m m o n o l y s i s . H o w e v e r , a p p l y i n g B o c 2 0 is m o r e e f f i c i e n t if its i n t e r a c t i o n with the c a r b o x y l i c a c i d p r o c e e d s in the p r e s e n c e o f an a m m o n i a d e r i v a t i v e with which the p y r o c a r b o n a t e r e a c t s much s l o w e r than it d o e s with c a r b o x y l a t e - i o n . Then the mixed a n h y d r i d e , f o r m e d as an i n t e r m e d i a t e , r e a d i l y r e a c t s with this a m m o n i a d e r i v a t i v e to give amide. A m m o n i u m h y d r o g e n c a r b o n a t e 4 is most s u i t a b l e for this p u r p o s e .
I f ammonium
h y d r o g e n c a r b o n a t e is a d d e d to a m i x t u r e o f c a r b o x y l i c acid, B o c 2 0 and p y r i d i n e in a s u i t a b l e s o l v e n t , a c a r b o x a m i d e is formed. Since the o r d e r o f r e a g e n t s ' a d d i t i o n was o f no i m p o r t a n c e for the yield o f the t a r g e t p r o d u c t , o n e - p o t p r o c e d u r e a p p e a r e d to be the most c o n v e n i e n t . P o s s i b l e p a t h w a y s o f initial r e a g e n t s ' c o n v e r s i o n to final r e a c t i o n p r o d u c t s are p r e s e n t e d in the scheme.
Scheme 1
R C O O H ' P y + (ButOCO)20
IRCO-O-COOBu t] + ButOH + CO2 l
+(NH3)
RCONH2 + ButOH + CO2
A l t h o u g h the a c t u a l r e a c t i o n p r o c e s s o c c u r s in a much m o r e c o m p l i c a t e d way and its p r e c i s e m e c h a n i s t i c d e t a i l s are not yet clear, the m e t h o d is simple ~, c o n v e n i e n t and the yields o f d e s i r e d p r o d u c t s are r a t h e r h i g h The r e s u l t s o b t a i n e d are d i s p l a y e d in T a b l e l . E a r l i e r it was shown that e s t e r i f i c a t i o n o f N - a l k y l o x y c a r b o n y l d e r i v a t i v e s o f chiral amino a c i d s with the B o c 2 0 - p y r i d i n e s y s t e m was not a c c o m p a n i e d by the r a c e m i z a t i o n i n d u c e d loss o f o p t i c a l a c t i v i t y 6. D e t a i l e d HPLC s t u d i e s also c o n f i r m e d the a b s e n c e o f r a c e m i z a t i o n u p o n a m i d a t i o n o f N - p r o t e c t e d amino a c i d s by the p r o p o s e d p r o c e d u r e . It a p p e a r s that Z - P r o - P h e - N H 2 is well s e p a r a b l e from Z - P r o - D - P h e N H 2 on RP HPLC C~8 in 30% w a t e r a c e t o n i t r i l e 7. In p a r t i c u l a r , HPLC o f L-L d i p e p t i d e , o b t a i n e d by a c y l a t i o n o f LPheNH2 with Z - p r o l i n e N - h y d r o x y s u c c i n i m i d e e s t e r (or by the mixed a n h y d r i d e m e t h o d ) , missed the p r e s e n c e o f any L-D d i a s t e r e o m e r a d m i x t u r e . H o w e v e r , a m i d a t i o n o f Z - P r o P h e - O H led to f o r m a t i o n o f up to 10% o f Z - P r o - D - P h e N H 2 .
W h e n Z - P r o - P h e O H was
a m i d a t e d in the p r e s e n c e o f c o p p e r ( I I ) c h l o r i d e ~, the r a c e m i z a t i o n was as low as 0.6% and it was s u p p r e s s e d a l t o g e t h e r by H O A t 9, h o w e v e r , amide yield was r a t h e r low (30%).
