Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~ ~7'7 ~ ~
A Process for Enzymatic Production of Peptldes
______________________________________________
Back~round of the Invention
1. Field of the Invention
The present invention relates to a process for enzymatic
production of peptides. More particularly, the invention
relates to a process for producing peptides by using a
specific group of enzymes as catalysts.
2. Description of the Prior Art
It is known to carry out syntheses of peptides by more or
less sophisticated coupling reactions, both in respect of
~lomogeneous synthesis and of heterogeneous solid phase
synthesis. All these chemical methods, ho~ever, involve
the risk o~ undesirable secondary reactions and raccmizat-
ions, and it is therefore necessary to control the chemical
reactions carefully to minimize or eliminate these problems.
Moreover, the amino acid side chains must often be pro-
tected, requiring deblocking as the last chemical step to
produce the desired peptides. Depending upon the size of
the synthetized peptide, the yields may be low, and the
secondary reactions frequently necessitate cumbersome
purification procedures to obtain the pure peptide. All
these inherent problems of chemical peptide syntheses plus
the high price of several of the coupling reagents and
the blocking amino acid derivatives mean that even small
synthetic peptides, e.g. pentapeptides, are relatively
expensive to produce.
~ince enzylrles are very specific catalysts, and proteolytic
enzymes are thus able to~hydrolyze peptide bonds in
proteins, studies have previously been made of the pos-
., ~
,
~ 7~ ,g
sibilities of reversing this hydrolysis reaction or, in other words,of utilizing enzymes as catalysts in the synthesis of peptide
bonds. Bergmann and Fraenkel-Conrat (ref. 1) and sergmann and
Fruton (ref. 2) made a detailed examination of this and showed
in 1937 that papain and chymotrypson c~uld catalyze the coupling
of certain acylamino acids and amino acid anilides. These studies
have been continued and intensified on the basis of two fundament-
ally different ap~roaches,viz. a thermodynamic and kinetic one.
Funda~ental features in the thermodynamic methods are
the use of a suitable protease for catalyzing thé establishment
of the thermodynamic equilibrium between the reactants, and
a removal of the reaction product from the reaction mixture. Thus,
Isowa et al (ref. 7-9) and U.S. patent No. 4,119,~93 and British
patents Nos. 1,523,546 and 1,533,129, ~uisi et al (ref. 12, 18)
and Morihara et al (ref. 14) found that se~eral serine, thiol and
metalloendoproteases catalyze the synthesis of peptides from
protected, but very easily soluble di-, tri- and tetrapeptides,
provided the final products precipitate from the reaction
mixture on account of their solubility being lower than the equi-
librium concentration. Examples of such syntheses are illustrated
in the follo\~ing reaction schemepapain
1) Z-leu-Phe-OH + Phe-ODPM _ ~ Z-leu-Phe-Phe-ODPM
ODPM = diphenyl methyl ester
g( 2) OH + phe_val_OBut Thermolysin
A-Arg(N02)Phe-Val-OBu
3) B05-Val-Tyr(Bz)OH + Val-His(Bz)-Pro-Phe-OEt
BOC-Val-Tyr(Bz)-Val-His(Bz -Pro-Phe-OH
(Z = carbobenzoxy, Bz = benzoyl, BOC = tert. butylox~carbonyl, all
amino acids being in the L-form).
However, this reaction principle can only be used as
general method of synthesis if, like in the conventional
chemical coupling procedure, all the amino side chains
with potentially ionizable groups are blocked before the
reaction and deblocked af-ter the reaction. The method is
also de~icient in that -the high specificity of the
various enzymes calls for the use of various enzymcs,
depending upon the type of the peptide bond to be
synthetized, or rather of the amino acids forming the
peptide bond. Even though these complications might be
overcome, the method involves several other problems
because it re~uires a very high concentration of enzyrne
(100 mg/mmole peptide), long reaction periods (1 to 3
days) and gives very varying yields ~typically 20 to ~O,o,
1~ cf.: theabove mentioned U.S. and British patents).
Klibanov et al (ref. 103 have proposed to ~ork in a
system consisting of water and an organic solvent
immiscible with water. This procedure, too, requircs a
high concentration of enzyme and a ]ong reaction period
of up to several days.
The other type of enzymatic syntheses rely on the kinetic
approach of the reaction. It has been shown for most
serine and thiol proteases that an acyl enzyme intermediate
is formed in one of the catalytic steps during the hydro-
lysis of peptides or pep-tide esters, which is then hydro-
lyzed by water in the subsequent step or steps. If other
nucleophiles than water are present during the hydrolysis,
they too will accept the acyl group from the acyl enzyme,
resulting in the formation of an amide bond. This has been
studied e.g. by Fastrez and Fersht (re~. 3) who rc-port
chymotrypsin-catalyzed hydrolysis of N-acetyl-L-phenyl-
alanine ethyl ester (Ac-Phe-OEt) in the presence of
various arnino acid amides. The reaction is sho~n in
scheme (4):
.
,
Y7'7 f~
, - ,
Ac-Phe-OEt + CT;~ Ac-Phe-OH + CT
k2
(Ac-Phe-OEt)-CT~ Ac-Phe-CT~''
+ EtOH ~ ~ ~
k~ Ac-Phe-NH-R + CT
Scheme (4) (CT = chymotrypsin)
First an enzyme substrate complex is for~ed followed by
the formation of the acyl enzyme intermediate (Ac-Phc-CT).
This is hydrolyzed by water to Ac-Phe-OH, but if a
nucleophile (R-NH2) is also present, the acyl enzyrne
intermediate will be subject to aminolysis in addition
to hydrolysis. Assuming that k~ k 3 and kL~>~ k_4, the
ratio of aminolysis to hydrolysis clearly depends on
k4/k3 as well as on the concentration of thé nuclcophile
which is in competition with 55 M water. Fastrez and
Fersht found that e.g. 1 M alanine amide at pH 10 is a
44 times stronger nucleophile than 55 M water, which
resulted in a predominant formation (larger -than 95%) of
the shown N-acyldipeptide-amide. Morihara and Oka (ref.
13-16) have further exploited this principle for enzymatic
peptide synthesis. Using chymotrypsin and trypsin they
synthetized a plurality of pep-tides from N-acyiamino
acid esters and amino acid derived nucleophiles and
demonstrated that the reaction was purely kinetic since
high yields could be obtained, independent of the
solubility of the product.
~he studies mentioned above secm to indicate that kinetic
approaches possess several advantages over the thermo-
dynamic methods:
1. Quicker reaction (in certain cases completed within few
minutes).
77~
.
2. Possibility of using low concentrations of enzyme.
3. Amino acid side chains must not necessarily be blocked.
4. Immobilized and insoluble enzymes may be used as the
peptides are in solution, permit'cing automatization.
As, however, the reaction products may be soluble the
synthesis must be quicker than the secondary hydrolysis
of the product so that they can be separated in time.
Moreover, the known kinetic approaches are limited in
their applicability because the specific properties of the
enzymes examined call for the use of various enzymes for
the synthesis of the various peptide bonds. Additionally,
synthesis of large peptide molecules invariably causes
the internal peptide bonds in the molecule to be hydro-
lyzed, independent of the esterolytic activity of the
cnzymes, due to the endopeptidase acti~i-ty of the en~yrnes
used till now.
