CA1046441A - Peptide synthesis using pepsin - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The present invention provides a process for producing a peptide having the formula X-A-B-C-Y (III) wherein A represents Ala , Gln., Asn., .omega.-BOC-Lys., Leu., Gly., Glu., or Pro; A may be A1 - A2 and A1 represents a hydrophilic amino acid residue and A2 represents Val., Met., Leu. or Gln.;
B represents Phe., Tyr., Leu., Met., Glu., Asp., Gln., Asn or Trp.;
C represents Phe., Leu., Ileu., Tyr., Cys-SBzl., Ser-OBzl., Trp.
or Met.; X represents an .alpha.-amino acid protective group;
an amino acid residue having N-terminal protective group or a peptide residue having N-terminal protective group;
Y represents a carboxyl protective group;
an amino acid residue having C-terminal protective group or a peptide residue having C-terminal protective group, which process comprises reacting a peptide having the formula X-A-B-OH (I) with an amino acid or a peptide having the formula H-C-Y (II) in the presence of enzyme pepsin. The peptides of formula III
have physiological activity.

Description

` ~10~6441 ..
The present lnvention xelates to a process for producing a peptide in the presence of pepsin. More particularly, the present invention relates to a process for producin~ a peptide -~ having the formula X-A-B-C-Y ....... ~........ (III) wherein A represents Ala., Gln, Asn., ~-BOC-Lys., Leu., Gly., ~ Glu., Glu. or Pro.; A can be Al-A2 and ~1 represents a hydrophilic ,, OMe ,~
amino acid residue and A2 represents Val., Met., Leu. or Gln.;

-~ 10 B represents Phe., Tyr., Leu., Met., Glu., Asp., Gln., Asn or Trp.;

~ C represents Phe., Leu., Ileu., Tyr., Cys-SBzl., Ser-OBzl., Trp., ;:
or Met.; X represents an ~-amino acid protective group, an amino ~ . ..
~ acid residue having N-terminal protective group or a peptide ;,-~ residue having N-terminal protective group; and Y represents a carboxyl protective group, an amino acid residue having C-terminal protective group or a peptide residue having C-terminal protective group.
, The hydrophilic amino acid residue of Al can be Ala., Gln., `.~ Asn., ~-BOC-Lys., Leu., Gly., Glu., Glu., or Pro.
~`' 20 OMe According to the present invention such a peptide of formula (III?
~- is produced by reacting a peptide having the formula X-A-B-OH ................. ~I) wherein X, A and B are as aforesaid with an amino acid or a peptide having the formula ~-, H-C-Y .................... (II) ; ~ wherein Y is as aforesaid in the presence of enzyme pepsi~3'~'2~' ~ arious peptides having physiological actiyity have recently been found, and syntheses of such peptides have been developed. Typical processes for producing peptides include an azide methodi a mixed acid anhydride method; a dicyclohexyl carbodiimide method; and an active ester method. However, in these :,:.
, , ., . ~ . . .

~ 469L~
...... .
conventional processes, disadvantages including racemization, side reactions, complicated temperature control and long reaction times, have been found. Especially, in the fragment condensation method, the disadvantage of inherent racemization has been found.
~ In the syntheses of peptides, the racemization problem is serious.
:~ When the racemization occurs, a purity of the product decreases , , and the separation of the isomer impurity is disadvantageous in an industrial operation.

-~ Certain peptide syntheses by using an enzyme papain have been disclosed in addition to the organic chemical processes.

[For example, O.K. Behrens and M. Bergmann; J. Biol. Chem., 129,587 (1939) and H.B. Milne and Warren Kilday; J. Org. Che., 30, 64 ... .
` (1965)] The term of "peptide" means that defined in Hary D. Law;

- "The Org. Chem. of Peptides" (Willy Interscience 1970) page 6, .
and it is preferable to use as reactants peptides (including : polypeptide and oligopeptide) having at least a slight solubility - in the medium, as a peptide which is insoluble is inactive in thP
medium. The term of "pepsin" especially pepsin titer is illus~
trated in Anson, M. "J. Gen. Physiol." Vol. 22, P. 79 1938.
v`- 20 The racemization problem has been solved for prepara-. . .
tions of di- or tri-peptide. When a tetra- or higher peptide is ,,, synthesiz~d using a papain having various substrate specificity, ~"~b' the side reactions of a hydrolysis of peptide, a transpeptidation .':.................................................................... ~ .and a formation of plastein have occurrëd. There is thus no peptide synthesis or an industrial saale.
. -, The inventors have studied on a formation of peptide ; bond in order to overcome the abova disadvantages, and have com-pleted syntheses of polypeptides especially higher than tripep-~` tides by using an enzyme pepsin which is different from those of the conventional processes. The proteolytic enzymes (protease) ;, .~
have characteristic hydrolyzing properties for peptides in good reproducible operation, and under mild conditions w~thout side ~ ,~r I ~., . :

., , :