7117
T a b l e 1. P r e p a r a t i o n of N - P r o t e c t e d A m i n o A c i d and D i p e p t i d e A m i d e s RCO-NH2 entry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
RCO
solvent
Boc-Ala Z-AIa Boc-Asp(OBzl) Glp Z-GIp (Z-Cys): Boc-Glu(OBzl) Z-Gly Boc-Leu Fmoc-Leu Boc-Met Boc-Phe Boc-D-Phe Z-Phe Fmoc-Phe Boc-Pro Boc-Trp Boc-Tyr(Boc) Boc-Tyr(Bzl) Z-Val Z-Ala-Pro Z-Ile-Pro Z-GIy-GIy Z-Gly-Pro Z - P r o - P h e TM Z-Trp-Pro Z-Met-Pro
DO DO DO DMF MeCN DMF DO DMF DO DO DMF DO MeCN MeCN DO DO MeCN MeCN DO DO DO DO DO DMF DO DMF DO
yield %b 83 78 95 92 84 87 90 80 82 70 88 80 77 92 73 82 93 78 76 78 91 83 50 90 75 92 76
M.p. °C ¢
[a]D. d
126-127 136-137 158-160 152-160 156-157 193-194 122-123 134-135 145-146 138-139 120-121 142-144 141-143 163-164 162-163 102-104 137-138 160-161 170-172 205-208 160-161 114-115 180-182 145-146 170-172 97-98 136-137
-1,4 -4,5* +7,8 -19.0 +1.4 -172 r +4.1
Lit. M.p.°C 124-125 130-131 157-160
-11.1 -17.6 -7.3 +16.7 -14.5 -8.5 -18.2 -49.4 +6.7 +9,8 +13.6 +22.9 f -40.2 -93.8
data [a]o -2,7 -4,5" -2,6
4 10 4
199-201 120-122 138-139 144-146
+4.0
11 4
-11.4
4
142-149
+16.7
4
161-162
-2.6
4
104-106 133-136
-43.4 +7.7
4 4
170-171 204-206
+16.0 +25.5 f
4 10
179-181 150-151
-43.0 -54.6 -16.3 -65.5
ref.
12 13
a) Each c o m p o u n d gave a single spot on TLC. Satisfactory data of m i c r o a n a l y s i s were obtained for each new c o m p o u n d within an error range of -+0.3%. b) Isolated yield; c) Uncorrected capillary m e l t i n g points, d) c 1, EtOH at 18 ° C. c) c 1, MeOH, f) c 1, DMF.
Acknowledgment: Foundation
This
of Fundamental
research
was
Investigations,
supported
by
a
grant
from
the
Russian
(grant 94-03-08640).
R e f e r e n c e s and N o t e s A b b r e v i a t i o n s u s e d are: DO, d i o x a n e ; M e C N , a c e t o n i t r i l e ; M e O H , m e t h a n o l ; E t O H , ethanol, EA, ethyl acetate; DMF, dimethylformamide; Boc, tert-butyloxycarbonyl; Fmoc, 9-fluorenylmethyloxycarbonyl; Z, b e n z y l o x y c a r b o n y l ; HOAt, l-hydroxy-7azabenzotriazole; RP HPLC, reversed phase high performance liquid chromatography; TLC, thin liquid chromatography. All a m i n o a c i d s a r e o f t h e L - c o n f i g u r a t i o n ( u n l e s s otherwise stated).
7118
1.
Pozdnev, V.F. Zh. Org. Khim. 1983, 19, 882.
2.
Pozdnev, V.F. Int. J. Peptide Protein Res. 1992, 42, 407-414
3.
Pozdnev, V.F. Int. J. Peptide Protein Res. 1994, 44, 36-48
4.
Nozaki, S.; Muramatsu, I. Bull. Chem. Soc. Jap. 1988, 61, 2647-2648.
5.
Typical procedure: to a stirred solution of N-protected amino acid (10 mmol), pyridine (0.5 ml) and Boc20 (3 g, 13 mmol) in an appropriate solvent (10-15 ml) ammonium hydrogencarbonate (1 g, 12.6 mmol) was added and the mixture was stirred for 4-16 h. Ethyl acetate or mixture chloroform with 10% n-propanol
was
added and after washings with water and 5% H2SO4 the solution was dried, the solvent was evaporated and the product was triturated with ether. In another variant the reaction mixture was diluted with water (30-40 ml), stirred until crystallization was completed, a residue was then collected by filtration, washed by water, dried and recrystallized as necessary. 6.
Pozdnev, V.F. Bioorg. Khim. 1984, 10, 912-920.
7. HPLC condition: column Silasorb C18, 6 mm, 250 x 4.6 mm, eluent: CH3CN/H200.01% KH2PO4, flow rate lml/min., UV detection at 254 nm., retention time L,Dform:13.03 min., L,L-form: 15.05 min. 8.
Miyazawa, T.; Donkai, T.; Yamada, T.; Kuwata, S. Int. J. Peptide Protein Res., 1992, 40, 49-53.
9.
Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397-4398.