Sum ary of the Invention
The object of the present invention is to provide an
enzymatic peptide synthesis which eliminates the draw-
backs mentioned in the foregoing, and more particularly asynthesis that is of a general nature so that it is not
limited to specific amino acid components, and which
~oes not involve any risks of subsequent hydrolysis of
the internal peptide bonds.
Briefly, this and other objects of the invcntion can be
attained in a proccss for producing a pcptide of the
gcneral formula
A-B
:
-- ~ ~ 7 7 ~ ~ 3
wherein A represents an N-terminal protected L-amino acid residue
or an optionally N-terminal protected peptide residue having a
C-terminal L-amino acid and B represents an optionally C-terminal
protected L-amino acid residue, which comprises:
S reacting a substrate component selected from the group
consisting of
(a) amino acid esters, peptide esters and depsipep-
tides of the formula
A-OR , A-SRl or A-SeRl
wherein A is as defined above and Rl represents
alkyl, aryl, heteroaryl, aralkyl or an c~-des-
amino fragment of an L-amino acid residue,
(b) optionally N-substituted amino acid amides and
peptide amides of the formula
A-NR2R2
wherein A is as defined above and R2 and ~ each
represent hydrogen, alkyl, aryl, heteroaryl or
aralkyl, and
(c) optionally N-terminal protected peptides of the
formula
A-X
wherein A is as defined above and X represents
an L-amino acid residue
with an amine component selected from the group
consisting of
(a) L-amino acids of the formula
H-B-OH,
wherein B is an L-amino acid residue,
(b) optionally N-substituted amino acid amides Oc
the formula
~7~
H-B-NR3R3
wherein B is an L-amino acid residue and R3 and
R each represent hydrogen, hydroxy, amino or
alkyl, aryl, heteroaryl or aralkyl, and
(c) amino acid esters of the formula
H-B-OR , H-B-SR- or H-B-SeR
wherein B is an L-amino acid residue and R4
represents alkyl, aryl, heteroaryl and aralkyl,
in the presence of a L-specific serine or thiol of a carboxypeptidase
~10 enzyme from yeast or of animal, vegetable or microbial origin in an
aqueous solution or dispersion having a pH from 5 to 10.5, preferably
at a temperature of from 20 to 50C, to form a peptide, and
subsequently cleaving a group H, -NR3R , -OR , -SR or SeR or an
N-terminal protective group, if desired.
Detailed description of the preferred embodiments
The invention is based on a fundame~tal change in
relation to the prior art, viz. the use of exopeptidases instead
of the enzymes employed till now which have all displayed predo-
minant or at any rate significant endopeptidase activity.
It has been found that besides being exopeptidases the
useful enzymes must display a peptidase activity of broad specific-
ity to thereby allow a synthesis activity of corresponding broad
specificity, which is not restricted to a single or a few types
of peptide bonds. The enzymes must be capable of forming acyl
enzyme intermediates of the type describded above under the kinetic
approaches, and must especially possess such characteristics as
will allow the aminolysis of the intermediate ur~er conditions
. .
.. : ~ ,, : .
'7~
- 8 -
where K 4 ~ K4, cf. scheme (4) above, to avoid immediate hydrolysis
of the peptides produced. The ability to form acyl enzyme inter-
mediates may also be expressed as the ability to cleave the
C-terminal protective group in the su~strate component used, i.e.
in the present case an esterase or ami~ase activity. For a systhe-
sis to take place, said activity must dominate the peptidase ac-
tivity of the enzyme under the reaction conditions used.
This multiplicity of characteristics is found in the
group of exopeptidases called carboxypeptidases, which are capable
of hydrolyzing peptides with free carboxyl groups. It has been
found that a plurality of such carboxypeptidases exhibit different
enzymatic activities ~hich are very depenaent on pH so that e.g.
in a basic environment at a pH from 8 to 10.5 they display pre-
dominantly esterase or amidase activity and at a pH from 9 to
10.5 no or only insignificant peptidase activity. These properties
can be advantageously used in the process of the invention
because they contribute to the achievement of good yields.
Further, it has been found that the ability to form
said acyl enzyme intermediates are particularly pronounced in
serine or thiol proteases. Accordingly the carboxypeptidases used
in the process for the conversion are L-specific serine or thiol
carboxypeptidases. Such enzymes can be produced by yeast fungi, or
they may be of animal, vegetable or microbial origin.
A particularly expedient enzyme is carboxypeptidase
from yeast fungi (CPD-Y). This enzyme is described by ~ayashi et
al. (ref. 5) and by Johansen et al. (ref. 6) who developed a
p~rticularly expedient purification method by affinity chromato-
graphy on an affinity resin comprising a polymeric resin matrix
with coupled benzylsuccinyl groups. CPD-Y, which is ~ serine
~ t~ , "
~ , . .
enzyme, is characterized by having the above relation between the
different enzymatic activities at pH~ 9 and by having no endopepti-
dase activity. In addition to its specificity for C-terminal
amino acids or esters with free ~-carboxyl groups, CPD-Y can
also hydrolyze peptides in which the C-terminal ~-carboxyl group
is blocked in the form of an ester group, e.g. an alkyl or aryl
ester or as an amide group or an N-substituted amide, e.g. an
anilide. Importantly, the enzyme hydrolyzes most substrates, inde-
pendent of the type of the C-terminal amino acid residue. Another
advantage of CPD-Y is that it is available in large amounts and
displays relatively great stability.
In addition to CPD-Y, which is the preferred enzyme
at present, the process of the invention is feasible with other
L-specific serine or thiol carboxypeptidases, such as those listed
in the following survey:
Enzyme Oriqin
Penicillocarboxypeptidase S-l Penicillium janthinellum
S-2 ~ "
Carboxypeptidase(s) from Aspergillus saitoi
" Aspergillus oryzae
Plants
Carboxypeptidase(s) C Orange leaves
Orange peels
Carboxypeptidase C~ Citrus natsudaidai Hayata
Phaseolin French bean leaves
Carboxypeptidase(s) from Germinating barley
Germinating cotton plants
-- 10 --
- (Plant Cont.)
Tora~toes
Watermelons
Bromelain pineapple po~der
Bovine carboxypeptidases A and B (CPD-A and CPD-B),
S however, are not suitable because they are metallocarboxypeptidases.
As explained above, the synthesis is based on a reaction
of a so-called substrate component also called acid component or
donor, and containing the moiety A with a so-called amine component,
also called nucleophile component or acceptor, and containing the
moiety B thereby to form a peptide A-B.
The moiety A, which is an L-amino acid residue or a
peptide residue having a C-terminal L-amino acid can, if desired,
be N-terminal amino protected to avoid undesirable secondary
reactions.
The need for amino protection diminishes with increasing
chain length of the peptide residue and is essentially non existent
when the peptide residue consists of three amino acids, depending,
however, upon their type and sequence.
Examples,of useful amino acids are aliphatic amino
acids, such as monoamino monocarboxylic acids, e g. Glycine (Gly),
alanine (Ala), valine (Val), norvaline (Nva), leucine (Leu),
isoleucine (iso-Leu), and norleucine (Nle), hydroxy amino acids,
such as serine (Ser), threonine (~nr) and hornoserine (homo-Ser),
sulfur-containing arnino acids, such as methionine (Met) or cystine
(CysS) and Cysteine (CysH), monoamino dicarboxylic acids and
amides thereof, such as aspartic acid (Asp), glutamic acid (Glu),
asparagine (Asn) and glutamine (Gln), diaminomonocarboxylic acids,
such as ornithine (Orn) and lysine (Lys), ar~inine (Arg), aromatic
amino acids, such as phenylalanine (Phe) and tyrosine (~yr), as
well as heterocylic amino acids, such as histidine (His) and
tryptophan (Trp).