46~41 reactions. Accordingly, the proteolytic enzymes have been utiliz--~ ed for varlous studies of chemical structures of polypeptides and proteins. The preliminary structures of various natural polypeptides such as insulin have been determined by using them.
Pepsin is classified in digestive enzymes of endopeptitases, i.e.
enzymes which are active to an inner bond of peptide chain as proteolytic enzymes.
;~ It has now been found that pepsin can be used for synthesis of desirable peptides in a simple operation in higher yield.
- The process of the present invention can be applied to ~ the sequential method and the fragment condensation method under - the conditions given hereinafter. When the process of the present invention is applied to the fragment condensation method which is markedly advantageous compared to the sequential method, the advantages of the process is quite significant.
' In the process of the present invention, the peptide having the formula (I) (hereinafter referring to C-component) is ~` used as a reactant. The C-component should have a partial conden-sation structure of two or three specific amino acids in a specific connection i.e. the bond of -A-B- in the formula (I) in the reactive position. As far as the specific partial structure - is included, various peptides can be used. The terminal group of the C-component, that is ~-position of amino group should be protected by a protective group for an amino group such as carb-`~ obenzoxy group (Z); substituted carbobenzoxy group e.g. p-methoxy-benzyloxycarbonyl group (pMZ); t-butyloxycarbonyl group (BOC) or tosyl group (TOS).
The amino acid or peptide having the formula (II) (here-inafter referred to as the N-component) which is the other react-; ant, should have, at the reactive position, the specific amino ~ acid such as phenylalanine, leucine, isoleucine, tyrosine, S-benzyl .... ..

.'' ` :

.: ::
. ~ . ~: : . .

~6~
;.' cysteine, O-benzyl serine, tryptophan, or methionine. The terminal ` carboxyl group of the N-component should be protected by a protective group for carboxyl group such as methyl ester, ethyl ester, benzyl ester, t-butyl ester or p-nitrobenzyl ester group;
amide group, substituted amide group e.g. 2,4,6-trimethyl benzyla-` mide group (TMB), hydrazide or a derivative thereof. The N-comp--- onent can be free base type as well as a salt type such as hydro-chloride, hydrobromide, trifluoroacetate, p-toluenesulfonate.
: The N-component and the C-component can be selected from the above-mentioned materials,as an object peptide unit. Desirable peptides can be produced by the fragment condensation.
In the process of the present invention, the C-component and the N-component may be used in equivalent amounts. However ::
it is possible to react them with an excess of one of them. The molar ratio of the N-component to the C-component is usually . . ~ .
, 1 : 5 ~ 5: 1 preferably 1: 2 ~ 2 : 1.
In the reaction, it is preferable to use the N-component as a solution in an alkaline medium containing as alkali such as alkali metal hydroxide or alkaline earth metal hydroxide. It is ` 20 necessary to react the reactants in a buffer solution having pH
;~- of 2 to 6 at a temperature from 20 - 50C. The buffer solution ,~ can be for example citric acid buffer solution, Michaelis buffer solution, Mcllvaine buffer solution. The use of a buffer solution ., .
having pH of lower than 2 or higher than 6 is not desirable as '~' the yield is low. The preferred pH is 3 - 5, and the optimum pH
:; is 4. When the reactant temperature is lower than 20C, a long time is required to complete the reaction. When the reaction temperature is higher than 50C, the activity of pepsin is substan- ;
tially decreased, and the yield is low. The preferred temperature is 30 40 C.
Pepsin used in the invention is digestive enzyme of endopeptitases. Pepsin having pepsin titer 1 : 5,000; 1: 10,000;

-- ~L --.~ ., .

! . : ` . ~ .

~`;
1(~4644~
:.,.
and 1 : 60,000 can be easily obtained. As far as high activity, a crude pepsin can be used. A catalytic amount of pepsin is used and preferably 0.4 - 400 mg of pepsin per 1 m mole of the reactants is used.
The reaction is smoothly affected in water or water miscible medium such as methanol, ethanol, dioxane and dimethyl-formamide, The product is sparingly soluble in water or -the water miscible medium and accordingly, it it precipitated as crystals ~; from the reaction system. The crystals precipitated are filtered and are desirably washed with weak alkali aqueous solution, weak acidic aqueous solution and water, to obtain thepure product. The terminal protective group of N-component and the terminal pro-.::
`~ tective group of the C-component of the product can be removed by the conventional manner if desirable.
The present invention will be further illustrated by way of the following examples.
[Example 1]

:: -- A solution of 0.753 g (1.5 m mol) of HCQ-H-Phe-G:Ly-Leu-Met-NH

(MW 502.1) in 40 mQ of citric acid buffer solution ~pH=4.0) was , ., :
added to a solution of 1.26 g (2.5 m mol) of ~,~-Boc-Lys-Phe-OH
,;, (MW 505.6) in 5 mQ of lN-NaOH 30 mQ of water was then added with -- stirring, and 0.2 g of pepsin (1 : 5000 manufactured by Mikuni Kagaku Sangyo K.K.) was then added to the mixture with stirring ; at 40C for 24 hours to cause a reactant. The resulting white `~
.,,j . .
precipitate was filtered by a glass filter (G-3) and was washed ;
with 5% ammonia water, 5% citric acid aqueous solution and water, sequentially and then the product was dried over P2O5, under `;
`- reduced pressure at 50C, to obtain 1.25 g of ~,~-Boc-Lys-Phe-Phe-.
t' Gly-Leu-Met-NH2 having melting point of 207 - 216 C (decomposition) and [~]D5 = -38.8 (c = 0.5 DMF). The yield was 88.2% Elementary i. analYsis (C47H72010N8 `:

