Note: Descriptions are shown in the official language in which they were submitted.
1059938
The present invention relates to a process for
producing a peptide. More particularly, it relates to a
process for producing a peptide by using a specific enzyme
as a catalyst.
Typical conventional processes for producing peptides
include the azide method, the mixed acid anhydride method, the
carbodiimide method, the active ester method and the acid
chloride method. However, various industrial problems are
encountered by these conventional processes, such as racemiza-
tion of the carboxyl component of the C-terminal amino acid
residue occurs. Other probIems include side reactions, temper-
ature control, selection of solvent, the properties of amino
protective groups and carboxyl protective groups and the
effects of functional groups on the side chains of amino acids.
The fragment condensation method of preparing peptides can be
applied to advantage for compounds which contain glycine (the
; only amino acid which cannot be racemized) at the carboxyl
terminal group. However, for compounds containing any other
amino acid at the carboxyl terminal group the racemization cannot
be prevented. In actuality, on any peptide synthesis, the
racemization problem is serious. When racemization occurs, the
purity of the product is decreased, and is necessary to
separate the impure isomer fromthe product. This is very
detrimental for any industrial operation.
Among the conventioanl methods for forming peptide bonds,
the azide method is the only method in which racemization is not
much of a problem and itis for this reason that it is a desirable
method. However, the azide method involves complicated
operational procedures and a urea derivative is produced in
a side reaction. Because of these features the azide method
is undesirable from the viewpoint of yield. In addition to
-- 1 --
~osssas,
the various organic chemical processes, for preparing peptides,
a particular peptide synthesis using the enzyme papain or
chymotrypsin has been disclosed (See, for example, J.S. Fruton
"Advances in Protein Chemistry", 5, Academic Press`Inc. New York,
N.Y. 1949).
The reactions of the method are as follows:
(1) Bz-Leu-OH + H-Leu NH0
(I) (II)
papain Bz-Leu-Leu-NH0
~ (III)
(2) Bz-Leu-OH + H-Gly-NH0
(I) (II)
papain Bz-Leu-Gly-NH0
) (III)
(3) Bz-Tyr-OH + H-Gly-NH0
(I) (II)
chymotrypsin Bz-Tyr-Gly-NH0
~ (III)
(4) Z-Phe-Gly-OH + H-Tyr-NH2
(I) (II)
papain Z-Phe-Gly-Tyr-NH2
> (III)
to remove the phenylamino group from the peptide (III) under
severe conditions because the phenylamino group which is bonded
to the C-terminal group of the amine component (II) cannot be
easily separated fromthe peptide, and thus cleavage of the
peptide chain is disadvantageous. Because of this deficiency,
this mode of peptide synthesis cannot be practically used for
peptide synthesis. On the other hand, reaction (4) is accompanied
by transamidation and transpeptidation side reactions and thus
is not practically suitable. (See, for example, R.B. Johnston et
al; J. Biol, Chem., 185, 629(1950) and J.S. Fruton et al; J. Biol.
Chem., 204,891(1953). In reaction (4), the primary amino group
of the acid amide bonded to the terminal group of the amine
component, promotes the papain catalyzed amidase reaction.
-- 2 --
~059938
Accordingly, these processes provide only a theoretical interest
in showing that papain and chymotrypsin act as catalysts for the
synthesis of peptide bonds in which the phenylamino group is
used as the protective group for the terminal carboxyl group
of the amine component.
A need, therefore, continues to exist for a method of
peptide synthesis in which the variety of difficulties
encountered by the prior art procedures can be overcome.
Accordingly, the present invention provides a process
for synthesizing a desired oligopeptide or polypeptide by a
simple operation in high yield.
According to the present invention there is provided
a process for producing a peptide having the formula:
X-A-B-Y
wherein A and B are the same or different and represent an amino
acid residue or a peptide residue, X represents an amino protective
group, Y represents a carboxyl protective group selected from
the group consisting of substituted or unsubstituted tertiary
alkoxy, and benzyloxy, benzylamino and benzhydrylamino by
reacting an amino acid or peptide having an N-terminal protective
group or a salt thereof of the formlula
X-A-OH
with an amino acid or peptide having a C-terminal protective group
or a salt thereof of the formula
H-B-Y
in the presence of thiol proteinase or serine proteinase in an
aqueous solution having a pH sufficient to maintain the enzyme
activity of said thiol proteinase or serine proteinase.
The thiol proteinase enzyme includes enzymes of papain,
Stembromelein, Ficin, Cathepsin B, Chymopapain, Streptococcal
proteinase, Asclepain, Clostridium histolyticum protenase B and
Yeast protenase B. Thiol proteinase is characterized by having
:
-- 3 --
1o59g38
the ability to hydrolyze a wide range of proteins and to promote
the cleavage of many peptide bonds, amido bonds and ester bonds
as well as promoting the decomposition of benzoyl alginine amide,
benzoyl glycylleucylglycine and benzoyl tyrosylglycine amide
thereby having the activity of exo and endo peptidase (J.R. Kimmel
and E.L. Smith, Advance Enzymology, 19, 267 (1957). Serine
proteinase includes enzymes of Substilisin, Aspergillus alkaline
proteinase, Elastase, ~-Lytic proteinase, Chymotrypsin,
Metridium protenase A, Trypsin, Thrombin, Tlasmin, Kininogenin,
Enteropeptidase, Acrosin, Phaseolus protenase, Altemaria
endopeptidase, Arthrobacter serine protenase and Tenebrio ~-
protenase. It has been reported that the serine proteinase has
substa..tial hydrolysis activity to the Leu(15) - Tyr (16) and
Tyr (16) - Leu (17) sequence of the insulin B chain and also
exhibits high esterase activity to acylamino acid ester. The
subtilisin produced by using B. Subtilis or analogous germs
includes types of Carsbery, Novo, BPN' and the like~ The
alkaline proteinase isolated from ray fungus or fungi is a known
material. The amino acid or peptide starting materials having
the formula
X-A-OH
wherein ~ represents a protective group for the terminal amino
group and A represents an amino acid residue or a peptide residue
which is used in the process of the invention are referred to as
the acid component. The radical A in the formula more defin-
itively represents an amino acid residue or peptide residue where-
in suitable amino acids include aliphatic amino acids such as
monoamino monocarboxylic acids, e.g. glycine (Gly), alanine
(Ala), valine ~val), norvaline (nor-Val), leucine (Leu),
isoleucine(iso-Leu), norleucine(nor-Leu); oxyamino acids, e.g.
serine (Ser), theronine (Thr), homo-serine(homo-Ser); sulfur-
containing amino acids, e.g. methionine (~et) or cystine (CysS)
10599~8
and cysteine (CysH); monoamino dicarboxylic acids, e.g.
aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn)
and glutamine (Gln); diamino monocarboxyiic acids, e.g. ornithine
(Orn), lysine (Lys), arginine (Arg); aromatic amino acids, e.g.
phenylalanine (Phe), and tyrosine (Tyr); and heterocyclic
amino acids, e.g. histidien (His), tryptophan (Try). (These amino
acids are designated by symbols which are commonly used in the
field.) (The peptides are also designated by combinations of
these symbols).
Suitable protective groups for the free terminal amino
group (an N-terminal protective group) of the acid component
include tertiary alkoxy-carbonyl groups such as t-butyloxycarbonyl
(BOC-), t-amyloxycarbonyl (t-Aoc-); benzyloxycarbonyl (Z-),
p-methoxybenzyloxycarbonyl (PMZ), 3,5-dimethoxybenzyloxycarbonyl
{Z(OMe)2-}, 2,4,6-trimethylbenzyloxycarbonyl (TMZ-),p-phenylazo-
benzyloxycarbonyl (PZ-); p-toluenesulfonyl (Tos-); o-nitrophenyl-
sulfenyl (Nps-); and the like. The other amino acid or peptide
starting material having the formula
H-B-Y
which is used in the process of the invention is referred to as
the amine component. In the formula B represents an amino acid `
residue or peptide residue which can be the same defined above
as A. The protective groups for the carboxyl group (C-terminal
protective groups) of the amino component include tertiary alkoxy
groups suchàs t-butoxy (-OBut), benzyloxy (-OBzl), p-nitrobenzyl-
oxy~{-OBzl (p-NO2)}, benzhydryloxy (-OBzh), benzylamino (-NHBzl),
2,4-dimethoxy benzylamino (-NHDMB), and benzhydrylamino (-NHBzh).