As protective groups may be used the amino protective
groups common within the peptide chemistry, such as benzoyl, (Bz),
acetyl (Ac) or tertiary alkoxycarbonyl groups, e.g. t-butyloxycar-
bonyl (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-nitrophenylsul-
1~ fenyl(Nps-), or the like.
Preferred protective groups are benzyloxycarbonyl and
t-butyloxycarbonyl since these derivatives are easy to produce,
economic in use and easy to cleave again.
As stated above the substrate component may be
selected from the group consisting of
(a) amino acid esters, peptide esters and depsipeptides
of the formula
A-ORl, A-sRl or A-SeRl
wherein A is as defined above and R represents
alkyl, aryl, heteroaryl, aralkyl or an ~-des-
amino fragment of an L-amino acid residue, and
(b) optionally N-substituted amino acid amides and
peptide amides of the formula
A-NR R
wherein A is as defined above and R2 and R2 each
represent hydrogen, alkyl, aryl, heteroaryl or
aralkyl, and
~c) optionally N-terminal protected peptides of the
formula
,
- 12 -
A-X
wherein A is as defined above and ~ represents an
an amino acid residue.
In this context "alkyl" means straight chain or
branched alkyl, preferably with 1 to 6 carbon atoms, e.g. methyl,
ethyl, propyl, isopropyl, butyl, isobytyl, tert. butyl, amyl, hexyl,
and the like.
"Aryl" means phenyl and the like.
"Aralkyl" means benzyl, phenethyl, and the like.
All of these groups may be substituted with substi-
tuents which are inert with relation to the enzyme, e.g. halo
(fluoro, chloro, bromo, iodo) nitro, alkoxy (methoxy, ethoxy, etc.),
or alkyl (methyl, ethyl, etc.), provided the amino acid residue
or the peptide derivative is a substrate for th~ carboxypeptidase.
Thus in case of esters the group ORl is preferably
selected from among alkoxy groups, such as methoxy, ethoxy or t-
butoxy, phenyloxy, and benzyloxy-group. m e groups may optionally
be substituted with inert substituents, such as nitro groups
(p-nitrobenzyloxy). Other groups may be used as well if the
amino acid residue or the peptide derivative is a substrate for the
carboxypeptidase. An example is the so-called depsipeptides ~here
the C-terminal amino acid is linked via an ester bond instead of
a peptide bond, e.g. benzoyl-glycine-phenyl lactate (Bz-Gly-O-des-
NH -Phe).
Preferred carboxylic acid protective groups are alkoxy
groups, in particular methoxy groups, or in other words: The
substrate component preferably contains or consists of an amino
acid methyl ester since these derivatives are easy to produce and
good substrates at the same time. However, e.g. ethyl esters,
~ ' ~
.
1~7~7~
- 13 -
propyl esters, isopropyl esters, bu~yl esters, t-butyl esters,
benzyl esters or tert. butyloxy esters may be used equally well.
It should be mentioned that ionizable groups which
may be present in the individual amino acids, which are constituents
of a peptide residue A, may, if desired7 be blocked in a manner
kno~n per se, depending upon the type of the group. However, this
is not always required, which is precisely one of the advantages
of the present process. If it is desired to protect the function-
al groups, suitable protective groups for the ~ -amino group N
are e.g. N~lJ-benzyloxycarbonyl (N ~-Z), t-butoxycarbonyl (N ~V-BOC)
or tosyl (N~ -Tos). Suitable protective groups for the N-guanidino
group (N ) in Arg are Nitro (N -NO2), N -benzyloxycarbonyl (N -Z)
and N , N -dibenzyloxycarbonyl tN -Z-Z). Suitable protective
groups for the imidazole ring (N ) in His are N -benzyl (N Bzl)
and Tosyl (N m-Tos). Suitable protective groups for theuJ-carboxyl
groups are ~ -benzyloxy (-OBzl). Suitable protective groups for
the hydroxyl group in aliphatic or aromatic hydroxy amino acids
are aralkyl groups, such as benzyl (Bzl). Suitable S-protective
groups for the mercapto group in CysH are e.g. the benzyl group
(Bzl). The protective groups must be stable during the primary
reaction and be easy to cleave from the final product without
causing any secondary reactions.
The process of the invention can in principle be carried
out with any amino acid as substrate component. In fact, the
preferred substrate component is an L-amino acid ester or peptide
ester selected from benzyl or Cl-C~ alkyl esters or a p-nitroanilide.
The second participant in the reaction is the so-called amine
component which is selected from the group consisting of
(a) L-amino acids of the form~la
H-B-OH
~-~'7'7~
- 14 -
wherein B is an L-amino acid residue, and
(b) optionally N-substituted amino acid amides of
the formula
H- B-NR3R3
wherein B is an L-amino acid residue and R and
R each represent hydrogen, hydroxy, amino or
alkyl, aryl, heteroaryl or aralkyl, and
(c) amino acid esters, thioesters or selenioesters of
the form~la H-B-OR, H-B-SR or H-B-Se~ respec-
tively wherein B is an L-amino acid residue and R4
represents alkyl, aryl, heteroaryl and
aralkyl,
The alkyl, aryl, heteroaryl and aralkyl groups may be
substituted and are defined as explained in connection with the
substrate component.
It is seen that when R3 = hydrogen, R3 = hydro~en,
H-B-NR3R3 represents the free amide while when R3 = OH, H-B-NR3R3
is a hydroxamic acid when R3 = amino H-B-NR3R3 is a hydrazide,
and when R3 = phenyl H-B-NR R3 represents an anilide.
m e preferred amine components are amides of the
above formula, wherein B is an L-amino acid residue, R3 = H and
R3 = H or Cl 3 alkyl. The preferred esters are H-B-OR~ wherein
B is an L-amino acid residue and R4 is Cl 3 alkyl.
The process of the invention thus possesses the
decisive advantage over e.g. Isowa et al and Morihara et al (op.
cit.) that it can be carried out both with free (not C-terrninal
protected) amino acids, and with amino acids which are C-terminal
protected e.g. by conversion into the corresponding arnides, anilides,
hydrazides, esters or other specified carboxyl derivatives of amino
acids.
~.
,,
,
: ~:
~ '7'74~
- 14a -
As regards the amine component, the reac~ion sequence,
yields, etc. depend very much upon whether the components are intro-
duced as free acid or a.s C-protected derivative, e.g. as amide.
This will be illustrated in greater detail below in connection with
the examples; the following general picture seems to emerge,
however:
It applies to the free acids that the hydrophobic
amino acids, such as alanine, leucine, valine and phenylalanine,
as well as acids with a positively charged side chain, such as
lysine and arginine, are relatively easy to incorporate in the
chain of the substrate component, while amino acids whose side
chains contain either carboxyl groups (aspartic acid or glutamic
acid), hydroxyl groups (serine and threonine) or amide groups
(asparagine and glutamine) are more difficult to incorporate.
Heterocyclic amino acids, such as proline and histidine,
are extremely difficult to incorporate as free acids.