10. Galaverna, G.; Corradini, R.; Dossena, A.; Marchelli, R. lnt. J. Peptide Protein Res. 1993, 43, 53-57 11. Hirotsu, Y.; Shiba, T.; Kaneko, T. Bull. Chem. Soc. Jap. 1970, 43, 1564- 1567 12. Fruton J. S.; Bergmann M. J. Biol. Chem. 1942, 145, 871-878 13 Smith, E L.; Bergmann, M. J. Biol. Chem. 1944, 153, 627-630. 14. Acylation of L- or D-PheNH2 with Z-proline N-hydroxysuccinimide ester gave Z° Pro-PheNH2, m.p. 180-181 °, [ a ] D - 6 3 , 7 ° ( c 1, EtOH) (lit. data: m.p. 180181°[Izumiya, N.; Fruton, J. S. J. Biol. Chem. 1956, 218, 59.]) and Z-Pro-DPheNH~, m.p.166-167 °, [a]D-47.4 ° (C i, EtOH).
(Received in UK 24 May 1995; revised 25 July 1995; accepted 28 July 1995)
Pergamon
ElsevierScienceLtd Printedin GreatBritain 0040.-4039/95$9.50+0.1)0
0040-4039(95)01412-8
A C T I V A T I O N OF C A R B O X Y L I C ACIDS BY P Y R O C A R B O N A T E S . A P P L I C A T I O N OF D I - t e r t - B U T Y L P Y R O C A R B O N A T E AS C O N D E N S I N G R E A G E N T IN THE S Y N T H E S I S OF A M I D E S OF P R O T E C T E D A M I N O ACIDS AND P E P T I D E S
Vladimir
F. P o z d n e v
Institute o f Biomedical Chemistry, Russian Academy o f Medical Sciences, Moscow 119832, Russia
Abstract: Amides formation from protected amino acids and peptides was achieved in an
easy and convenient one-pot procedure using di-tert-butyl pyrocarbonate as activating agent in the presence of pyridine and ammonium hydrogencarbonate. The method gave good yields and did not induce racemization during the amidation of urethane protected amino acids.
C a r b o x y l i c acid amides is a large class of n a t u r a l and s y n t h e t i c c o m p o u n d s , i n c l u d i n g , drugs and p e p t i d e h o r m o n e s . Wide a p p l i c a t i o n o f c a r b o x a m i d e s s t i m u l a t e d an i n t e n s i v e search for m e t h o d s o f c a r b o x y l i c acids amidation. A l t h o u g h much has been done in this field o f s y n t h e t i c o r g a n i c chemistry, there is as yet no efficient method for c a r b o x y l i c acid amidation. The key p r o b l e m o f amide synthesis is the d e v e l o p m e n t of an a d e q u a t e method for carboxyl g r o u p a c t i v a t i o n . Indeed, it is the choice o f the carboxyl g r o u p a c t i v a t i n g r e a g e n t that d e t e r m i n e s a m e t h o d ' s efficiency - in p a r t i c u l a r , its c o n v e n i e n c e , o c c u r r e n c e o f side r e a c t i o n s and f o r m a t i o n of b y - p r o d u c t s . As an e x t e n s i o n o f the p r e v i o u s studies on c a r b o x y l i c acids a c t i v a t i o n by dialkyl p y r o c a r b o n a t e s 1"3, we have a t t e m p t e d to employ these c o m p o u n d s as c o n d e n s i n g r e a g e n t s in the s y n t h e s i s o f amides o f p r o t e c t e d amino acids and peptides. The work was done with c o m m e r c i a l l y a v a i l a b l e d i - t e r t - b u t y l p y r o c a r b o n a t e ( B o c 2 0 ) , a r e a g e n t widely used for the i n t r o d u c t i o n o f the B o c - p r o t e c t i n g group.
7115
7116
S e v e r a l a p p l i c a t i o n s o f B o c 2 0 are p o s s i b l e for the s y n t h e s i s o f c a r b o x y l i c acid amides. One
approach
is to
synthesize
symmetrical
anhydrides
or
active
e s t e r s 2 with
their
s u b s e q u e n t a m m o n o l y s i s . H o w e v e r , a p p l y i n g B o c 2 0 is m o r e e f f i c i e n t if its i n t e r a c t i o n with the c a r b o x y l i c a c i d p r o c e e d s in the p r e s e n c e o f an a m m o n i a d e r i v a t i v e with which the p y r o c a r b o n a t e r e a c t s much s l o w e r than it d o e s with c a r b o x y l a t e - i o n . Then the mixed a n h y d r i d e , f o r m e d as an i n t e r m e d i a t e , r e a d i l y r e a c t s with this a m m o n i a d e r i v a t i v e to give amide. A m m o n i u m h y d r o g e n c a r b o n a t e 4 is most s u i t a b l e for this p u r p o s e .