These protective groups for the terminal carboxyl group of the
amine component are resistant to esterase and amidase reactions
which are caused by the thiol proteinase and serine proteinase
enzymes.
The acid component and the amine component used in the
1059938
process of the invention include the amino acid residues and
peptide residues which have a functional group on a side chain.
In most of these cases, it is prefexable to protect the function-
al group with a protective group. Suitable protective groups for
~-amino group (N~) can be N~-benzyloxycarbonyl (N~-Z), t-butoxy-
carbonyl (N~-BOC) and tosyl (N~-Tos). Suitable protective groups
for the N-guanidino group (NG) of ARg include nitro (NG-NO2),
NG-benzyloxycarbonyl (NG-Z) and NG. NG-dibenzyloxycarbonyl
(N -Z-Z). Suitable protective groups for imidazole rings (Nim)
of His include N m-benzyl (N m-Bzl) and tosyl (N m-Tos). Suitable
protective groups for the ~-carboxyl group include ~-benzyloxy
(-OBzl). Suitable protective groups for the hydroxyl group of
a liphatic or aromatic oxyamino acids include aralkyl groups
such as -benzyl (Bzl). Suitable S-protective groups for the
mercapto group of CysH include the benzyl group (Bzl). The prot-
ective groups should possess the characteristics of being stable
in the main reaction and that they can be easily separated from
the product without being involved in side reactions. The acid
component and the amine component starting materials can have
protective groups or the N~ amino group of the amine component
can be free or in the form of an inorganic or organic salt such
as a hydrochloride, hydrobromide, oxalate, p-toluenesulfonate
or acetate. In the process of the invention, the condensation
reaction in which the peptide bond is formed can be conducted
in an aqueous solution having a pH which maintains enzyme
activity which is about 4 to 7.5 for the thiol proteinase and
6 to 9 for the serine proteinase.
There are two methods which can be employed to achieve
the proper pH to maintain enzyme activity. One method is to
conduct the condensation reaction in a buffer solution such as
a citric acid buffer solution, McIlvaine buffer solution, Kolthoff
~059938
buffer solution, tris-HCl buffer solution, or veronal buffer
solution in which the acid component and the amine component
are dissolved and the enzyme is added. The other method is to
conduct the condensation reaction by maintaining the pH of the
reaction mixture in the proper range to maintain enzyme activity
by adding the acid or the base to the reaction mixture depending
upon the pH detected.
The starting materials are usually used in a ratio of
0.8 to two moles, preferably one to 1.5 moles of the acid
component per one mole of the amine component. If the starting
materials are not too soluble in the aqueous medium, it is possible
to improve the solubility of the reactants by adding a solvent
such as an alcohol, e.g. methanol, or ethanol; dimethylformamide;
dioxane; tetrahydrofuran; dimethylsulfoxide, or the like to the
aqueous solution. The amount of the added solvent should be
limited so as not to inhibit the activity of the enzyme in the
reaction of the invention. If a solvent is employed, it is
usually used in an amount of less than 1 part by weight,
preferably 0.2 to 1.0 part by weight per one part by weight
of water. The reaction of the invention is performed in an
aqueous medium, and it is necessary to decrease the relative
solubility of the reaction product preferably to a sparingly
soluble or insoluble state in the system.
The amount of thiol proteinase or serine proteinase
enzyme employed is in a range of 10 to 500 mg, preferably
10 to 400 mg, especially 50 to 300 mg per 1 mmole of the amine
component. An enzyme activator such as CysH, or a salt thereof
or 2-mercaptoethanol or a salt the.reof can also be added to the
solution. The reaction temperature employed is usually in a
range of 20 to 55C, preferably 30 to 40C which is sufficient
to maintain enzyme activity. The reaction proceeds smoothly
under these conditions for 1 to 24 hours. The reaction product
-- 7 --
~059~38
precipitates from the reaction system and the reaction product
can be easily isolated.
In accordance with the process of the invention, a
minimum size dipeptide, oligo-peptide or polypeptide having
the formula
X-A-B-Y
can be easily produced by appropriately selecting the desired
A and B radicals in the starting materials having the formulas
X-A-OH and H-B-Y.
When the dipeptide derivative of lysyl lysine which is produced
by the following reaction
Z-Lys(Z)-OH + H-Lys(Z)-OBut
) Z-Lys(Z)-Lys(Z)-OBut
wherein the Z-derivative produced by lysine whose ~-amino group
is protected with the carbobenzoxyl group (Z-), is used as the
acid component and a t-butyl ester is used as the amine component
the lysyl lysine whose amino group in the side chain is protected,
can be obtained. When the dipeptide derivative containing
arginine i.e. arginyl leucine which is produced by the following
reaction
Z-Arg(Z,Z)-OH + H-Leu-OBzh
~ Z-Arg(Z,Z)-Leu-OBzh,
wherein the tri-Z-arginine is produced by protecting the guanyl
group and the amino group of arginine with Z, is used as the
acid component and the benzhydryl ester of leucine is used as
the amine component, a dipeptide derivative can be easily obtained
by the reaction in the presence of papain. When an amino acid
having a functional group in th~ side chain is used, the amino
acid can be reacted with the functional group protected or
unprotected as in the examples. Various reactions of histidine
will be shown by the following reaction sequences.
lOS9938
BOC-His(Bzl)-OH + H-Leu-OBzh
) BOC-His(Bzl)-Leu-OBzh ...(a)
BOC-His-OH + H-Leu-OBzh
- > BOC-His-Leu-OBzh --(b)
Reaction (a) represents the situation in which the side chain
is protected, and the reaction (b) represents the case in
which the side chain is unprotected.
The dipeptide situation has been illustrated. Another
situation is the case of the acid component which has one
peptide bond and is represented by the reaction of an acyl
dipeptide with an amino acid amide derivative. The reaction
can be smoothly performed without the occurence of side reactions
in the presence of papain. This situation is shown by the
~ollowing reaction .
Z-Pro-Gly-OH + H-Leu-NHBzh
) Z-Pro-Gly-Leu-NHBzh
The above reaction proceeds smoothly to form carbobenzoxy-
prolyl-glycyl-leucine-benzhydrylamide.
Examples of the synthesis of tripeptides are shown in
Table 3. The situation in which the acid component is a
dipeptide and the amine component is a dipeptide will now be
illustrated. The following reaction can be applied for the
synthesis of fragment (3-6) tetrapeptide of Val5-angio-tension-II
which is known as a polypeptide hormone
BOC-Val-Tyr(Bzl)-OH + H-Val-His(Bzl)-OBzl
~ BOC-Val-Tyr(Bzl)-Val-His(Bzl)-OH
When valyl histidine ester is used as the amine component of the
reaction and the reaction is performed in the presence of
papain, a tetrapeptide containing a benzyl ester group cannot
be obtained. This is believed to be the result of an esterase
action which is one of the characteristics of papain. The above
lOS9938
reaction is especially advantageous when the tetrapeptide
is used as the acid component in the next step of a polypeptide
synthesis. When the carboxyl group of the amine component
is the benzhydryl amide, a tetrapeptide benzhydrylamide
derivative can be obtained in high yield. Examples in
which a dipeptide derivative is used as the acid or amine
component of the reaction are shown in Table 4. The same
reaction can be performed by using a serine proteinase such as
Subtilisin BPN'. As stated above, the synthesis of oligopeptides
and polypeptides can be achieved by selecting the appropriate
protective group for the carboxyl group of the amino component
by using the thiol proteinase or the serine proteinase though
they exhibit esterase and amidase action. The catalytic effect
of the present enzymes for peptide synthesis is completely
unexpected. As it is clear from the foregoing description, in
the process of the present invention, the synthesis of peptides
having the desired amino acid sequence can be attained by
utilizing the characteristics of an endopeptitase. Only a
catalytic amount of the enzyme is sufficient and the enzyme can
be repeatedly used. The reaction proceedssmoothly under mild
conditions in a buffer solution or in a soluiton having a
desired pH. The yields are relatively high and the purity of the
products is substantially high. The process of the invention
can be utilized both in stepwise elongation of peptide chains
and in condensation of peptide fragments. Both reactions are
effective for industrial purposes. Moreover, racemization of
the peptides does not occur, which is a result which could not
be attained by the conventional synthesis methods.