, .
:::
_ 5 _ '~

- ~09~6~4~L
.:-C H N S
Calculated (~)59.98 7.71 11.91 3.41 .: .
Found (%) 59.74 7.71 11.74 3.42 (Reference) A 4.0 g of ~ Boc~Lys-Phe~Phe~Gly~Leu~Met~NH2 was dissolved in -~ 100 mQ of glacial acetic acid, and HCQ gas was injected into the solution with stirring for 40 minutes to remove the protective group of Boc. After the reaction, the reaction mixture was added to 500 mQ of cooled ether to form precipitate. The precipitate was filtered and dried above NaOH, under reduced pressure, to obtain 2.70 g of 2HCQ H~Lys~Phe~Phe~Gly~Leu~Met~NH2 having melting point of 168 ~ 170C (decomposition) and [~]D5 = ~16.0(c = 0.5 50~ MeOH).
- The yield was 78.0%.
~- Elementary analysis (C37H56O6N8S.2HCQ-1 2 H2O = 841.915) ,~ C H N S CQ
Calculated (%) 52.97 7.04 13.31 3.52 8066 Found (%) 52.84 7.33 13.33 3.81 8.42 [Example 2]
A solution of 0.753 g (1.5 m mol) of HCQ H~Phe~Gly~Leu~Met~NH
(MW502.1) in 40 mQ of citric acid buffer solution (pH = 4.0) was added to solution of 1.0 g (2.5 m mol) of pMZ-Ala~Phe~OH(MW 400.4) in 5 mQ of lNNaOH 30 mQ of water was then added with stirring, and -then,0.2gof pepsin (1 : 5000 manufactured by Mikuni Kagaku Sangyo K.K) was added to the mixture with stirring in an incubator at 40C for 24 hours to react them. The resultingwhite precipitate was filtered, and was washed with 5% ag.NH40H, 5% citric acid aqueous solution and water sequentially and then the product was dried above P2O5, under reduced pressure at 50C, to obtain 1.14 g ;
j~ of pMZ~Ala~Phe~Phe~Gly~Leu~Met~NH2 having melting point of 229 ~
'~!, 30 233 C and [~]D5 = ~55 0 (c = 0.5 DMF).
The yield was 89.2%

~! Elementary analysis (C43H5709S = 847.031) ~ 6 ~

~09169~41 '.:
C H N S
Calculated (%) 60.81 6.92 11.43 3.92 Found (%) 60.90 6.77 11.56 3.78 [Example 3 ]
A 1.14 g ~2.5 m mol) of pM2-Gln-Phe-OH (MW 457.5) and a 0.75 g (1.5 m mol) of HCQ~H-Phe-Gly-Leu-Met-NH2 (MW 502.1) were used in accordance with the process of Example 1, the reaction was ' carried out at 40C for 24 hours to obtain 1.05 g of pMZ-Gln-Phe-Phe-Gly-Leu-Met-NH2 having melting point of 236 - 237 C, and [~]D5 = -22.8 (c = 1, DMSO). The yield was 77.5%.
Elementary analysis (C45H60OloN8 i - C H N S
~ Calculated (%) 59.71 6.68 12.38 3.54 :;:
Found (%) 59.89 6.58 12.31 3.46 - [Example 4 ]
-;~ In accordance with the process of Example 1, the reaction was :
~; carried out by using 1 g (2.5 m mol) of pMZ-Ala-Phe-OH (MW 400.4) and 0.93 g (1.5 m mol) of HCQ-H-Ileu-Gly-Leu-Met-NHDmB(MW 618.2) to obtain 1.30 g of pMZ-Ala-Phe-Ileu-Gly-Leu-Met-NHDmB having melting point of 225 - 227 C and [~]D = -25.4(c = 1, DMF) wherein DmB represents 2,4-dimethoxybenzyl. In this example, 0.4 g of , pepsin was used.
:.
~ The yield was 82.2%.
::~
Elementary analysis (C49H69N7OllS = 964.192) C H N S
Calculated (%) 61.04 7.21 10.17 3.33 ` Found (%) 61017 7.35 10.09 3.33 :.
[Examples 5 - 7.¦
In accordance with the process of Example 1, except reacting '~ 30 C-component (2.5 m mol) and N-component (2.5 m mol) of Table 1, ;

., ,; the corresponding peptides were obtained. The results are shown in the following Table 1.