I f ammonium
h y d r o g e n c a r b o n a t e is a d d e d to a m i x t u r e o f c a r b o x y l i c acid, B o c 2 0 and p y r i d i n e in a s u i t a b l e s o l v e n t , a c a r b o x a m i d e is formed. Since the o r d e r o f r e a g e n t s ' a d d i t i o n was o f no i m p o r t a n c e for the yield o f the t a r g e t p r o d u c t , o n e - p o t p r o c e d u r e a p p e a r e d to be the most c o n v e n i e n t . P o s s i b l e p a t h w a y s o f initial r e a g e n t s ' c o n v e r s i o n to final r e a c t i o n p r o d u c t s are p r e s e n t e d in the scheme.
Scheme 1
R C O O H ' P y + (ButOCO)20
IRCO-O-COOBu t] + ButOH + CO2 l
+(NH3)
RCONH2 + ButOH + CO2
A l t h o u g h the a c t u a l r e a c t i o n p r o c e s s o c c u r s in a much m o r e c o m p l i c a t e d way and its p r e c i s e m e c h a n i s t i c d e t a i l s are not yet clear, the m e t h o d is simple ~, c o n v e n i e n t and the yields o f d e s i r e d p r o d u c t s are r a t h e r h i g h The r e s u l t s o b t a i n e d are d i s p l a y e d in T a b l e l . E a r l i e r it was shown that e s t e r i f i c a t i o n o f N - a l k y l o x y c a r b o n y l d e r i v a t i v e s o f chiral amino a c i d s with the B o c 2 0 - p y r i d i n e s y s t e m was not a c c o m p a n i e d by the r a c e m i z a t i o n i n d u c e d loss o f o p t i c a l a c t i v i t y 6. D e t a i l e d HPLC s t u d i e s also c o n f i r m e d the a b s e n c e o f r a c e m i z a t i o n u p o n a m i d a t i o n o f N - p r o t e c t e d amino a c i d s by the p r o p o s e d p r o c e d u r e . It a p p e a r s that Z - P r o - P h e - N H 2 is well s e p a r a b l e from Z - P r o - D - P h e N H 2 on RP HPLC C~8 in 30% w a t e r a c e t o n i t r i l e 7. In p a r t i c u l a r , HPLC o f L-L d i p e p t i d e , o b t a i n e d by a c y l a t i o n o f LPheNH2 with Z - p r o l i n e N - h y d r o x y s u c c i n i m i d e e s t e r (or by the mixed a n h y d r i d e m e t h o d ) , missed the p r e s e n c e o f any L-D d i a s t e r e o m e r a d m i x t u r e . H o w e v e r , a m i d a t i o n o f Z - P r o P h e - O H led to f o r m a t i o n o f up to 10% o f Z - P r o - D - P h e N H 2 .
W h e n Z - P r o - P h e O H was
a m i d a t e d in the p r e s e n c e o f c o p p e r ( I I ) c h l o r i d e ~, the r a c e m i z a t i o n was as low as 0.6% and it was s u p p r e s s e d a l t o g e t h e r by H O A t 9, h o w e v e r , amide yield was r a t h e r low (30%).
7117
T a b l e 1. P r e p a r a t i o n of N - P r o t e c t e d A m i n o A c i d and D i p e p t i d e A m i d e s RCO-NH2 entry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
RCO
solvent
Boc-Ala Z-AIa Boc-Asp(OBzl) Glp Z-GIp (Z-Cys): Boc-Glu(OBzl) Z-Gly Boc-Leu Fmoc-Leu Boc-Met Boc-Phe Boc-D-Phe Z-Phe Fmoc-Phe Boc-Pro Boc-Trp Boc-Tyr(Boc) Boc-Tyr(Bzl) Z-Val Z-Ala-Pro Z-Ile-Pro Z-GIy-GIy Z-Gly-Pro Z - P r o - P h e TM Z-Trp-Pro Z-Met-Pro
DO DO DO DMF MeCN DMF DO DMF DO DO DMF DO MeCN MeCN DO DO MeCN MeCN DO DO DO DO DO DMF DO DMF DO
yield %b 83 78 95 92 84 87 90 80 82 70 88 80 77 92 73 82 93 78 76 78 91 83 50 90 75 92 76
M.p. °C ¢
[a]D. d
126-127 136-137 158-160 152-160 156-157 193-194 122-123 134-135 145-146 138-139 120-121 142-144 141-143 163-164 162-163 102-104 137-138 160-161 170-172 205-208 160-161 114-115 180-182 145-146 170-172 97-98 136-137
-1,4 -4,5* +7,8 -19.0 +1.4 -172 r +4.1
Lit. M.p.°C 124-125 130-131 157-160
-11.1 -17.6 -7.3 +16.7 -14.5 -8.5 -18.2 -49.4 +6.7 +9,8 +13.6 +22.9 f -40.2 -93.8
data [a]o -2,7 -4,5" -2,6
4 10 4
199-201 120-122 138-139 144-146
+4.0
11 4
-11.4
4
142-149
+16.7
4
161-162
-2.6
4
104-106 133-136
-43.4 +7.7
4 4
170-171 204-206
+16.0 +25.5 f
4 10
179-181 150-151
-43.0 -54.6 -16.3 -65.5
ref.