It is a different matter if C-terminal protected amino
acids e.g. amino acid amides, or N-substituted amides, e.g. hydra-
~ides or anilides are u~ed as amine component. This will be
elaborated below, but it may be said quite generally that in this
case the reaction is highly independent of the structure and
allows even very high yields (up to 90%) to be obtained, however,
here too, heterocyclix amino acids are more difficult to incor-
porate than aliphatic ones.
7~
.
. 15
As stated above, the process of the ~invention is carried
at pH 5.0 to 10.5, preferably at pH 8.0 to 10.5. I'he
preferred pH-value, which is often within a very narrow
range, depends upon the pH-optima and plH-minima,
respectively, for the different enzyrnatic activities of
the enzyme used, it being understood that the pH-value
should be selected so that the activities are counter-
balanced as explained in the foregoing.
Generally, the peptidase activity increases with dccreas-
ing pH-values below about 9.0 thereby rendering the process
less advantageous in terms of yield. However, in some
cases, e.g. with Bz-Ala-Gly or Bz-Ala-Gly-NH2, the formed
peptides or peptide amides are very poor substrates for
the carboxypeptidase, so tha-t the esterase activity versus
the amino acid ester or peptide cster preferably used as
substrate component still dominates, thereby making a
synthesis possible even at pH-values about or below 5Ø
Generally speaking, the optimum pH depends on the ac-tual
starting materials, the formed peptides and the enzyme.
If CPD-Y is used as enzyme, the pH-value is preferably
8.0 to 10.5, particularly 9.0 to 10.5, as explained below~
This pH-value should be maintained throughout the coupling
reaction, and may then be ch~nged for precipitation of the
reaction product, cleavage of protective groups, etc. This
may be provided for by incorporating a suitable buffer
for the selected pH-range in the reaction medium, such as
a bicarbonate buffer.
However, the selected bu~fer is not critical to the
reaction, provided the proper pH is maintained.
The pH-value may also be maintained by adding an acid,
such as HCl, or a baser such as ~aOH, during the reaction.
'
3 - : :
.
- 16 -
The reaction is, as mentioned, carried out in an aqueous
reaction medium which, if desired, may contain up to 50% by volume
of an organic solvent. Preferred organic solvents are alkanols,
e.g. methanol and ethanol, glycols, e.g. ethylene glycol or poly-
ethylene glycols, dimethyl formamide, dimethyl sulfoxide, tetra-
hydrofuran, dioxane and dimethoxyethane.
The selection of the composition of the reaction medium
depends particularly upon the solubility, temperature and pH of
the reaction components and the peptide product, and upon the
stability of the enzyme.
The reaction medium may also comprise a component that
renders the enzyme insoluble, but retains a considerable part of
the enzyme activity, such as an ion exchanger resin. Alternatively
the enxyme may be immobilized in known manner, cf. Methods in
Enzymology, Vol. 44, 1976, e.g. by bondins to a matrix, such as a
cross-linked dextran or agarose, or to a silica, polyamide or
cellulose, or by encapsulating in polyacrylamide, alginates or
fibres. Besides, the enzyme may be modified by chemical means to
improve its stability or enzymatic properties.
~ne concentration of the two participants in the reaction
may vary within wide limits, as explained below. A preferred
starting concentration for the substrate component is 0.01 to 1
molar and for the amine component 0.05 to 3 molar.
The enzyme concentration may vary as well, but is prefer-
ably 10 6 to 10 4 molar.
Accor~ing to the invention the reaction temperature is
preferably 20 to 50C. The most appropriate reaction temperature
for a given synthesis can be determined by
'7'7~
. .
; -_ 17
.
experiments, but depends particularly upon the used amine
component and enzyme. An appropriate temperature will
usually be about ~5 to 45C, preferably about 35C. At
temperatures lower than 20C the reaction time will
usually be inappropriately ]ong~ while -temperatures above
50C often cause problems with the stabili-ty of the
enzyme and/or reactants or o~ the reaction products.
Similar variations occur for the reaction time ~jhich
depends very much upon the other reaction parameters, as
explained below. The standard reac-tion time in the
process o~ the invention is about 10 minutes, but may take
up to a few hours.
It should be added that when using an amide or substituted
amide as the amine component, it is often advantageous or
even necessary to cleave the amide group specifically ~rom
the formed peptide amide in order to continue the synthesis.
Also in this respect the carboxypeptidase, especially
CPD-Y is very suitable since as described above CPD-Y
exhibits amidase activity at pH ~ 9 while the carboxy-
peptidase activity is negligible.
By the same token the carboxypeptidase might generally beused to cleave the groups H-, NR3~3 , -OR , -SR or SeR
as defined from the formed peptide whether it is desired to
continue the synthesis or just to obtain a final peptide
which is not C-terminal protected.
As will be further illustxated below the process of the
invention is applicable for the formation of an unlimited
number of dipeptides, oligopeptides and polypeptides, based
3Q on the same inventive principle, viz. reacting a substrate
cornponent with an amine component in the form of an amino acid
or amino acid derivative in the presence of a carboxypeptidase.
- 18
Examples of well-known oligopeptides or polypeptides which
may be produced accordingly are enkephalins, somatostatin 7
somatostatin analo~s and peptides with similar biological
activities, and the so-called "sleep peptide" (Trp-Ala-
Gly-Gly-Asp-Ala-Ser-Gly-Glu).
In certain cases, e.g. when dealing with peptides consist-
ing of more than five amino acids, it might be advantageous
to synthesize parts of the desired peptide in the form of
oligopeptide fragments e.g. pentapeptides with the suitable
amino acid sequences and subject the oligopeptides to
fragment condensation in any manner known per se.
It might also be advantageous in order to improve e.g.
the solubility characteristics of the peptides formcd to
use as the N-terminal amino acid arginine or another
ionizable amino acid during the synthesis steps and then
cleave the arginine from the pep-tide with a specific
enzyme e.g. trypsin, when the dcsired amino acid
sequence is otherwise in order.
These and other modifications also form part of the
invention.
Before the process of the invention will be illustrated
by examples, starting materials, methods of measurement, etc.
~rill be explained in general terms.
Startin~ materials
Carboxypeptidase Y from baker's yeast was isolated by
the affinity chromatography proccdure of Johansen ct al.
(ref. 6) and obtained as a lyophilized powder (16,~ enzy~c
in sodium ~ trate). Before use the enzyme was desaltcd
on Scphadex G-25 fine (1~5 x 25 cm) equilibrated and
clutcd with distilled water. The conccntration of thc
~. ~
~'7~
.
enzyme was determined spectrophotome-trically using E18~o nm
= 14.8 (ref. 6). A s-tock solution of 7 mg/ml (110 /uM)
was prepared and stored in aliquots of 250-500 /ul
at -21C. Benzoylalanine methyl ester (Bz-Ala-OMe) was
purchased from Bachem, Liestal, Switzerland. Boron tri-
fluoride etherate complex (for synthesis), solven-ts and
reagents (all analytical grade) were from Merck,
~armstadt, West Germany. All amino acids and amino acid
amides and their derivatives were from Sigma Chemical
Company, St. Louis, USA. Carbobenzyloxy-phenylalanine
methyl ester (Z-Phe-OMe) was prepared according to the
procedure of Yamada et al. (ref. 20) and used as a syrup.