The present invention will be further illustrated by
way of the following Examples.
-- 10 --
1059938
_XAMPLE 1
A mixture of 20 ml of McIlvaine buffer solution having
a pH of 6.2 and 3 ml of methanol was added to 497 mg (1.20 mmol)
of Z-Lys(Z)-OH and 366 mg (1.09 mmol) of H-Lys(Z)-OBut. Then,
130 mg of papain (titre 1200 CSU/g manufactured by Green-cross
K.K.) and 0.05 ml of 2-mercapto ethanol were added to the
mixture with stirring at 38C for 24 hours, and the reaction
was conducted. The resulting oily product was extracted with
ethyl acetate and the extracted solution was sequentially
washed with water, 0.5 N HCl, 7% ammonia water and water. The
ethyl acetate solution was condensed and petroleum ether was
added to the solution whereby 385 mg (48%) of crude crystals
of Z-Lys(Z)-Lys(Z)-OBut were obtained. A portion of the crystals
was recrystallized from ethyl acetate-petroleum ether whereby
a pure product having a melting point of 62 to 66C and an
[~]25= 14.2O (C=0.5 methanol) was obtained.
ELEMENTAL ANALYSIS
C H N
Calculated(%) 65.557.15 7.65
Found (%) 65.557.22 7.67
EXAMPLE 2
A mixture of 20 ml of McIlvaine buffer solution having
a pH of 7.0 and 4 ml of methanol was added to 692 mg (1.20 mmol)
- of Z-Arg(Z,Z)-OH and 470 mg (1.00 mmol) of H-Leu-OBzh TosOH.
Then, 150 mg of papain and 0.1 ml of 2-mercapto ethanol were
added to the mixture with stirring at 38C for 10 hours and
the reaction was conducted. The resulting colorless precipitate
was filtered and was sequentially washed with water, 7~ ammonia
water, and water whereby the product Z-Arg(Z,Z)-Leu-OBzh was
obtained.
yield 850 mg (g9%)
melting point 161 to 168C
~059938
[~]D5= -9.4C (C=1.0 N,N-dimethylformamide)
Elemental Analysis C H N
Calculated (%) 68.76 6.24 8.18
Found (%) 68.43 6.18 8.38
EXAMPLE 3
A 20 ml amount of McIlvaine buffer solution having
a pH of 6.6 was added to 691 mg (2.00 mmol) of soc-His(szl)-oH
and 599 mg (1.80 mmol) of H-Leu-NHbzh-HCl. Then, 250 mg
of papain and 100 mg of cysteine hydrochloride were added to
the mixture with stirring at 38C for 24 hours and the reaction
was conducted. The resulting colorless precipitate was
filtered and sequentially washed with water, 7% ammonia water
and water whereby 750 mg of crude crystals were obtained. The
product was dissolved in 50 ml of hot methanol and the hot
solution was treated with activated c~rbon to remove protein.
The solution was concentrated, water was added to the residue
and the product was recrystallized whereby the crystalline
product, BOC-His(Bzl)-Leu-NHBzh was obtained.
yield 686 mg(62%)
melting point 113 to 115C [~]25= -8.1(C-1.0 chloro-
D form)
Elemental Analysis C H N
Calculated (%) 71.24 7.27 11.23
Found (%) 71.15 7.30 11.31
EXAMPLE 4
A 20 ml amount of McIlvaine buffer solution having a
pH of 6.6 was added to 511 mg (2.00 mmol) of BOC-His-OH and
599 mg (1.80 mmol) of H-Leu-NHBzh-HCl. Then, 250 mg of papain
and 100 mg of cysteine hydrochloride were added to the mixture
with stirring at 38C for 24 hours and the reaction was
conducted. The resulting colorless precipitate was filtered and
sequentially washed with water, 7% ammonia water and water
whereby crude crystals of product were obtained. The product
1059938
was dissolved in methanol-water and the hot solution was treated
with activated carbon to remove protein. The solution was
concentrated whereby the crystalline product, BOC-His-Leu-NHBzh
was obtained.
yield 336 mg (35%)
melting point 221 to 223C
[~]25= _ 23.6 (C=0.5 chloroform)
Elemental Analysis C H N
Calculated (%) 67.52 7.37 13.12
Found (%) 67.39 7.38 13.21
EXAMPLE S
A 40 ml amount of McIlvainebuffer solution having a pH
of 6.2 was added to 531 mg (2.00 mm~l) of BOC-Phe-OH and 935 mg
(2.00 mmol) of H-His-(Bzl)-NHDMB~2HCl and then 4 ml of lN NaOH
was added to the solution. Then, 480 mg of papain and 240 mg of
cysteine hydrochloride were added to the mixture with stirring
at 38C for 24 hours and the reaction was conducted. The
resulting colorless precipitate was filtered and was sequentially
washed with water, 7% ammonia and water, wherein 960 mg of
crude crystals of product were obtained. The product was
dissolved in 100 ml of hot methanol and the hot solution was
treated with activated carbon for 30 minutes to remove protein.
The solution was condensed and then water was added to the solution
whereby the crystalline product of BOC-Phe-His(Bzl)-NHDMB~1/2H2O
was obtained.
yield 650 mg (50%)
melting point 128 to 132C
[~]D = +1.6 (C=1.0 chloroform)
Elemental Analysis C H N
Calculated (%) 66.44 6.82 10.76
Found (%) 66.34 6.66 10.78
- 13 -
1059938
EXAMPLES 6 to 23
The process of Example 1 was repeated except that the
acid component of N~-acylamino acid and the amine component
of the amino acid ester of H-Val-OBzh were used as shown in
Table 1. The results are shown in Table 1. The process of
Example 1 was repeated except that the acid component of the
N~-acylamino acid of Z-Ala-OH and the amino component of the
amino acid ester or amide were used as shown in Table 2. The
results are shown in Table 2.
1059938
_
CO~`
U~ ~
U~ 0 ~ ~0 O Ltl ~9 O ~57 N ~ O ~1 t~ ~ ~ ~r a~ O CO
~ 1 t~ r` ~ Cl:~ Il-) ~D ~ ~ ~1 ~1 a~ ~ ~1 o~ ~ ~ Ll~ ~1 (~
tO Z . . . . . . . . . . . . . . . . .
~ ~ _ ~ ~ er er u~ ~r In U~ In U~I` r`In er ~ r~ ~
oP oP
~C ~ ~ ~ ~ ~ ~ ~ ~ cn ~ O ~ ;- I~ ~ r~ ~ a~ ~ r~ w
r~ ~ r~ er ~D Ln ~D ~ ~ ~ ~ ~ ~r ~ ~ ~
. . . . . . . . . . . . . . . . . . . .
~D ~ ~D ~D ~ ~D ~D ~D~ ~D~ ~D ~D ~D~9 ~D
~1
~ ~ ~;r co ~1 ~ ~ co a~ ~ o~ OD~ ~ ~r ~ ~ o~ I` o~
a) ~ o ~r In ~ ~ O ~ ~r ~ oo OD1--~1 ~ ~ ~ ~ ~ ~ ~ ~
~ ~.. .. .. .. .. .. .. .. .. ..
a) ~ ~ a~ ,1 a~ ~ ~ ~ t- r~1~ coco ~oo~ ~ ~ ~ o o
~D ~D ~D~ ~ D ~D ~D ~D I~ r~
~ w`~ - ~ - ~ ~ -- ~ - ~ -~ ~ ~ ~
n ~ ~ ~ r~
~_ ~ O ~ In C~ ~ r~ OD
~J
~ oo ~,l ~ ~,l l l l l l l l
~ ~ O O O ~ U~ ~ U~ OD ~ O
~ ~ co a~ ~ ~ ~ ~1 ,'
~ -
~1 0~ ~ ~ ~r ~D ~ U~ ~1 ~ ~ O
,a~ - ~ ~ ~ ~ co ~ ~D ~ ~0 r~
~ _ ~
N N N N N N N N O O
:q m m m m m m m ~ ,
o o o o o o o o l 0
l l l l l l l l ~- ~
~ ~1 _I ~-1 _I ~1 ~1 ~1 ~-1 N
_~ o ,a td ~ al (d ~d a~ a~ O _
~S ~ ~ ~ ~> ~ ~ ~ ~ ~; N
O ~S l l l l l l l l _ _
o ::~ a~ h ~1 ~ ~: ~ :~ t
h O S a) ~ a) u~ ~1 ~1 ~1 :~
.~ ~ ~ ~ ~n E~ ~ ~ t~ W ~ :1
E~ l l l l l l l l l
N ~ ~ 1~;1 N ~;1 N N ~;1 ~
.. _.. _ .... _ ...