12 13
a) Each c o m p o u n d gave a single spot on TLC. Satisfactory data of m i c r o a n a l y s i s were obtained for each new c o m p o u n d within an error range of -+0.3%. b) Isolated yield; c) Uncorrected capillary m e l t i n g points, d) c 1, EtOH at 18 ° C. c) c 1, MeOH, f) c 1, DMF.
Acknowledgment: Foundation
This
of Fundamental
research
was
Investigations,
supported
by
a
grant
from
the
Russian
(grant 94-03-08640).
R e f e r e n c e s and N o t e s A b b r e v i a t i o n s u s e d are: DO, d i o x a n e ; M e C N , a c e t o n i t r i l e ; M e O H , m e t h a n o l ; E t O H , ethanol, EA, ethyl acetate; DMF, dimethylformamide; Boc, tert-butyloxycarbonyl; Fmoc, 9-fluorenylmethyloxycarbonyl; Z, b e n z y l o x y c a r b o n y l ; HOAt, l-hydroxy-7azabenzotriazole; RP HPLC, reversed phase high performance liquid chromatography; TLC, thin liquid chromatography. All a m i n o a c i d s a r e o f t h e L - c o n f i g u r a t i o n ( u n l e s s otherwise stated).
7118
1.
Pozdnev, V.F. Zh. Org. Khim. 1983, 19, 882.
2.
Pozdnev, V.F. Int. J. Peptide Protein Res. 1992, 42, 407-414
3.
Pozdnev, V.F. Int. J. Peptide Protein Res. 1994, 44, 36-48
4.
Nozaki, S.; Muramatsu, I. Bull. Chem. Soc. Jap. 1988, 61, 2647-2648.
5.
Typical procedure: to a stirred solution of N-protected amino acid (10 mmol), pyridine (0.5 ml) and Boc20 (3 g, 13 mmol) in an appropriate solvent (10-15 ml) ammonium hydrogencarbonate (1 g, 12.6 mmol) was added and the mixture was stirred for 4-16 h. Ethyl acetate or mixture chloroform with 10% n-propanol
was
added and after washings with water and 5% H2SO4 the solution was dried, the solvent was evaporated and the product was triturated with ether. In another variant the reaction mixture was diluted with water (30-40 ml), stirred until crystallization was completed, a residue was then collected by filtration, washed by water, dried and recrystallized as necessary. 6.
Pozdnev, V.F. Bioorg. Khim. 1984, 10, 912-920.
7. HPLC condition: column Silasorb C18, 6 mm, 250 x 4.6 mm, eluent: CH3CN/H200.01% KH2PO4, flow rate lml/min., UV detection at 254 nm., retention time L,Dform:13.03 min., L,L-form: 15.05 min. 8.
Miyazawa, T.; Donkai, T.; Yamada, T.; Kuwata, S. Int. J. Peptide Protein Res., 1992, 40, 49-53.
9.
Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397-4398.
10. Galaverna, G.; Corradini, R.; Dossena, A.; Marchelli, R. lnt. J. Peptide Protein Res. 1993, 43, 53-57 11. Hirotsu, Y.; Shiba, T.; Kaneko, T. Bull. Chem. Soc. Jap. 1970, 43, 1564- 1567 12. Fruton J. S.; Bergmann M. J. Biol. Chem. 1942, 145, 871-878 13 Smith, E L.; Bergmann, M. J. Biol. Chem. 1944, 153, 627-630. 14. Acylation of L- or D-PheNH2 with Z-proline N-hydroxysuccinimide ester gave Z° Pro-PheNH2, m.p. 180-181 °, [ a ] D - 6 3 , 7 ° ( c 1, EtOH) (lit. data: m.p. 180181°[Izumiya, N.; Fruton, J. S. J. Biol. Chem. 1956, 218, 59.]) and Z-Pro-DPheNH~, m.p.166-167 °, [a]D-47.4 ° (C i, EtOH).
(Received in UK 24 May 1995; revised 25 July 1995; accepted 28 July 1995)