Phenylalanine hydrazide and alanine hydrazide hydro-
chloride were prepared from the esters as described by
Losse et al. (ref. 11). The uncorrected melting points
were 86-88C (Lit.: 82-8~C (11)) and 182-185C (Iit.:
184-185C (21)), respectively. Asparagine amide dilly(lro-
chloride was obtained from aspartic acid via the diethyl
ester according to Fischer (ref. 4) by classical amino-
lysis (m.p.: 210-214C, Lit.: 214-215C (19)). Other
starting materials were provided from the above companies
or produced in analogous manner.
Determination of product yields
The purity was determined qualitatively by TLC on Silica
Gel 60 F254 (Merck). The solvent system used was CHC13/
CH3(CI~)30H/CH3COOH/H20 (11:5:2:1) and the spots ~Jcre
visualized by fluorescence quenching for estimation of the
reactant composition.
The reactant compositions wcre determined quantitatively
by reversc-phase l~PLC using an RP-8,10 /urn ~Merck) colu-nn
and a Hewlett Packard 1084 Chromatogr~ph equipped with a
variable wavelen~th W detector (Model 7~875 A). S~par-
ation was achievcd using suitab]c spccific ~radicnts of
.
~ ~L'7'~
-
`~ 20
elution systems from 10/o CH3CN in 10 mM--NaAc, pH 4 to
100/o CH3CN or from 10 mM-NaAc, pH 4 to lOG~ CH3CN. The
latter system was used for compounds such as Bz-Ala-
Gly-OH, Bz-Ala-Ala-OH, Bz-Ala-Ser-OH and ~heir respective
amides, cf. below. The flow rate was 3 ml/min, the
column temperature 47C and the monitoring wavelength
260 nm. The yields were determined on the basis of the
molar ratio of the reactants which was obtained from the
integrated areas under the peaks in the elution profile.
Identification of products
The spots were identified by thin-layer cochromatography
with suitable standard compounds. Several products wére
identified by a combination of HPLC and amino acid ana-
lysis. For this purpose, 1 ml-aliquots were taken from
the reaction mixture after 10 minutes, and the reaction
was discontinued by adding 250 /ul 6 M-HCl: The pH was
then adjusted to 4 with NaOH and the mixture separated
by HPLC using Waters equipment including two pumps, a
Model 660 Solvent Programmer, a Model U6K Injector, a
Model 450 Variable Wavelength Detector combined with a
Recorder (Radiometer REC 61) or a Hewlett Packard Recorder/
Integrator Model 3380A. Elution was monitored by scanning
at a suitable wavelength between 255-280 nm. The chroma-
tography was reverse-phase using a ~aters C-18 /u-Bondapak
column with the elution system TEAP- 20% (v/v) TEAP
(triethylammoniumphosphate buffer) in methanol under
suitable gradients and flow rates of 1.5-2.0 ml/min.
The TEAP-buffer was prepared according to Rivier (ref.
17). In many cases the system 0.1 M-HAc, pH 3 - 20/~
(v/v) 0.1 M-HAc, pH3 in methanol gave also sufficient
resolution.
The effluent containing the N-acyldipeptides was collected
manually and taken to ~ryness by lyophilization or on a
Buchi Rotovap at 35-45C. Small samples of the residues
were hydrolyzed in 6 M-HCl at 110C in vacuo for 36 h.
e~ C3
,.
- - . 21
.
The evaporated hydrolyzates were then analysed on a
Durrum D-500 amino acid analyzer.
Synthesis with free amino acids as amine component
EX~MPLE 1
Asolution of 2 ml of 0.6 M Valinc-O.l M KCl-lmM EDTA,
pH 9.8 was mixed with 100 /ul (0.1 mmole) of a 1 M
Bz-Ala-OMe (dissolved in 96~ ethanol) solution. The react--
ion was carried out in a pH-stat at 35C and pH 9.8, the
pH-value being kept constant by automa-tic addition of 0.5 M
NaOH~ The reaction was initiated by a~ding 0.7 mg of
carboxypeptidase Y (150 U/mg, prepared by De ~orenede
Bryggerier). After a reaction time of 30 minutes, the
reaction was discontinued by adjusting pH to about pH = 1
with 6 M HCl. The reaction product was purified and
isolated by means of high pressure chromatography. The
yield of Bz-Ala-Val-OH was 40%. Quantitative amino acid
analysis after hydrolysis of the product in 6 M HCl for
24 hours gave relatively 1.0 mole of Alanine and 1.0 mole
of Valine.
The effect of pH, temperaturc and concentration of
substrate cornponent, amine corrlponent and CPD-Y on -the
yield in the above reaction:
Bz-Ala-OMe + H-Val-OH CPD ~ Bz-Ala-Val-OH + CH30H ~5
~las studied in five separate series of experiments
analo~ously with the above-mentioned procedure. One of
the pararneters mentioned belo~ was varied in each e~peri-
ment, while the four others ~Jere kept constant: t3.6 M
valine, 55 mM Bz-Ala-OMe, 4.5 mM CPD3-Y, pH 9.7, 35C. The
results are shown in fig. 1. It ~li]l be scen Irom fig.
lA that the p}~-range for optirnal yield is rather n.3rro~,
~ '7~
.
; 22
:
extending over 0.5 pH units only. It will be seen from
fig. lB that an increase in the reaction temperature
caused an almost linear increase of synthesis on account
of relatively less hydrolysis. At temperatures above 45C,
enzyme inactivation and non-enzymatic ester hydrolysis
became prohibitive. At lower tempera-tures and pH-values
up to 10.0, the ester hydrolysis was negligible within the
10 minutes standard reaction time. It became significant,
however, when the enzymatic reaction rate was inhibited
and called for reaction times up to 2 to 5 hours.
While the yields of Bz-Ala-Val-OH increased with higher
amine component concentration (fig. lD), the reverse was
the case for the relationship between yield and concen~
tration of the substrate component Bz-Ala-OMe (fig. lC).
The latter observation, taken together with the depen-
dency of yield on high enzyme concentration (fig. 1~),
suggests an optimal ratio of substrate to enzyme concen-
trations.
The time course of a typical reaction, illustrated by the
above reaction under conditions little short of optimum is
illustrated in fig. 2. In the presence of 0.5 M valine,
the substrate Bz-Ala-OMe was rapidly converted (within
20 minutes) to 38% Bz-Ala-Val-OH and 62% Bz-Ala-OH. The
figure also shows that the dipeptide was not hydrolyzed
at pH 9.7 in the presence of excess valine. If, however,
pH was adjusted to 5 -to 8, all Bz-Ala-Val-OH was hydrolyzed
within seconds. This selective behaviour of CPD-Y at high
pH is an important property for its usefulness in peptide
syntheses.
EY~LE 2
A solution of 2 ml of 3M lysine - 0.1 M KCl - lmM EDTA,
pH 9.8 ~las mixed ~lith 400 /ul of 100~' methanol and
7'~
~' 23
~- -. ,; .............. .
100 /ùl (O.l mmole) of a IM Z-Phe-OMe (dissolved in 100/o
methanol). The reac-tion was carried out as described in
example 1 and initiated by adding 0.7 mg of carboxypep-ti-
dase-Y. After a reaction -time of 30 minutes, pH was ad-
justed to 1 with 6 M HCl. The reaction product waspurified and isolated by means of high pressure chromato-
graphy. The yield of Z-Phe-Lys-OH-was 60/o. Quantitative
amino acid analysis, as described in example 1, showed
that the product relatively contained 1.0 mole of lysine
and 1.0 mole of phenylalanine.
Analogously with examples 1 and 2, the peptides listed in
table I were prepared from the starting materials stated.