~ S S S S S S S S S S
~ N N N N N N N N N N
m m m m m m m ~n m m
o o o o o o o o o o o
~ ~ l l l l l l l l l
,~ ~ ~ _~ _~ ~ ~ ~ ~ ~ ,~
o
o ~ D m P m__:c __ _~ :: _ _
o m
m m m m m m m m oN o
O O O O O O O O O Z N
Ql l l l l l l l l _ _
3 ~ O ~ ~1 ~ ~:: ~ ~ tJ~ tQ
u o ~ m a) s
P~ U~ E~
l l l l l l l l l
_._ N N M ____ N N _____ N N _~
~D I_ 00 0~ O ~--1 N ~ ~r L~
X ~1 ~ ~1 ~1 ~1
1~
~ ;~ ~; . . .
-- 15 --
1059938
. __ ~ __ .
~r~ ~ co~r ~r u~ ~ CO~ u~r~
~- 00~ ~1 In ~1 ~ ~r ~1 ~ ~ ~ ~r
Z . .. . . . . .. . . . . . . .
~ u~ ~ ~ u~ L~ ~ a~ o ~9 ~ 1` 1-
~n
_ _
d~ o\O O ~r ~ ~n ~ ~ ~ ~ ~ o c~ ~ ~D O
oo oooo ~ o ,, o~ ~
~D 00 OD ~ U~ ~ ~9 ~D ~D ~D ~D ~ ~D I_ t_
U ~ ~ co ~D In ~ ~ ~ r- o~ ~ o ~ ~9
~D 00 ~ ~ In ~r ~ I~ ~ o Ln In
o ~, . . . . . . . . . . . . . . . .
0~ ~9 ~D 00 LnLt~
a.) 1` ~ ~D ~D r- r~ ~ r- I- ~ ~D ~D ~D ~D
O
r~
~ ~1 ~ ~ ~ ~ L~.IJ O_ ~D l t~l ~D ~ 1~ t'~l ~D
~I Q~ U a~ 1, ) ~ ~ ~i ~I r-l ~D
l Ll~ l l l l l
C5~ C~ ~I 00 ~) Il') ~D
.
_
d~ ~ ~ ~ ~ ~ ~ CO
. ~_ 0~ ~ 00 C~ ~D U~ L~ ~r
~ ~~) ~ ~ N N _ ~ O
N::1 N N m m ~
Q 0~O O O Z Z Z _
d~ ~1::~ a~ (1) ~1~ ~ ~ ,_, u~
E~ U 1~ O ~ r-l _1 U~ ~1
--~ ~ ~¢ I N
o ~~ ~ ~ ~ ~ ~ m
s~ ~ ~ ~ ~ ~ ~ ~ $ ~I
~ ~ ~ ~ ~ ~ ~ ~ Z
~ . 1~;1 M 1~ ~;11!~ C;l ~
_
~ ~ l m
5~ ~ ~ ~ N N _ O
~J N ~1 N N m m ~
m m m m m m o _
~ O O O O Z~ Z -- N
o ~ ~ c) ~ ~ ~ ~ m ~a
n S~ m
''~ R ~ 1~ ~ H ~¢ '¢ I¢ Z 1- 1
,~ U $ m m m m m m x
_ _ _ . _
CO O ~c o~ o~ 1o 1o ~C o
' '~ R --I~1 ~1 ''I r-l ~ ,_1 ~1
~ U ~ ~ ~ ~ ~ ~ ~ ~
~_ ~ C~ C~ ~ ~ ~ ~
a)
X ~r~ co a~ o ~ ~ ~
~1 ~ ~1
_
-- 16 --
lOS9938
XAMPLE 24
A 1.5 ml amount of N,N-dimethylformamide was added
to 370 mg (1.20 mmol? of Z-Pro-Gly-OH and 305 mg (1.00 mmol) of
H-Leu-NHBzh l/2H2O with stirring, and then 15 ml of citric acid
buffer solution having a pH of 5.5 was added to the mixture. Then,
200 mg of papain and 0.2 ml of 2-mercapto ethanol were added to
the mixture with stirring at 38C for 24 hours and the reaction
was conducted. The resulting colorless precipitate was filtered
and was sequentially washed with water, 5% citric acid aqueous
solution, water and 7% ammonia solution whereby 310 mg (51~)
of crude crystals having a melting point of 146 to 149C were
obtained. The product was dissolved in methanol and a small
amount of insoluble material was separated by filtration. The
solution was concentrated and water was added to the residue
whereby the product, Z-Pro-Gly-Leu-NHBzh H2O having a melting
point of 148 to 150C and an ~]25= 41.0(C=0.5 methanol) was
obtained.
Elemental Analysis C H N
Calculated (%)67.75 7.02 9.30
Found (%) 68.05 6.96 9.24
EXAMPLES 25 to 32
_. .
The process of Example 24 was repeated except that an
acid component of the N~-acyldipeptide of Z-Phe-Ala-OH and an
amine component of the amino acid or amide shown in Table 3
were~used. The results are shown in Table 3.
lOS9938
. _ _ O 0~- ~ ~ __ O D _ _
~ ~r er ~ ~ ~o OD U) ~ ~D ~ ~ ~ ~D U7 a~ o
.U~ ~ ~ ~ a~ o~ a~ ~ a~ a~ ~ ~D ~D ~1
~o`P ~
In ~o ~ t~ cn o ~ o~ ~ u~ ~o a~ ~ ~ o
~l~l ~ ~ ~D D ~ 00 r~ ~ ~ ~ ~ o o
~9~ ~ ~D ~D ~D ~ ~ ~ ~D ~ ~ ~ ~O ~D
1~ ~ ~n o 1- ~r ~ 1~ 1` ~D ~ O I
u ~ ~ ~ ~ ~ a~ ~ o~ ~ ~ I~ I~ D 1` 9
. . . .. . . . . . . .
a~ ~ o ~ o ~1 o o~ _1 c~ oo ~ou~ u~ O O U~ In
~ r~ I~ I~ ~1- 1~ ~ ~D ~D ~D ~ ~ ~ ~ ~
~ ~ ~ ~ ~3 ~ ~3 ~ ~3 ~ ~3 ~ ~ ~ ~ ~
._ . _ . .
o cn ~ a~ u~ O
u~ ~ ~ ~ ~r
~ U ~1 N N ~ r-l ~1 tN
rl o
~-1 0-- O~ ~ CO ,_1 1_ O O
~ ~ ~ ~ Lr ~ ~ ~ ~ ~
.. _ .. _ __ . . ___ ._
_l ~ ~r ~D I~ O _l ~ ~O O
.a~ d`l) ~1 o~ ~ ~ 1~ a~ 1~ oo
~ .. .__ ._ . -
N N N N N ~ S
m m m m m ~ m
m m m m m o m
Z Z Z N Z ~Z; Z Z
m`' ~ m ~ a~ ~:
~m~ ~, ~N ~,
o o~ o om o
~m h
O ~ ~ P~ ~ ~ ~ ~Z ~
.. _ .. _ _._ __ .
~ ~ ~ ~ ~ ~ l ~
N N N N N N ~ N
mz mz Z Z Z Z Z~ Z
--,~::
~ _I .--1 _~ .--1 ~ ~ ~ mN Ul
7m m m m m m ~mZ m
m m m m m m m m
O O O O O O O O
~: ~ ~ d >1 >1 ~1
Y
o a) a) ~ o o o o o
S~ ~ S~
rl ~; ~ P~ ~ P~
U
I¢ U ~ N 1~;1 W ~;1t~ 1!1 ~
. . _
k u~ ~D I~ co ~ o ~1 ~
X t`l ~ ~ ~ ~`1 ~ ~ ~
__ .
-- 18 --
1059938
EXAMPLE 33
.