:,~
, . ; , . : ;. ~.. ,, ,,,~ , ~:

lC14G441 o\O I ~ ~ ~ ~ ~ ~
r ~ ~1 ~D 00 " I ,u~ O ~ ~r ~
~n ~ ~ 1~ ~- ~ I~
U~ N N ~ CC) ~ ~ n ~' ~i O ~ U ~ U~ ~
.. ,.r-l ~ I 00 t~ ~I ~
.,,~,`~ ~1 ~ 1, ~ I~ ~I r-l `';; ~ Z~r n Z~
~. ~ a) m ~ ~ m ~g m x O ~
~ ~r - ~ ~ ~ - ~ o ~ .~ oo co .. , . a) ~ ~ . . ~ ~ . . ~ ~ . .
c~ ~ co ~ ~, a) ~ ~ c~ a) ,~ I~
,.` i ~ .......... ~ ........... ~
`` ! ~ ~ ~ `;~ ~ o ~ ~g ~ ~D ~ co '.. -,' O ~ ~: ~ O ~ ~: ~ O ~ In `~; ~ . ~ . ~ . ~ . ~ . ~ .
~, ~ ~ O :CO~ i~ o I`~ In O Ln . ,~ ~ ~ U~ ~ . U~C~ In ~4 In C) u~ :4 u~
~ ~ .
.. ~ O
~;. ~ ,~ o u~ oIn ou~
O ~u~ ~ ~ ~ . ~
o~ o_~ o1:~
~'- ~ ~ L~ ; ~ 11 ELI ~ 11 ;: . . . .. _ t`l O ~ N U ~ ~ .
~'';; tJ~
,. ~ ~ ~ ^
'.......... ~'0 oC) C' ~D c~ 00 C' 1n : ~ ~ -- ~ ~ ~ ~ ~r ~
,., , ~ ~I ~ _ .
.,j`,'' a)O\ô ~r t~
,.; ....... .,1 _~ ~r ~ ~o ,"'.,. . . ._. _ . .
;'.,l ~ I ~ ~ ~
~ 1l ~ Z 11~ ~ ~ ~
,~ ' ~ ~ m ~ a) ~ c~ m ~ c~ m :;, :j Q S-l I H ~ l¢ ~ Z 1 l ~ Z .:
Ql O ::~ V O ~ 1~ 0 ~, . .. _ m H ~ H ~
~-'i ' ~) I N ~ :
~ ~ z '' :

~ i O H ~ _- : ` `;
Z ~ :1 _ . '' .
.. " . ..

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m X ~ O
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.~, ~ ~., X U~ ~ I~
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' ' ' .~ ` ` ' . ~.

10~ 4~
,'.', o\ ~ J ~ co o ,~,, u~ ~ ~ O ~ ~ ~r ~r ',;~, ~ CO 1` U~ ~ ~ O ~ ~ U~ O
~-~ ~ Z CO ~ ~ ~ . . ~ o . ~ o~ mcO o~
. ~ ~ ~ CO X ~ O X ~ ~ ~, ~ CO
':' ~ ~ ~ ~ ~ a~ ~ ~ ~ D
,-........... ~ X ~ r- ~ ~ 1~ 1~ O 1 U D ~ D ~D D O o ~ ul U o~ O ~ ~ D

D ~ ~ r~ D ~ ~ ,~ ~ ~ ~ o u ___ _ ___ ~

~ O ~D cn In O ~r r~J
~
~0 ~D~ ~r~ ... ~
. ' ~:.- a~ ~
,.
; ~ a)O,o . .
~ ~ ~ CO N . . 00 _ Z ~ ~ Z ~ ~ ~ :~ .

m H ~ m p, ~ .C Ql m ~ ~ I m ~.` - ~ l ~ ~
~ ~ ~ ~ ~ ~ ~ ~ Z 1, ' ~ ~ ~ ~: ~ l ~ ~ ~
D H :~ ~ ~ ~

a ~ o O

o o~ ~ om o~aJ !
1~ ~ _ ,.,,.,.,,, ~0 00 O~ ~l ~
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[Example 12]
A solution of 0.539 g(2.5 m mol) of HCQ H-Phe-OCH3(MW = 215.67) in 40 mQ of citric acid buffer solution (pH = 4.0) was added to a solution of 1.00 g (2.5 m mol) of pMZ-Ala-Phe-OH(MW = 400.42) -~ in 5 mQ of lN-NaOH. 20 mQ of water was then added with stirring, ~ i .-' and then, 0.2 g of pepsin (1 : 5000) was added to the mixture . with stirring at 40C for 24 hours to react them. The resulting ` white precipitate was filtered by a glass filter (G-3), and - was washed with 5% ammonia water, and water, sequentially, and ~' 10 then the product was dried under reduced pressure and recrystalli-~- zed from methanolto obtain pMZ-Ala-Phe-Phe-OCH3 having melting ~- point of 189 - 192 C, and [~ ~ = -17.2 (c = 1, DMF).

.~; The yield was 59.3%

~ Elementary analysis (C31H35N3O7 = 561.641 , C H N
Calculated (%) 66.30 6.28 7.48 Found (%) 66.23 6.23 7.53 ,.................................... . .
[Examples 13 - 21]

. In accordance with the process of Example 12 except reacting f~' 20 C-component (2.5 m mol) and N-component (2.5 m mol), the corres-: ponding peptides were obtained. The results are shown in Table 2.
,.................................... `
:,,, , ~'' 'f"
' :

. . ~ .
'" "' ' ' - 1- ,',.
'` .
''~
~ 30 / ~
,~
,, ., 'f~
, :-. .