The experiments were carried out in a Radiometer pH-stat
and the yields were determined by HPLC ( cf. the foregoing).
The conditions were 4.5 /uM CPD-Y; pH 9.7 and 35C.
.~ ~
. .
~ >7~
24
~ _ . _ . . .
Table I
:
Carboxypeptidase-Y ca-talyzed synthesis of peptides with
free amino acids as amine component.
.
Substrate Amine component - Product Yield %
(conc.) (concentration)
. ._
Bz-Ala-OMe Glycine (3.0 M) Bz-Ala-Gly-OH 62
Alanine (1.9 M~ Bz-Ala-Ala-OH 65
Valine (0.6 M) Bz-Ala-Val-OH 40
(Ex. 1)
Leucine (0.17 M) Bz-Ala-Leu-OH 24
. Phenylalanine (0.16 M) Bz-Ala-Phe-OH 27
Serine ~3.2 M) Bz-Ala-Ser-OH 5o
Threonine (0.7 M) Bz-Ala-Thr-OH 24
Methionine (0.6 M) Bz-A].a-Met-OH 46
Lysine ~1.5 M) Bz-Ala-Lys-OI~ 56
Arginine (0.8 M) Bz-Ala-Arg-OH 26
Aspartic acid (1.0 M) Bz-Ala-Asp-OH O
Asparagine (0.6 M) Bz-Ala-Asn-OH 5
Glutamic acid (1.2 M) Bz-Ala-Glu-OH O
Glutamic acid-r-methyl Bz-Ala-
_ ester (1.0 M) Glu(OMe)-OH 3o
Z-Phe-OMe Valine (0.6 M) Z-Phc-Val-OH 6
Lysine (3.0 M) Z-Phe-Lys-OH 60
Bz-Phe- . (Ex. 2)
Cly-OMe Valine (0.6 M) Bz-Phe-Gly- 40
(30 mM) Val-OH
~ .. .
Z-Ala-OMe Glycine (2.0 M) Z-Ala-Gly-OH 3o
(10 mM) Alanine (0.7 M) Z-Ala-Ala-OH 60
Leucine (0.15 M) Z-Ala-Leu-OH 21
Lysine (1.5 M). _ Z-Ala-Lys-OH 60
Bz-Gly-OJ~e Glycine (2.0 M) Bz-Gly-Gly-OH 63
(20 mM) Leucine (0.15 M) Bz-Gly-Leu-OH¦ ?l
- . . . :
.
Table I (continued)
. ~?--~ -
I
Substrate I Amine component Product Yield %
(conc.) ¦ (concentration)
_ _
Bz-Tyr-OEt Valine (0.6 M) . Bz-Tyr-Val-OH 3o
( 25 mM) Alanine (1.9 M) Bz-Tyr-Ala-OH 46
Ar~inine (0.8 M) Bz-Tyr-Arg-OH 35
_ -~ _
Ac-Phe-OEt Alanine (0.8 M) Ac-Phe-Ala-OH 85
(50 mM)
..__
Z-Ala-Ala- Glycine (2.0 M) Z-Ala-Ala- 40
OMe Gly-OH
(10 mM) Leucine (0.15 M) Z-Ala-Ala- O
Leu-OH
Phenylalanine (O.15 M) Z-Ala-Ala- - O
Phc-OH
Z-Ala-Phe- Glycine (2.0 M) Z-Ala-Phe- 35
OMe Gly-OH
(10 mM) Leucine (0.15 M) Z-Ala-~hc- O
Leu-OH
_......... .
Z-Ala-Ser- Leucine ~0.15 M) Z-Ala-Scr- ~ 40
OMe Leu-OH
~10,mM) ............ _ . .... _
Z-Ala-Val- Leucine (0.15 M) Z-Ala-Val- 25
OMe Leu-OH ..
(10 mM) Lysine (1.5 M) Z-Alu-Val- 60
__ Lys-OH
Z-A].a-NLeu- Glycine (2.0 M) Z-Ala-NLeu- 60
OMe Gly-OH
(10 mM) Leucine (0.15 M) Z-Ala-NLeu- 16
.. . -- --- _ Leu-OH
Z-Ala-Met- Glycine (2.0 M) Z-A]a-Met- 5o
OMe Gly-OH
(10 mM) Leucine (O.15 M) Z-Ala-Met- 15
Lcu-OH
_ .. __ . . _ . _
Z-Ala-Trp- Glycine (2.0 M) Z-Ala-Trp- ~ 23
OJYe Cly-OH
(10 mM~ Lellcine (O.15 M) Z-Ala-Trp- 3
Leu-OH
:
.
;
. .
- . ~
~ 6
e .
Table I (continued)
.. .
Substrate Amine component Product Yield
(conc.) (concentrati~n)
_
; Z-Ala-Trp- Phenylalanine (0.15 M) Z-Ala-Trp- O
OMe Phe-OH
(10 mM) Lysine (1.5 M) Z-Ala-Trp- 36
Ly s- OH
Z-Ala-Ala- Leucine (0.15 M) Z-Ala-Ala- O
Tyr-OMe Tyr-Leu-OH
(10 mM) Lysine (1.5 M) Z-Ala-Ala- 23
Tyr-Lys-OH
Alanine (1.7 M) Z-Ala-Ala- 31
Tyr-Ala-OH
:
- : '` . `
`
.
- 27
.~. .
Synthesis wi-th amino acid amides as amine component
EX~PLE 3
A solution of 2 ml of 0.6 M methionine amide (Met-NH2) -
O.1 M KCl - 1 mM EDTA, pl~ 9.~, was r,lixed with 100/ul
(0.1 mmole) of a 1 M Bz-Ala-OMe solution in 96S' methanol.
The reaction was carried out as described in e~ample 1
and initiated by adding O.7 mg of carboxypeptidase-Y.
After a reaction time of 30 minutes, pH was adjusted to
1 with 6 M HCl. The reaction product was purified and
isolated by means of high prcssure chromato~raphy. The
y~eld of Bz-Ala-Mct-NH2 was 95%. Quantitative amino acid
analysis, as described in example 1, showed that the
product relatively contained 1.0 mole of Ala, 1.0 mole
of Met and 1.0 mole of NH3.
The course for the reaction is shown in fig. 3, from
which it appears that within 20 minutes the substrate is
converted almost completely to about 95% dipeptide, 5%
being hydrolyzed to Bz-Ala-OH.
EXAMPLE 4
.
A solution of 2 ml of 0.4 M valine amide (Val-NH2) -
0.1 M KCl - 1 n~ ~DTA, pH 9.5, was rllixed with 100/ul
(0.1 mmole) of a 1 M Bz-Ala-OMe solution in 96~ ethanol.
The reaction was carried out as dcscribed in exarnple 1.
~`he rcaction product Bz-Ala-Val-NH2 precipitated during
the reaction. After 20 minutes' reaction pl~ was adjustcd
to 1, and the precipitate was isolatcd by ccntri~u~ation.
The product ~.as dissolved in 1 ml o~ 96% ethanol and
purified and iso]ated by hieh pressurc chromato~raphy.
The yield of Bz-Ala-Val-~1~2 was 95~', while, as shown in
c~ample 1, it was only 4~' whcn the free acid was used
.
. .
7~
28
as amine component. Quanti-tative amino acid analysis,
~-- as described in example 1, showed that the product
relatively contained 1.0 mole of Ala, 1~0 mole of Val
and 1.0 mole of NH3.