A mixture of 15 ml of McIlvaine buffer solution having
a pH of 8.0 and 15 ml of methanol was added to 564 mg (1.2 mmol)
of BOC-Val-Tyr-(Bzl)-OH and 516 mg (1.0 mmol) of H-Val-His(Bzl)-
OBz1-2HCl. Then, 300 mg of papain and 150 mg of cysteine hydro-
chloride were added to the mixture with stirring at 38C for
24 hours and the reaction was conducted. The resulting colorless
precipitate was filtered and washed with water whereby 450 mg
of crude crystals were obtained. The product was dissolved in
40 ml of ethanol and the hot solution was treated with activated
car~on to remove protein. The solution was cooled and the
resulting precipitate was filtered and the filtrate was condensed.
The residue was recrystallized by adding ether to the residue
whereby the product BOC-Val-Tyr(Bzl)-Val-His(Bzl)-OH was
obtained.
yield 100 mg (12%)
ninhydrin test negative
melting point 159 to 165C
[~]25= -8.16 (C-0.5 N, N-dimethylformamide)
Elemental Analysis C H N
Calculated (~) 63.44 7.26 10.00
Found (~) 63.67 7.04 10.24
EXAMPLE 34
A mixture of 20 ml of McIlvaine buffer solution having
a pH of 6.1 and 10 ml of methanol were added to 518 mg (1.1 mmol)
of BOC-Val-Tyr(Bzl)-OH and 583 mg (1.0 mmol) of H-Val-His(Bzl)-
NHBzh 2HCl and then 2 ml of lN NaOH was added to the solution.
Then, 240 mg of papain and 120 mg of cysteine hydrochloride
were added to the mixture with stirring at 38C for 48 hours
and the reaction was conducted. The resulting colorless
precipitate was filtered and was sequentially washed with 0.5
N HCl, 7% of ammonia water, and water whereby 950 mg of crude
-- 19 --
lOS9938
crystals were obtained. The product was dissolyed in 300 ml
of methanol and the hot solution was treated with activated
carbon to remove protein. The solution was concentrated and
the residue was recrystallized from N,N-dimethylforamide-water
whereby BOC-Val-Tyr(Bzl)-Val-His(Bzl)-NHBzh was obtained.
yield 410 mg (42%)
- melting point 237 to 239C
[a]D =-6.1 (C=1.0 N,N-dimethylformamide)
Elemental Analysis C H N
Calculated (%) 69.84 7.10 10.00
Found (~) 70.18 7.01 9.94
EXAMPLES 35 to 41
The process of Example 34 was repeated except that an
acid component of N~ acyldipeptide and an amine component of
a dipeptide amide of H-Phe-Ser-NHBzh as shown in Table 4 were
used. The results are shown in Table 4.
~ 20
:,~
- 20 -
--- lOS9938
_ ~ ~D _ .
U~ ~
~r
O ~D ~ ~D ~0 0 ~` ~Q ~ r~ o ~r r- ~
~ ~') ~ ~1 ~1 0 N ~r ., . --1 0 Ltl I`
u~ Z O o o o o o a~ ~ ~ ~ ~ ~ c~ o
~1 -I ~ ~1 ~1 ~\ ~1 ~1
:~^
,1-- oo ~r ~ In oo ~ o ~ ~ ~9 In ~ ~ ~
o a~ ~ o ~ o o ~ ~ ~ ~ ~ co ~n
~: . . . . . . . . . . . .
a~ ~ ~D n r~ I~ ~ ~ ~D ~D ~D u~ u~ n n
:~ ~
t~ ~ I` t~ o ~ ~ o ~r
~) O ~: ~1 0~ N ~ ~1 a~ ~ O t` ~ 1--l 1~
~ . . . . . .. ,. . . . . .
o C) I~ I~ ~ ~r ~D ~ a~ a~ co l_ ~ ~ o~ a~
~D ~D ~D ~O \D ~D U~ ~ ~9 ~O ~D ~D ~ D
. _
~J) ID ~ N O O O N
~ ~ In ~ O ~ ~ ~ n
rl ~ ~ N N N N N N N
~ ~ l l l l l l l
,-1 0 o et'CO IS~I_ CO O CO
a) ~-- n ~ ~ r) N O ell
~ N N ~1 N N N N
N
~P C~ ~ ~D ~ ~D
~ ~ l b a) ~ ~
cn ~ O ~ u~ u~ ~ ~n
Pl ~ a) ~ a~
,
r:4 ~1 N ~ p~ ~1 ~ ~4
~ ~ ~? ~ ~ ~ ~
~ ~, ~ ~ , ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
:~ I N 1~ N ~ N I N I N a) N I N
~ ~ ~m ~m ~m ~m om ~m ~m
,1 o ~ p: I ~::I p: ~ ~ ~ ~ I p:: ~ m
R ~ ~ Z t~ Z C~ ZP~ Z ~:4 Z ~ Z E~ æ
~ ~ l o o
E~ ~ ~ m
.
N N N N N N N
m m m m m m m
h S~ ~ S~
a~ c) a
~ u~ ~n u~ u~ u~ u~ u~
~ o ~ ~ ~ ~ a~ a)
'~ ~0 ~ ~ ~ ~ ~ ~ ~
~ ~ ~: ~r: ~ ~ X m ~c
o o ~ o
.~: l l ~ X l ::~
~ 0~ ~ ~ 01 0~, :~ 01 -
~ ~ l l ~ ~ l ~
O ~!) ~1 ~ t~ ~!) ~ C~
a) l l .
rl ~ ~ ~ ~1 ~ O
~¢ UO ~ g O ~ ~1 ~ El-l
~ m m ~ ~ ~ . .
. _
u~ ~ ~ oo a~ o ~
~r
. _ _
~OS9938
EXAMPLE 42
A mixture of 7.5 ml of McIlvaine buffer solution having
a pH of 7.0 and 7.5 ml of methanol was added to 282 mg (0.6 mmol)
of BOC-Val-Tyr-(Bzl)-OH and 345 mg (0.5 mmol) of H-Val-His(Bzl)-
Pro-Phe-OEt-2HCl and then 1 ml of lN NaOH was added. Thereafter,
150 mg of papain and 70 mg of cysteine hydrochloride were added
to the mixture with stirring at 38C for 24 hours and the reaction
was conducted. The resulting colorless precipitate was filtered
and was sequentially washed with water, 0.5 y HCl, 7% ammonia
water and water whereby 400 mg of crude crystals of product were
obtained. The product was dissolved in 50 ml of ethyl acetate
and the hot solution was treated with activated carbon to remove
protein. The solution was concentrated and the residue was
recrystallized from methanol-water whereby Boc-Val-Tyr(Bzl)-Val-
His(Bzl)-Pro-Phe-OEt was obtained.
yield 226 mg (42%)
ninhydrin test negative
melting point 167 to 173C
[~]D = ~34 0 (C=1.0 N,N-dimethylformamide)
= -56.1 (C=l.Q methanol)
Elemental Analysis C H N
Calculated (%) 66.82 7.19 10.39
Found (%) 66.72 7.16 10.55
The same product was produced by a solution method. The
properties of the product are as follows:
melting point: 164 to 174C
comelting test: 161 to 167C
[~]D = ~35 3 (C=1.0 N,N-dimethylformamide)
-59.2 (C=1.0 methanol)
The racemization of the product produced by the enzyme
method was not found in comparison with the product obtained by
the solution method.
- 22 -
1059938
EXAMPLE 43
A mixture of 15 ml of McIlvaine buffer solution having
a pH of 8.0 and 15 ml of methanol was added to 943 mg (1.20 mmol)
of BOC-Asn-Arg(NO2)-Val-Tyr)Bzl)-OH and 690 mg (1.00 mmol) of
H-Val-His(Bzl)-Prop-Phe-Oet 2HC1. Thereafter, 300 mg of papain
and 150 mg of cysteine hydrochloride were added to the mixture
with stirring at 38C for 4 hours and the reaction was conducted.