~046~41 ~ ..... _ . _ __ .. d U~U~ UlU~ ::1 n~ 0~ ~ oo~ 0~1 U)~ u~,~ ODO~ ~ 0~ ..
,' ~ ~ I~ I~ ~O ~ ) O~ I~ I~ ~D ~ O ~ Ot) ~ ~ ~1 i i ~ ,' h `D ~ ,~ a) oo O O U) ~ Cl~ I~ ~ O~ ~ `I `;t ~
5 P~ ~ ~D ~ `D r~ ~ ~D ~ ~ 7 ~D ~ ~D ~9 ~1:) `D t~ ,.
~ o~ o ~ c~ o ~1 a~ ~ ~ ~ ~ a~ `D O O `D ~ `D ~ ~
. . ~I ~ ~ ~ ~ ~ ~) ~ ~ U'l n co o~ ~J ~r) ~ ~ t~
. ~1 ~D ~D ~D ~D ~D ~D ~D ~D ~D ~) ~D ~D ~D ~ ~D ~D ~D ~C) ~' C~ ~ ~4 ~ ~ ~ LI ~) ~ t~ ~ C.) 1:4 ~) ~4 ~ C~
'.''' _ .. _ . -. :
., O ~ O o ~ 0~ C~l I ~ X O `O ^ 0!~ o ~

~.. ._ . .. __ ._ _ ___ ,:

.a. U ~ .-1 ~ ~D ~ ~ ~ ~ 1~ ~D ~ t~l ~ ~ N N N ~1 .;0 ~1~ ~D ~ O 00 J ~D ~ a~ ~.
p~ ~ ~ t- oo O u~ ~1 ~i ~c~ c~l ; ~ o 3 H 1~ U r @ ~ P~ ~4 O
~ ~ ~
.. O O N O O ~ ~ ~ @ t.') ~ ~ O . H CJ--C O ,!:1~ ~: rC Djl ~ ~ N

~ z t~ ~ ~ o ~ ¢ $ ~ ~ ~Q ;
i . ' ~ _ . _ _ : 8 ~ ~ :
O N _ _ _ _ _ _ _ _ Z

.~ t-d~l .................. ..... .. _ _. '':, " X ~ ~ ,1 ~ , ~ . ~ ~1 CO ~ O c~

.': .

,, .
'-' --11-- , ' .. . ... . .

['Example 22] 1 4~
The process of Example 12 was repeated except using 30 mQ of water and 0.1 g of pepsin (l : 10000), and reacting for 18 hours to obtain pMZ~Ala-Phe-Phe-OCH3 having the melting point of 189 -193C. The yield was 51.3%.
Example 23~
- The process of Example 12 was repeated except using 30 mQ of J''i~` water and reacting for 18 hours to obtain pMZ-Ala-Phe-Phe-OCH3 having the melting point of 187 - 192C. The yield was 59%.
[Example 24]
The process of Example 22 was repeated except using 0.2 g of pepsin (1 : 1000) to obtain pMZ-Ala-Phe-Phe-OCH3 having the melting point of 188 - 193C. The yield was 65.8%.
[Example 25]
The process of Example 12 was repeated except using 30 mQ of ?:`:
water and reacting for 10 hours to obtain the same object product.
The yield was 50.0%.
~Example 26] -~
A solution of 2.5 m mol of HCQ H-Leu-Met-NH2 in 40 mQ of citric acid buffer solution (pH = 4.0) was added to a solution of 2.5 m mol of pMZ-Ala-Phe-OH in 5 mQ of lN-NaOH, and then 0.2 g of pepsin of Example 8 was added at 40C for 24 hours to cause a reaction The resulting white precipitate was washed and dried in accordance with the process of Example 8, and recrystallized from MeOH-H2O
to obtain 1.02 g of pMZ-Ala-Phe-Leu-Met-NH2 having melting point of 218 - 219 C and [~]D = 31.6 (c=0.5 DMF). The yield was '~
63.3%.
Elementary analysis (C32H4 N O S = 643 809) C H N S
Calculated (%) 59.70 7.0510.88 4.98 Found (%) 59.94 7.1910.78 4.92 : . .

.
!;~,.-- . .. .
~",~.~ , : . ', ', ",.
... .

~046~4~
` [Example_27]

A solution of 0.995 g(2.5 m mol) of 2HBr-H-Phe-Gly-NHNH2 (MW = 398.105) in 40 mQ of citric acid buffer solution of Example 12 was added to a solution of 1.50 g (3.75 m mol) of pMZ-Ala-Phe-OH in 7.5 mQ of lN-NaOH, 30 mQ of water was then added with t!:
stirring, and then the reaction was carried out in accordance ;~ with the process of Example 12. The resulting precipitate was washed and dried and recrystallized from DMF-MeOH-Et2O to obtain 0.945 g of pMZ-Ala-Phe-Phe-Gly-NHNH2 having the melting point of 208 - 210 C, and [~]D = -31.8 (c-l DMF). The yield was 61.2~.