The dependency o~ the reaction sequence on pH and the
valine amide concentration is S}lOWn in fig. 4. The react-
ion was carried out analogously with the synthesis above,
the constant parameters being: Valine amide 0.6 ~5,
B~-Ala-OMe 55 mM, CPD-Y 4.5 /uM, pH 9.7, 35C.
It will be seen from ~ig. 4B that the yield is essen-
tially insensitive to the variation in the valine amide
concentration in contrast to fig. lD which shows the
yield with varying valine concentration, at least a
concentration e~uimolar to or higher than the substrate
concentration (55mM).
It will be seen from fig. 4A tha-t the effective pH-
range of valine amide extends down to 9.0, thcre being
a sharp upper limit at pH 9.8 above which the yield
decreases heavily.
Analogously with examples ~ and 4, the peptides listed
in table II were prepared from -the starting materials
s-tated. The experiments were carried out under the
following conditions: 4.5 /uM CPD-Y; pl~ 9.6 and 35C,
while the substrate concentration is stated in the table.
- ` ~
;~ 29
~ Table ~I
:
Carboxypeptidase-Y catalyzed synthesi.s of peptides ~ith
amino acid amides as amine component.
Substrate Amine component Product IYield ~0
(conc.) (concentration)
.~ __ . .. ___
Bz-.Ala-OMe Glycine amide (0.3 M) Bz-Ala-Gly-NH2 90
(55 mM) Serine amide (0.3 M~ Bz-Ala-Ser-NH290
(Ex. 4) Bz-Ala-Val-NH2 95b)
Leucine amide (0.3 M) Bz-Ala-Lcu-NH285
. Methionine amide(O.6 M) Bz-Ala-Met-NH2 95
Phenylalanine amide Bz-Ala-Phe-NH2gob)
Tyrosine amide (0.6 M) Bz-Ala Tyr-NH2 9o
Asparagine amide(O.3M) Bz-Ala-Asn-NH2 80
Proline amide (0.3 M) Bz-Ala-Pro-NH2O
Glutamic acid amide Bz-Ala-Glu-NH O
(0.25 M) 2
Histidine amide (0.2 M) Bz-Ala-His-NH2 89
Threonine amide (0.2 M) Bz-Ala-Thr-NH2 ~8 ..
._ _ .......... . . _. ._ ._ . .
Z-Phe-OMe Valine amide (0.5 M) Z-Phe-Val-NH2 9,7b)
; (55 mM) Serine amide (0.4 M) Z-Phe-Ser-NH2 60
Tyrosine amide (0.4 M) Z-Phe-Tyr-NH2 64
. . ._. ... _
Bz-Phe- Valine amide (0.4 M) Bz-Phe-Gly-Val- 90
Gly-OMe NH
(30 mM) 2
__ _ __ .... __ ...................... ..
Z-Phe-Ala_ Valine amide (0.4 M) Z-Phe-Ala-Val- 90b)
(20 ~1~ Glycine amide (0.4 M) Z-Phe-Ala-Gly- 95
~H2
Histidine amide (0.4 M) Z-Phe-Ala-Hi~- 50
NH2
.. . . ._ - -- .. .... ._
b) Product precipitated
~ ~ 7t~c3
~o
.;; .
Table II (continued)
Substrate Amine component Product ¦Yield %
(conc.) (Concentrati~n)
. .
Z-Leu-Gly- Valine amide (0.4 M) - Z-Leu-Gly-Gly- 80
Gly-OEt Val-NH2
(10 mM)
.
Bz-Tyr-OEt Valine amide (0.25 M) Bz-Tyr-Val-NH2 94
(50 mM) Glycine amide (0.55 M) Bz-Tyr-Gly-NH282
Z-Ala-OMe Leucine amide (0.15 M) Z-Ala-Leu-NH2 90
(10 mM) Glycine amide (0.15 M) Z-Ala-Gly-NH2 20
(75 mM) Tyrosine amide (0.2 M) Bz-Arg-Tyr-NH252
Bz-Tyr-OEt Valine amide (0.25 M) Bz-Tyr-Val-NH2 94
(50 mM) Glycine amide (O.55 M) Bz-Tyr-Gly-NH282
~-Pro-OMe Leucine amide (0.25 M) Z-Pro--Leu-NH2 O
(50 mM) .. .
Bz-Gly-OMe Histidine amide (0.2 M) Bz-Gly-His-NH2 20
(100 mM) Glycine amide (0.55 M) Bz-Gly-Gly-NH2 95
Leucine amide (0.25 M) Bz-Gly-Leu-NH2 _
Z-Ala-Phe- Leucine amide (0.15 M) Z-Ala-Phe-Leu- 95
OMe NH2
(10 mM) Glycine amide (0.15 M) Z-Ala-Phe-Gly- 84
NH2
.. . ~ ..
Z-Ala-Ala- Glycine amide (0.15 M) Z-Ala-Ala-Gly- 100
OMe NH
(10 mM) Leucine amide (0.15 M) Z-Ala-Ala-Leu- 100
._ _ ._ NH2
Z-Ala-Ser- Leucine amide (0.15 M) Z-Ala-Ser-Leu- 95
OMe NH
(10 mM) Glycine amide (0.15 M) Z-Ala-Ser-Gly- 70
. . ___ _ 2 .
Z-Ala-Val- Leucine amide (0.15 M) Z-Ala-Val-Leu- 84
OMe NH
(10 mM) Glycine amide (0.15 M) Z-Ala-Val-Gly- 100
._ _...... _ ._ . .. _ NH2 _ _
7 -,
; 31
: Table II (continued
. ~ .
Substrate ¦ Amice component Product Yield %
(conc.) (concen~ration)
Z-Ala-NLeu- Leucine amide (0.15 M) Z-Ala-NLeu- 100
OMe . ~Jeu-NH
(10 mM) Glycine amide (0.15 M) Z-Ala-NLeu- 100
Gly-NH2
_
Z-Ala-Met- Glycine amide (0.15 M3 Z-Ala-Met-Gly- 95
OMe NH
(10 mM) . 2
. ..
Z-Ala-Trp- Glycine amide (0.15 M) Z-Ala-Trp-Gly- 84
OMe NH
(10 mM) Leucine amide ~0.15 M) Z-Ala-Trp-Leu- 89
. NH2
Z-Thr-Pro- ~aline amide (0.2 M) Z-Thr-Pro-Val- 95
OMe NH
(25 rnM) .
Z-Ala-Ala- Glycine amide (0.15 M) Z-Ala-Ala-Tyr- .1.00
Tyr-OMe ~ly-NH2
(10 mM) Leucine amide (0.15 M) Z-Ala-Ala-Tyr- 100
L~U-NH2
__ .__ ._.__ .......... . __ _
Z-Tyr-Gly- Phenylalanine amide Z-Tyr-Gly-Gly- 40
r20 mM) (~-2 M)
,. _ - __ _
7'7~
.
_ 32
EX~MPLE 5
Synthesis wi-th amino acid hydrazides as amine com~onent
Analo~ously with examples 3 and 4 the peptides listed in
Table III were prepared from the s~arting ma-terials
stated. The experiments were carried out under the follow-
ing conditions: 4.5 /uM CPD-Y, pH 9.6 and 35C.
Table III
Carboxypeptidase-Y catalyzed synthesis of peptides with
amino acid hydrazides as amine component
-- .
...