The resulting colorless precipitate was filtered and was
sequentially washed with water, 0.5 N HCl, 7% ammonia and water
whereby 1.20 g of crude crystals were obtained. The product
was dissolved in 200 ml of ethanol and the solution was treated
with activated carbon to remove protein. The solution was
concentrated and the residue was recrystallized by adding ether
whereby the crystalline product BOC-Asn-Arg(NO2)-Val-Tyr (Bzl)-
Val-His(Bzl)-Pro-Phe-OEt was obtained.
Yield 530 mg (37%)
melting point 185 to 197C
[~]D = ~47-3 (C = 1.0 methanol)
[~]D5 = -25.3 (C = 1.0 N,N-dimethylformamide)
Elemental Analysis C H N
Calculated (%) 59.17 6.88 14.79
Found (%) 59.11 6.62 15.02
The same product was produced by the solution method. The
properties of the product are as follows.
melting point 185 to 198C
comelting point 185 to 190C
¦~]D = -48.4 (C = 1.0 methanol)
-26.5 (C = 1.0 N,N-dimethylformamide)
The racemization of the product produced by the enzyme
method was not found in comparison with the product obtained
by the solution method.
105~938
EXAMPLE 44
.
A 2 ml amount of N,N-dimethylformamide was added to
420 mg ~1.50 mmol) of Z-Gln-OH and 480 mg (1.25 mmol) of H-Leu-
OBzh-(COOH)2 with stirring and then 20 ml of citric acid buffer
solution having a pH of 5.5 was added to the mixture. Thereafter,
300 mg of Stembromelein and 0.2 ml of 2-mercapto ethanol were
added to the mixture with stirring at 38C for 24 hours and
the reaction was conducted. The resulting colorless precipitate
was filtered and was sequentially washed with water, 5% citric
acid, water, 7% ammonia water and water whereby 680 mg (97%) of
the product, Z-Gln-Leu-OBzh having a melting point of 158 to 163C
were obtained. After drying, the product was dissolved in methanol
and the hot solution was treated with active carbon. The solution
was concentrated and the residue was crystallized by adding water
to the concentrate whereby a pure product having a melting point
of 160 to 163C and an [~]25 = -38.1 (C=1.0 methanol)
was obtained.
Elemental Analysis C H N
Calculated (%) 68.67 6.66 7.51
Found (%) 68.65 6.65 7.61
EXAMPLE 45
A 15 ml amount of citric acid buffer solution having
a pH of 5.5 was added to 403 mg (1.20 mmol) of Z-Leu-Ala-OH and
310 mg (1.00 mmol) of H-Phe-OBut (COOH)2. Thereafter, 200 mg
of Stembromelein and 0.2 ml of 2-mercapto ethanol were~added
to the mixture with stirring at 38C for 24 hours and the reaction
was conducted. The resulting colorless precipitate was filtered
and was sequentially washed with water, 5% citric acid, water
7% ammonia water and water whereby 310 mg of crude crystals of
product were obtained. After drying, the product was dissolved
with ethyl acetate and the solution was treated with activated
carbon. The solution was concentrated and the residue was
- 24 -
~059938
recrystallized from petroleum ether whereby the product,
Z-Leu-Ala-Phe-Osut was obtained.
yield 250 mg (4.6%)
melting point 73 to 77C
~]25= _40 oo (C=0.5 methanol)
Elemental Analysis C H N
Calculated (~) 66.77 7~66 7.79
Found (%) 66.76 7.71 7.90
EXAMPLE 46
A 2 ml amount of N,N-dimethylformamide was added to 470 mg
(1.40 mmol~ of Z-Leu-Ala-OH and 585 mg (1.25 mmol) of H-Ile-OBzh
TosOH with stirring and then 20 ml of citric acid buffer solution
having a pH of 5.5 was added to the mixture. Thereafter, 300 mg
of Stembromelein and 0.2 ml of 2-mercapto ethanol were added
to the mixture with stirring at 38C for 24 hours and the reaction
was conducted. The resulting colorless precipitate was filtered
and was sequentially washed with water, 5% citric acid water,
7% ammonia solution and water whereby 610 mg (79%) of the product,
Z-Leu-Ala-Ile-OBzh having a melting point of 167 to 170C were
obtained. The product was dissolved in ethyl acetate and the
solution was treated with activated carbon. The solution was
concentrated and the residue was recrystallized b~ adding ether
to the concentrate whereby a pure product having a melting point
of 170 to 171C and an [~]25= -60.2 (C=1.0 methanol) was
obtained.
Elemental Analysis C H N
Calculated (%) 70.22 7.37 6.82
Found (%) 69.95 7.40 6.91
EXAMPLES 47 to 56
The process of Example 46 was repeated except that the
acid component of an N~-acyl dipeptide and an amine component
of the amino acid ester or amide shown in Table 5 were used.
The results are shown in Table 5.
- 25 -
- lOS9938
. . ~ ~ O ~ ~ ~r ~ x
æ a~ a~ ~ ~o 0~ co
U~ _ ~ A ~1 ~O ~D CO CO ~1 ~1
~o~ ~ ~1
q5 It~ O IS ) CO I_ I_ ~r ,1 ,~ N
1: ~ ~ C~. ~ _l ~) ~ O~ ~
. . . . . . . .
~ ~D ~O ~ ~ I~ I~ ~O ~D ~
~r ~~ O ~ n o a~ ~ er .
~ ~ ~ ~ 1- ao . ~ ~1 ~ ~ ~ _1
a~ ~ o ~ . . . . . . . . . .
t~ ~ 1-- r~ ~ r~ o o
~D ~D ~D ~D I` I` I` r~
~ (3)(~3 ~) ~) ' ~)~ ~)~) ~(~ (~
~ ~ ~ ~D O ~D
~ ~ ~ I_ ~ ~ U~
.~ ~ ~ ~I ~ ~I
~ l l l l . I
,1 o O r /~ u~ I~ u~
a~ ~ ~J r- ~r ~ u~
~ ,~ ~ ~ ~
,~ _~ ~ ~r o r~ x
.~ a~. u~ ~ ~D U~
_ N . ~
o ~ o m m
~ U~ o t l l
S '
0 ~ ~ ~ ~ ~
Il~ ~ _I ~I N ¢ ~
~ o ~: ~ m
_l ~ lI ~: ~ a
E~ P. ~: ~ æ
. :
,L N ~ N N
J~ m m m m ~
a~ o z o z z
'~ o ~1 ~1 ~1 ~ ~1
O D~ :C :~ :t
~ O 0l :1~ O ~
~ t --I ~: 1 S
~ O l l ~ Q) ~'
1 N W 1-1
~ ~_ ~r er U~ _l
~ .
-- 26 --
~ ~0~9938
~ U) 1~ ~ Ll~ ~) ~ N ~
oo ~o ~ ~) ~ u~ r-
co ao ~o ~o ~ a~ ~ ~P ~D
I~a~ ~r~ ~o ~o 1_<~
~D ~D ~ ~9 ~ 1~ ~ 1- r~
o t~ I` co cn ~~ r~ oo t~ ,1
a~ C~ 1~ ~D O ~1 --1 X N ~
~1 ~ ~) cs~ a~ co 1~ o o
r I_ I_ ~D ~D ' ~ ~D ~ I_
(~3 ~) ~ ) (~)(3 ~ .
~ ~ ~ ~11--~
_
~ ~1
~ S O ., O
:r: ~ P.~ ~ ::C
~~ O O
N 14 N ~1 ~1
L~ 4~ Ul ~ Ul
- 26a -
~059938
EXAMPLE 57
A 2 ml amount of N,N-dimethylformamide was added to
380 mg (1.50 mmol) of Z-Thr-OH and 480 mg (1.25 mmol) of H-Leu-
OBzh (COOH)2 with stirring and then 20 ml of citric acid buffer
solution having a pH of 5.5 was added to the mixture. Thereafter,
300 mg of Ficin and 0.3 ml of 2-mercapto ethanol were added to
the mixture with stirring at 38C for 24 hours and the reaction
was conducted. The resulting colorless precipitate was filtered
and was sequentially washed with water, 5% citric acid, water, 7%
ammonia water and water whereby 330 mg (50%) of the product, Z-Thr-
Leu-OBzh having a melting point of 114 to 117C were obtained.