` Elementary analysis (C32H38N6 7 3 2 C H N
` Calculated (%) 61.52 6.2313.45 -- Found (~) 61.38 6.2513.38 : [Example 281 , . : .
` A solution of 2.5 m mol of HCQ H-Phe-OCH3 in 40 mQ of citric acid buffer solution of Example 8 was added to a solution of 1.47 g i (40 m mol) of pMZ-Ala-Leu-OH(MW =366.4) in 5 mQ of lN-NaOH, and ' 20 then the reaction was carried out in accordance with the process of Example 26. The resulting precipitate was washed and dried ... .
to obtain 0.302 g of pMZ-Ala-Leu-Phe-OCH3 having melting point of 187 - 191 C, and [~]D = 18.0(c=0.5 DMF). The yield was 22.9~-Elementary analysis (C28H37N3O7. 3- H2O = 533.628).
" ,~ :
, C H N
` Calculated (~) 63.02 7.11 7.87 .. .j . .
Found (~) 62.906.977.97 [Example 29]

`,A solution of 0.i93 g (2.5 m mol) of HBr H-Phe-Gly-OCH3(MW =

317.18) in citric acid buffer solution of Example 8 was added to , l ::
~ ~L046~
.
"~ a solution of 1.50 g (3.75 m mol) of pMZ-Ala-Phe-OH ln 5 mQ
.;, -; of lN-NaOH and then 20 mQ of water was added. The reaction was , .:
~i: carried out in accordance with the process of Example 8. The .. . .
resulting precipitate was washed, dried and recrystallized to obtain 1.46 g of pMZ-Ala-Phe-Phe-Gly-OCH3 having melting point of 227 - 229 C and ~]D = -26.7 (c = l DMF). The yield was 94.9%.

. . . .
" Elementary analysis (C33H35N4O8 = 618.693).

~ C II N
i,.',, .
Calculated (%)64.07 6.19 9.06 Found (%) 63.79 6.32 8.96 ~- [Example 30]
:- :
~ In accordance with the process of Example 28, the reaction was -~ carried out by using 4.0 m mol of pMZ-Ala-Met-OH and 2.5 m mol `~ of HCQ H-Phe-OCH3 to obtain 0.3413 g~of pMZ-Ala-Met-Phe-OCH3 ,j having melting point of 153 - 167 C and [~]2 = -17.4 (c=0.5 DMF).
~, .
-~- The yield was 24.6%. Elementary analysis (C27H35N3O7S = 545.660).

`~ C H N S

Calculated (%)59.43 6.47 7.70 5.88 Found (%)59.82 6.38 7.78 5.54 [ Example 31]

The reaction of Example 28 was repeated except using HCQ-H-Phe-Gly-OEt (2.5 m mol) instead of HCQ-H-Phe-OCH3(2.5 m mol). The ~

~ product was washed with 5% ammonia water/ and then water, and ~;

; then dried above P2O5 to obtain 0.8153 g of pMZ-Ala-Leu-Phe-Gly-OEt having melting point of 175 - 192 C and [~]D3 = -23.4 , (c=0.5 DM~). The yield was 54.5%.
~ ~
Elementary analysis (C31H42N4O8 = 598.703).

C H N

Calculated (%)62.19 7.07 9.36 Found (%) 62.06 7.01 9.20 ~: .
,1 ;, '"~' -. .
;.
. ~'.' ` . "

CExample 32]
A solution of 1.5 m mol of HCQ~H-Ileu-Gly-Leu-Met-NH2(MW = 468.1) in 40 mQ of citric acid buffer solution of Example 12 was added to a solution of 2.5 m mol of pMZ--Ala-Phe-OH in 5 mQ of lN-NaOH
and then was reacted for 48 hours at 40C in the presence of 0.2 g of pepsin of Example 12. The resulting precipitate was dried above P2O5 at 60C for 18 hours to obtain 1.10 g o~ pMZ-Ala-Phe-Ileu-Gly-Leu-Met-HN2 having melting point of 253.5 - 255 C
and [~]D ~ -42.4 (c=l, AcOH). The yield was 90%.
[_xample 33]
A solution of 0.702 g(l.5 m mol) of HCQ H-Ileu-Gly-Leu-Met-NH2 ~MW = 468.1) in 40 mQ of citric acid buffer solution of Example 12 was added to a solution of 1.15 g (2.5 m mol) of BOC-Asn-Ala-Phe-OH(MW = 450.5) in 5 mQ of lN-NaOH in the same manner with Example 22 and then, the precipitate was washed with 5% ammonia ; -water, 5% citric acid aqueous solution and water sequentially , and then the product was dried above P2O5 to obtain 1.052 g of -~
BOC-Asn-Ala-Phe-Ileu-Gly-Leu-Met-NH2 having melting point of 256 - 260 C and ~]23 = -36.2 (c=0.5 DMF). The yield was 81.0%.
Elementary analysis (C40H65NgOllS = 864.082).
C H N S
Calculated (%) 55.60 7.58 14.59 3.71 Found (%) 55.43 7.69 14.31 3.65 ~Example 34]
The process of Example 28 was repeated except using 1.32 g (2.5 m mol) of pMZ-Gln-Ala-Phe-OH (MW = 528.5), and 0.702 g(l.5 m mol) of 2HCQ H-Ileu-Gly-Leu-Met-NH2(MW = 468.1) instead of pMZ-Ala-Leu-OH and HCQ-H-Phe-OCH3 to obtain 1.04 g of pMZ-~n-Ala-Phe-; Ileu-Gly-Leu-Met-NH2 having the melting point of 258 - 260 C
;i~ 30 (decomposition). The yield was 74.5%.
Elementary analysis (C74H67N7011S
.. ;,,~