The product was dissolved in ethyl acetate and the solution was
concentrated. The residue was recrystallized by adding ether
to the concentrate whereby a pure product having a melting point
of 120 to 122C and an [~]25= -41.4 (C=1.0 methanol) was
obtained.
Elemental Analysis C H N
Calculated (%) 69.90 6.81 5.26
Found (%) 70.02 6.87 5.16
EXAMPLE 58
:
A 1.5 ml amount of N,N-dimethylformamide was added to
320 mg (1.20 mmol) of BOC-Met-OH and 254 mg (1.00 mmol) of H-Ala-
NHBzh with stirring. Then, 15 ml of citric acid buffer solution
having a pH of 5.5 was added to the mixture. Thereafter, 300 mg
of Ficin and 0.3 ml of 2-mercapto ethanol were added to the mixture
with stirring at 38C for 24 hours and the reaction was conducted.
The resulting colorless precipitate was filtered and was sequent-
ially washed with water, 5% citric acid, water, 7% ammonia water
and water, whereby 400 mg (82%) of the product BOC-Met-Ala-NHBzh
having a melting point of 163 to 164C were obtained. The
product was dissolved with ethyl ether and the solution was
concentrated. The residue was recrystallized from ether-petroleum
1959938
ether whereby a pure product having a melting point of 159 to
161C and an [~]D5=-29.6O (C=1.0 methanol) was obtained.
Elemental Analysis C H N
Calculated (%) 64.30 7.26 8.65
Found (%) 64.13 7.20 8.77
_AMPLE 59
A 2 ml amount of N,N-dimethylformamide was dissolved
in 516 mg (1.40 mmol) of Z-Phe-Ala-OH and 480 mg (1.25 mmol) of
H-Leu-Obzh (COOH)2 with stirring and then 20 ml of citric acid
buffer solution having a pH of 5.5 was added. Thereafter,
200 mg of Ficin and 0.2 ml of 2-mercapto ethanol were added to
the mixture with stirring at 38C for 24 hours and the reaction
was conducted. The resulting colorless precipitate was filtered
and was sequentailly washed with water, 5~ citric acid, water,
7% ammonia water and water whereby 540 mg (67%) of crude crystals
having a melting point of 156 to 158C were obtained. The
product was dissolved in methanol and a small amount of insoluble
material was removed. The solution was concentrated and the
residue was recrystallized by adding water whereby the product,
Z-Phe-Ala-Leu-OBzh having a melting point of 151 to 152C and
[~]25 =_47 4O (C = 0.5 methanol) was obtained.
Elemental Analysis C H N
Calculated (%) 72.09 6.67 6.47
Found (%) 71.84 6.66 6.63
EXAMPLE 60
A mixture of 20 ml of McIlvaine buffer solution having
a pH of 6.2 and 3 ml of methanol was added to 370 mg (1.26 mmol)
of Z-Phe-OH and 360 mg (1.07 mmol) of H-Lys(Z)-OBut. Thereafter,
130 mg of papain and 0.05 ml of 2-mercapto ethanol were added
to the mixture with stirring at 38C for 20 hours. The resulting
colorless precipitate was filtered and was sequentially washed
with water, 0.5 M citric acid, 7% ammonia water and water. The
- 28 -
lOS9938
resulting crude crystals were dissolvedin ethyl acetate and the
hot solution was treated with activated carbon. Thereafter, n-
hexane was added to the filtrate whereby the product, Z-Phe-Lys(Z)
-OBUt was obtained.
yield 531 mg (80%)
melting point 118 to 121C
~]D = -13.5 (C = 1.0 methanol)
Elemental Analysis C H N
Calculated (%) 68.057.02 6.80
Found (~ 68.196.98 6.85
EXAMPLE 61
-
A mixture of 20 ml of McIlvaine having a pH of 6.2 and
3 ml of methanol was added to 359 mg (1.20 mmol)of Z-Phe-OH and
173 mg (1.00 mmol) of H-Val-OBut. Then, 130 mg of papain and 0.05
ml of 2-mercapto ethanol were added to the mixture at 38C for 20
hours and the reaction was conducted. The resulting oily product
was dissolved in ethyl acetate and the solution was sequentially
washed with water, 0.5 N HCl, 7% ammonia water and water. The
solution was dried and treated with activated carbon to remove
protein. The solution was concentrated and the petroleum ether
was added to the concentrate whereby the crystalline product
Z-Phe-Val-OBut was obtained.
yield 315 mg (69~)
melting point 104 to 106C
~]D = -19.0C (C=1.0 methanol)
The melting point and [~]25 were the same with the data
disclosed in (Chem. Ber. 100,160 (1967) ).
The following are reference procedures for converting
the peptides produced by the process of the invention to the
corresponding free compounds.
- 29 -
1059938
REFERENCE EXAMæLE 1
A 2.00 g (3.20 mmol) amount of Z-Phe-Lys(Z)-OBut was
dissolved in 15 ml of ethyl acetate and was admixed with 20 ml
of 6.0 N HCl-ethyl acetate. The reaction was conducted at room
temperature for 2 hours and the solution was concentrated. Dried
ether was added to the residue whereby Z-Phe-Lys (Z)-OH was
obtained.
yield 1.69 g (94%)
melting point 94 to 96C
~]D = -6.2 (C = 0.5 methanol)
Elemental Analysis C H N
Calculated (%) 66.29 6.28 7.48
Eound (%) 65.93 6.02 7.71
REFERENCE EXAMPLE 2
A 909 mg (2.00 mmol) amount of Z-Phe-Val-OBu was
dissolved in a mixture of 8 ml of methanol and 0.12 ml of acetic
acid and was admixed with 100 mg of 10% Pd-C. The reaction
was carried out for 2 hours in a hydrogen atmosphere and then the
catalyst was filtered. Methanol was removed by distillation and
the residue was dissolved in ethyl acetate. The solution was
washed with an aqueous solution of sodium bicarbonate and
water. The ethyl acetate solution was dried and concentrated
and petroleum ether was added to it whereby the crystalline
product H-Phe-Val-OBut was obtained.
yield 612 mg (96%)
melting point 65 to 67C
[~]25= _30.0o (C=1.0 methanol)
The melting point and [~]25 were the same as the
data disclosed in (Chem. Ber. 100, 160 (1967).
EXAMPLE 62
A 20 ml amount of Kolthoff buffer solution having a ph
of 8.5 was added to 409 mg (1.10 mmol) of BOC-Tyr(Bzl)-OH and
- 30 -
~OS9938
303 mg (1.00 mmol) of H-Val-NHDMB HCl and then, 1.0 ml of lN NaOH
was added to the mixture. Thereafter, 100 mg of serine proteinase
(titre 100 x 104 PU ~g sold by Nagase Sangyo K.K.) was added to
the mixture with sitrring at 38C for 24 hours and the reaction
was conducted. The gel precipitate was filtered and was sequentially
washed with water, 0.5 N HCl, 7~ ammonia water and water, whereby
361 mg of crude crystals of product were obtained. The product
was dissolvedinmethanol and the hot solution was treated with
activ~ted carbon to remove protein. The solution was concentrated
and the product was recrystallized by adding water whereby the
pure product BOC-Tyr(Bzl)-Val-NHDMB was obtained.
yield 297 mg (48%)
melting point 165 to 168C
-~ [~]25 = -0.8 (C=0.25 chloroform)
Elemental Analysis C H N
Calculated (%) 67.83 7.32 6.78
Found (%) 67.71 7.35 6.60
EXAMPLE 63
A 20 ml amount of citric acid buffer solution having
a pH of 7.5 was added to 518 mg (1.10 mmol) of BOC-Val-Tyr(Bzl)-
OH and 517 mg (1.00 mmol) of H-Val-His(Bzl)-OBzl 2HCl and then
2.0 ml of lN NaOH was added to the solution. Then, 100 mg of the
; serine proteinase of Example 62 was added to the mixture with
stirring at 38C for 24 hours and the reaction was conducted.