~ - 15 -'' ' . ~,o~6~4~ .
C H N S
Calculated (~) 56.82 7.26 13.55 3.45 Found (~) 56.56 7.16 13.49 3.25 [Example ~
A 1.60 g (4m mol) of pMZ-Ala-Phe-OH was solved in lN-NaOH and on the other hand, 0.54 g (2.5 m mol) of HCQ H-Phe-OCH3 was solved in each 40 mQ of citric acid buffer solution having various pH values. Both solutions were mixed and30 mQ of water was added and then 0.2 g of pepsin of Example 12 was added to the mixture at 40 C for 24 hours to react them. The resulting precipitate was washed with 5% ammonia water! 5~ citric acid aqueous solution, and water sequentially, and then the product was dried above . . .
~ P2O5 at 50 C to obtain pMZ-Ala-Phe-Phe-OCH3. The relation of ; pH of citric acid buffer solution and the yield is shown in the following Table 3.

Table 3 ,~

` Yield m.p. [~]25 (~) (C) (c = 1, DMF) - - ~

2.38 61.1 170 - 173 -18.4

3.28 74.6 185 - 189 -16.7

4.08 99.9 186 - 189 -18.0

5.02 64.0 189 - 190 -17.2 ~Example 36]

A solution of 2 m mol of 2HBr H-Phe-Gln-NHNH2 in 40 mQ of citric acid buffer solution (pH = 4.0) was added to a solution of 3.75 m mol of pMZ-Ala-Tyr-OH in lN-NaOH, and then, 0.2 g of pepsin was added to the mixture 25C for 48 hours to react them in accordance with the process of Example 8, to obtain pMZ-Ala-Tyr-Phe-Gln-NHNH2 havin~ melting point 245 - 248C (decomposition) and[~]D2 = -4.6 (c=l, DMSO). The yield was 57.8~.

.. " , ~ . . .
. .
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Elementary analysiS (C35H45N708 H20 C H N
Calculated (%) 57.92 6.52 13.52 Found (%) 57.98 6.35 13.54 [Example 37]
The process of Example 12 was repeated except using 2.5 m mol of pMZ-Ala-Phe-Ala-Phe-OH and 2.5 m mol of HCQ-H-Leu-Met-NH-DMB and reacting for 14 hours to obtain p~Z-Ala-Phe-Ala-Phe-Leu-Met-NH-DMB having melting point of 250 - 268C and [~]D5~ -21.0 (c=0.4 DMF). The yield was 70%.
[Example 38]
The process of Example 12 was repeated except using 2.5 m mol of pMZ-Ala-Phe-Ala-Phe-OH and 2.5 m mol of HCQ-H-Ileu- Gly-OEt and reacting for 9 hours to obtain pMZ-Ala-Phe-Ala-Phe-Ileu-Gly-OEt having melting point of 248 - 252C. The yield was 63%.
[Example 39] -A solution of 0.216 g (10 m mol) HCQ H-Phe-OCH3(MW 215.5) in 40 mQ
of citric acid buffer solution (pH = 4.0) was added to a solution of 0.495 g(l0 m mol) of Z-Pro-Val-Phe-OH(MW 495) in 10 mQ of lN-.
NaOH, and then, 30 mQ of water was further added with stirring, and then, 0.1 g of pepsin (1 : 10000) was added to the mixture with stirring at 40C for 24hours to react them.
The resulting white precipitate was filtered by a glass filter (G-3) and was washed with 5% ammonia water, 5~ citric acid a~ueous solution and water sequentially, and then, the product was dried above P2O5, under reduced pressure at 50C to obtain 0.244 g of Z-Pro-Val-Phe-Phe-OCH3 having melting point of 199 - 201C and [~]D0 = -33.2 (c = 1, DMF). The yield was 37.2%.
[Example 40]
- --~ solution 0.216 g (10 m mol) of HCQ H-Phe-OCH3(MW 215.5 in 40 mQ
`~ of citric acid buffer solution (pH = 4.0) was added to a solution of 0.421 g of BOC-Gly-Val-Phe-OH(MW 421) in 10 mQ of lN-NaOH and , . :

. ... . . . .. . . . . . .

~ 6~4~

then 30 mQ of water was further added with stirring, and then 0.1 g of pepsin (1 : 10000) was added to the mixture with stirring in an incubator at 40C for 24 hours to react them. 0.414 g of BOC-Gly-Val-Phe-Phe-OCH3 having meIting point of 193 - 196C and ~- [~]D = -14.8 (c = 1, DMF) was obtained, The yield was 71.2%.
[Example 41]
The process of Example 39 was repeated except changing the molar ratio of Z-Pro-Val-Phe-OH: HC~-H-Phe-OCH3 -to 2 : 1 to obtain 0.297 g of the same product with Example 39. The yield was 51%.
. 10 [Example 42]
. _ The process of Example 40 was repeated except using tartaric acid-tartaric buffer solution (pH=4~ instead of citric acid buffer solution to obtain 0.426 g of the same product with Example 40.
The yield was 73.3%.