The resulting colorless precipitate was filtered and was washed
with water whereby 620 mg of crude crystals were obtained. The
product was dissolved in 300 ml of hot methanol and the hot solu-
tion was treated with activated carbon for 1 hour to remove protein.
The solution was concentrated and water was added to the residue
whereby the crystalline product BOC-Val-Tyr(Bzl)-Val-His(Bzl)-OH
H2O was obtained.
- 31 -
1059938
yield 450 mg (56%)
melting point 176 to 180C
[~]25= -6.6-(C=0.5 methanol)
Elemental Analysis C H N
Calculated (%) 64.84 7.17 10.31
Found (%) 64.84 7.16 10.90
EXAMPLE 64
A 7.5 ml amount of McIlvaine buffer solution having
a pH of 8.97 was added to 282 mg (0.60 mmol) of BOC-Val-Tyr(Bzl)-
OH and 345 mg (0.50 mmol) of H-Val-His(Bzl)-Pro-Phe-OEt-2HCl
and then 1.2 ml of lN NaOH was added to the solution. Thereafter,
40 mg of serine proteinase was added to the mixture with stirring
at 38C for 24 hours and the reaction was conducted. The result-
ing gel precipitate was filtered and was sequentially washed
with water, 0.5 N HCl and water whereby 400 mg of crude crystals
of product were obtained. The product was dissolved in hot
ethanol and the hot solution was treated with activated carbon
to remove protein. The solution was concentrated and the
residue was recrystallized from ethyl acetate whereby the product
of BOC-Val-Tyr(Bzl)-Val-His(Bzl)-Pro-Phe-OH 2H2O was obtained.
yield 334 mg (62~)
melting point 163 to 168C
[~]D = -26.9 (C=1.0 N,N-dimethylformamide)
Elemental Analysis C H N
Calculated (%) 64.66 7.11 10.40
Found (%) 64.87 6.80 10.52
EXAMPLES 65 to 70
The process of Example 64 was repeated except that an
acid component of an N~-acylamino acid or N~-acyl dipeptide
and an amine component of the dipeptide t-butyl esters shown in
Table 6 were used.
- 32 -
1059938
tn o ~ _ ~ ~ .
Z ~r ~ oo ~n ~ o a~ o a~ ~ oo r~
r~ ~ r~ r~ r~ r~ r~ co ~ ~ r. r~
a~ o~ _1 ~D O ~ ~0 0 00 ~ ~ O
~ ~ m . r; ~D ~ ~ ~ u~ r~ r~ o ~D
~ 0 r~ r~ r~ r~ D ~ r~ r~
0 ~ ~o i~ ao u~ ~ ~ u~ ~ ~ ~ oo ~
~ r~ co oo r; ~ er u~ ~ ~ ~r o co
0 0 o r- r~ x c~ co co oo co ~ ~ _
t~ ~ ~D ~D ~ ~D ~ ~ ~D ~ ~D ~O
~ ~ ~ ~ ~3~ ~ ~D ~ ~
1: O ~r ~D X ~1 r~
~) l o l ~t o l
: ~ 0~o ~o _l _l
u~
~1 l _1 S ~'
m $ ~' ~' ~ ~q m
o~ ~ o
m ' ~ ~Zr o I ~n
s s~, ~-1 ~' o ~: I s~
o ~ a) ~ c
sl O ~ s s
....
s
Q0 . 0 0 m m m ~
E~ ~ a' :'~ ~' :'~ ~' o
o ~ ~ ~ ~ a
~ ~ s s s s s ~m
~ ~ ~ P~ ~ P~ P~ ~ o
~ u m m m m m m
~ . 1 m o o o
o m s ~ _, h h
'U ~0 O ~ E~ ~ ~ u~
¢ U 3 a) S~ pS, ~0 S~
.__ ~1 _. C~ ~ ._ ~
~ ~ ~ r~ oo a~
: ~3 ~D ~D ~ D ~D r~
~' X
-- 33 --
1059938
EXAMPLE 71
In a flask, 280.2 mg (1 mmol) of Z-Gln-OH and 368.5 mg
(1 mmol) of H-Phe-Phe-OBut were suspended in 10 ml of water. The
glass electrode of a pH meter was inserted into the suspension.
A 150 mg amount of papain and 0.1 ml of 2-mercapto ethanol were
added to the suspension with stirring while the pH of the mixture
was adjusted to about 5.0 by adding 1/10 N NaO~ to dissolve
the solid components. The mixture was stirred at 38C for 20
hours and the reaction was conducted. During the reaction, pH
of the reaction mixture was maintained at 5 to 6 by adding 1/10 N
NaOH while measuring the pH of the reaction mixture with a pH
meter. The resulting precipitate was filtered and was sequentially
washed with water, lN HC1, water, 7~ ammonia water and water
and dried. The product was recrystallized from ethyl acetate
whereby 510 mg of the product of Z-Gln-Phe-Phe-OBut having a
melting point of 197 to 200C were obtained.
yield 80.8~
[~]D5= -18.3 (C= 1.0 N,N-dimethylformamide)
Elemental Analysis (C35H42O7N4) C H N
Calculated (~) 66.65 6.71 8.80
Found (~) 66.51 6.71 8.74
EXAMPLE 72
In a flask, 398.5 mg of Z-Phe-Val-OH and 320.4 mg of
- H-Phe-Val-Osut were suspended in 10 ml of water. The glass elec-
trode of a pH meter was inserted into the suspension. A 150 mg
amount of papain and 0.1 ml of 2-mercapto ethanol were added
to the suspension with stirring while adjusting the pH of the
mixture to about 4.5 to 5.0 by adding a diluted HCl solution
to dissolve the solid components. The mixture was stirred at
38C for 20 hours and the reaction was conducted. During the
reaction, the pH of the reaction mixture was maintained at 4.5
to 5.0 by adding a diluted HCl solution to the reaction mixture.
- 34 -
~059938
The resulting precipitate was filtered and was sequentially
washed with water, lN HCl, water, 7% ammonia water, and water
and was dried. The product was recrystallized from ethyl acetate-
petroleum ether whereby 30 mg ~yield 4.3~) of the product
Z-Phe-Val-Phe-val-Osut having a melting point of 130 to 145C
were obtained.
EXAMPLE 7 3
In a flask, 398.5 mg (1 mmol) of Z-Phe-Val-OH and 320.4
mg (1 mmol) of H-Phe-Val-OBut were suspended in 10 ml of water.
The glass electrode of a pH meter was inserted into the suspension.
- A 150 mg amount of serine proteinase was added to the suspension
with stirring while adjusting the pH of the mixture to 7.5 to
8.0 by adding a 1~0 N NaOH solution dropwise to the solution to
dissolve the solid components. The mixture was stirred at 38C
for 20 hours and the reaction was conducted. During the reaction,
the pH of the mixture was maintained at 7. 5 to 8 by adding 1/10
N NaOH to the mixture while measuring the pH of the reaction
mixture with a pH meter. The resulting precipitate was filtered
and was sequentially washed with water, lN HCl, water, 7% ammonia
water and water and was dried. The product was recrystallized
from ethyl acetate-petroleum ether whereby 190 mg (yield of
27.1%) of the product of Z-Phe-Val-Phe-Val-OBut having a melting
point of 130 to 145C were obtained.
EXAMPLE 74
In a flask, 471.5 mg (1 mmol) of Z-Phe-Tyr-OH and
320.4 mg of H-Phe-Val-OBut were suspended in 10 ml of water.
The glass electrode of a pH meter was inserted into the suspension.
A 150 mg amount of serine proteinase was added to the suspension
with stirring while adjusting the pH of the mixture to 7.5 to
8.0 by adding l/10 N NaOH to the mixture to dissolve the solid
components. The mixture was stirred at 38C for 20 hours and
the reaction was conducted. During the reaction, the pH of the
1059938
reaction mixture was maintained at 7.5 to 8.0 by adding 1/10
N NaOH to the mixture. The resulting precipitate was filtered
and sequentially washed with water, lN HCl, water, 7% ammonia
water and water and was dried. The product was recrystallized
from ethyl acetate-petroleum ether whereby 230 mg (yield of 30%)
of the product, Z-Phe-Tyr-Phe-Val-OBut having a melting point of
155C was obtained.
- 36 -