[Example 43]
_ The process of ~xample 40 was repeated except using disodium hydrogen phosphate-citric acid buffer solution (pH = 4) instead of citric acid buffer solution to obtain 0.396 g of the same product with Example 40. The yield was 68%.
:,...
, 20 [Example 44]
., .
The process of Example 39 was repeated except reacting for 20 hours , with 0.2 g of pepsin (1 : 5000) to obtain 0.230 g of the same product with Example 35. The yield was 35%.

[Examples 45 - 65]
.~
In accordance with the process of Example 39, by reacting C-compo-..
nent (10 m mol~ and N-component (10 m mol) of the following Table 4, the corresponding peptides were obtained. The results are ; shown in Table 4.
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'~ [Exa~ple 66]
A solution of 0.47 g (10 m mol~ of HCQ-H-Ileu~Gly-Leu-~et-NH2 - (MW 468.1) in 40 mQ of citric acid buffer solution was added to a solution of 0.38 g (10 m mol) of pMZ-Ala-Glu-OH (MW 382.4) OH
in 10 mQ of lN NaOH, and then 30 mQ of water was further added ^ with stirring, and then, 0.1 g of pepsin (1 : 10000) was added to ~ the mixture with stirring at 40C for 24 hours to react them.
-~ The resulting white precipitate was filtered by a glass filter ~' 10 (G-3) and was washed with 5% ammonia water, 5% citric acid -- aqueous solution and water sequentially, and then, the product was dried above P2O5 under reduced pressure at 50C to obtain 0.48 g of pMZ-Ala-flu-Ileu-Gly-Leu-Met-NH2 having melting point ;- OH
of 214 - 217C and [~]DO = -23.8 (c = 0.5, DMF). The yield was -60.3%. The elementary analysis of the product corresponded to the theoretical value within allowance.
~` [Example 67]
. .
The process of Example 66 was repeated except changing the molar ~ 20 ratio of pMZ-Ala-lGlu-OH and HCQ-H-Ileu-Gly-Leu-Met-NH2 to 2 ''~' OH

; to obtain 0.68 g of the same product with Example 66. The yield ''' was 85.3%.
~, ~ [Example 68']
;~;
The process of Example 67 was repeated except using disodium hydrogen phosphate-citric acid buffer solution (pH=4) instead of citric acid buffer solution to obtain the same product with Example 67. The yield was 84%.
[Example 69''- '7'5]

.,. ~ ..... _ _ '~l 30 In accordance with the process of Example 66, by reacting C-comp-~-~ onent (10 m mol) and N-component (I0 m mol) of Table 5, the corres-ponding peptides were obtained. The results are sho~n in Table 5.
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Claims (9)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for producing a peptide having the formula X-A-B-C-Y (III) wherein A represents Ala., Gln., Asn., .omega.-BOC-Lys., Leu., Gly., Glu., or Pro; A may be A1 - A2 and A1 represents a hydrophilic amino acid residue and A2 represents Val., Met., Leu. or Gln.;
B represents Phe., Tyr., Leu., Met., Glu., Asp., Gln., Asn or Trp,;
C represents Phe., Leu., Ileu., Tyr., Cys-SBzl., Ser-OBzl., Trp.
or Met.; X represents an .alpha.-amino acid protective group; an amino acid residue having N-terminal protective group or a peptide residue having N-terminal protective group; Y represents a carboxyl protective group; an amino acid residue having C-terminal pro-tective group or a peptide residue having C-terminal protective group, which process comprises reacting a peptide having the formula X-A-B-OH (I) with an amino acid or a peptide having the formula H-C-Y (II) in a weight ratio of the compound of formula (II) to the compound of formula (I) from 1 : 5 to 5 : 1 in the presence of enzyme pepsin in an amount from 0.4 to 400 mg. per mole of reactants.

2. The process according to Claim 1 in which the peptide of formula I has the formula X-A-B-OH
wherein A represents Ala., Gln., Asn., .omega.-BOC-Lys., or Gly.; and B represents Phe., Tyr., Leu., or Met.;
X is defined in Claim 1.

3. The process according to Claim 1 in which the peptide of formula I has the formula X-A-B-OH
wherein A represents Glu., , Pro.; and B and X are defined in Claim 1.

4. The process according to Claim 1 in which the peptide of formula I has the formula wherein A1 represents a hydrophilic amino acid residue and A2 represents Val., Met., Leu. or Gln.;
B and X are defined in Claim 1.

5. The process according to Claim 1 in which in the amino acid or a peptide having the formula H-C-Y
C represents Phe., Leu., Ileu., Tyr., Cys-SBzl., or Ser-OBzl; and Y is defined in Claim 1.

6. The process according to Claim 1 wherein the reaction is effected at a temperature from 20 - 50°C in a buffer solution having pH of 2 - 6.

7. A process according to Claim 6 in which the temperature is from 30°C to 40°C and the pH of the buffer solution is from 3 - 5.

8. A process as claimed in Claim 6, 7 or 8 in which the reactant is effected in water or a water miscible medium.

9. A process as claimed in Claim 6, 7 or 8 in which the ratio of the compound of formula (II) to the compound of formula (I) is 1 : 2 to 2 : 1.