Substrate Amine component Product Yield ~'
(conc.) (concentration)
. ,
~z-Ala-OMe Alanine-hydra~ide . Bz-Ala-Ala-NH- 37
(55 ml~) (0.6 M) NH2
Phenylalanine Bz-Ala-Phe-NH- ~0
_ hydra~ide (0.3 M) N~2
...._ .
~MPLE 6
Synthesis with amino ~cid esters as amine com~onent
The experiments with amino acid esters were carried out
analo~ously with examples 1, 2, 3 and 4, in a Radiometer
pH-stat at pH 9.0 ~ 9.7 and 23-35C. Products and yields
were determined by HPLC. The CPD-Y eoncentration was froM
4.5 to 11 /uM.
Table IV states the peptides produced from the mcntioned
starting materials.
i ., . -
:
It is seen that in some cases a certain oligomerizationis obtained. Since the yields ~enerally are very hi~h,
amino acid esters appear to be extremely useful if
oligomerization can be further limited.
.
Table IV
Carboxypeptidase-Y catalyzed syn-thesis of peptides with
amino acid esters as amine cornponent.
Substrate Amine component ProductIYield %
(conc.) (concentration)
._
Bz-Ala-OMe Glycine ethyl ester ~ Bz-Ala-Gly-OH~
(50 mM) (0.5 M) ~ Bz-Ala-Gly- ~ 100
~Gly-OH J
Glycine propyl ester Bz-Alu-~ly-OH 85
Glycine isopropyl Bz-Ala-Gly-OH 90
ester (0.5 M)
(0-5 M) Bz-Ala-Gly-OH 80
. fBz~Ala-Leu-OH ~
¦ Bz-Ala-Leu-Leu- ..
(O 25 M) ~ OH 85
. IBz-Ala-Leu-Leu-
Leu-OH
Bz-Ala-Leu-Leu-
Leu-Leu-OH
I.eucine ethyl ester ~ Bz-Ala-Lcu-OH ~
(0-~5 M) ~Bz-Ala-Lcu-Leu~ 5o
Leucine-t-butyl Bz-Ala-Leu-OH O
ester (0.25 M)
rBz-A] a-pnc-oH
Phenylalani~Je methyl ) Bz-Ala-Phe-Phe-
cster (0.25 M) OH ~0
Bz-Ala-Phe-Phc-
r),c-oll ,
77~
-- 34
.
- Table IV (continued)
Substrate Amine component Product Yield %
(conc.) ~concentration)
_ _ ,_
Bz-Ala-OMe Phenylalanine ethyl -(Bz-Ala-Phe-OH ~
(50 mM) cster (0 15 M) ~ Bz-Ala-Phe- ~ 70
LPhC-OH - J
Glutamic acid Bz-Ala-Glu
(~--t-Bu~ methyl (~-t-Bu)-OH 35
ester (0.5 M)
Glutamic acid (y-t-Bu~ Bz-Ala-Glu O
t-butyl ester (0.25 M) (~-t-Bu)-OH
. ~Bz-Ala-Met-OH
¦Bz-Ala-Met-
Methionine methyl J M~t-OI~ ¦
ester (0.2 M) ~ Bz-Ala-Met- ' ~6
Mct-Mct-OH
~z-Ala-Mct-.
Met-Mct-Met-OH¦
Bz.-Ala-Met- ¦ .
Met-Met-Met-
Met-OH -J
Methionine ethyl Bz-Ala-Met-OH 40
ester (0.2 M)
Methionine isopropyl Bz-Ala-Met-OH 8
ester (0.2 M)
Valinc methyl ester ~Bz-Ala-Val-OH ~
(0.7 M) ~ Bz-Ala-Val- ~ ~5
~Val-OH J
(0.5 M) Bz-Ala-Ser-OH 50
Tyrosine methyl Bz-Ala-Tyr-OH~5
e~ter (0.5 M)
Arginine methyl Bz-Ala-Arg-OH O
ester (0.5 M)
ArGinine (NO ) methyl Bz-Ala-Ar~(N02)- ~0
cster (0.5 ~ OH
l~istidine methyl Bz-Ala-His-OH 2
cster (0.5 M)
Threonine rncthyl Bz-Ala-Thr-OH
cstcr tO.5 l~)
. , _
~ - -- 35
s ~
-- - ~- Table IV (continued)
. . _ .
Substrate Amine component Product Yield %
(conc.) (concentration)
___
Ac-Phe-OEt Alanine me-thyl ester Ac-Phe-Ala-OH
(50 mM) (0.5 M) Ac-Phe-Ala- 60
Ala-OH
Bz-Gly-OMe Histidine methyl Bz-Gly-His-OH 40
(50 mM) ester (0.5 M)
EXA~LE 7
L- and D-stcreoisomers of the a_ _o comPonent
Analogously with cxamples 1, 2, 3, 4, and 6, the pep~idcs
stated in Table V wcre pro~uccd from the listed starting
materials.
It is seen that only the L-isomers are incorporated. This
is a very interesting fcature in tcrrns of process economy
since it is consequently not necessary to purify the
starting amino acids with a view to obtain the pure
L,isomer.
. , '
_.
-. Table V
~ . . . .
Carboxypeptidasc-Y catalyzcd ~ynthcsis of peptides with
L- and D-isomers of the amine component.
_
Substrate Amine component ProductYield ~,o
( conc . ) ( concentration) L-isomcrlD-isomer
. ._ ___
Bz-Ala-OMe Valine (0.6 M) Bz-Ala-Val- 42 O
(50 mM) OH
Alanine (1.8 M) Bz-Ala-Ala- 65 O
OH
Bz-Tyr-OEt Valine (o.6 M) Bz-Tyr-Val- 30 O
(30 mM) OH .
Alanine (1.8 M) Bz-Tyr-Ala- 46 O
01~ ,
Bz-Ala-OMe Val-NH2 (0.25 M) Bz-Ala-Val- 78 O
(50 m~l) NH2
Bz-Tyr-OEt Val-NH2 (0.25 M) Bz-Tyr-Val- 95 O
(30 mM) NH2 ..
Ac-Phc-OEt Ala-OMe (0.5 M) ~Ac-Phe-Ala-
(50 1~) 01~ 1 60 O
~c-Phe- I
... (Ala)2-OH J
. . ~
.
3~ ~1 '7'7~Z~
.
~- ~7
~ .
EXAMPLE 8
. . ._
Variation of the substrate ester ~roup
_
Analogously with examples 1, 2~ 3, and 4 the peptides
stated in Table VI were produced from -the ]isted starting
materials.
The results prove tl~e f]exibility of the process of the
invention as re~ards applicable substrates.
Table VI
Yield (%3 of carbox~peptidase-Y catalyzed synthesis of
peptides with different ester groups on the substrate
component.
Substrate I I
A ~ Bz Gly-OMe Bz-Gly-OEt Bz-Gly-OiPr Bz-Gly--OBz
component ~ (20 mM) (20 mM) (20 mM) (20 TT~)
. . . . _ _ .
Glycine _. . 45 47
Glycine amide 59 ! 95 ~ 88 ¦ 91
Leucine 21 59 74 80
(O 15 M) _ _
100 95 ~ 95
.
.. . ~
. .
- 38
EXAMPLE 9
Depsipeptides as substrates
Analogously with examples 3 and 4 the peptides stated in
Table VII were produced from the listed starting materials.
The conditlons were 4.5 /uM CPD-Y, pH = 7.6 and 25C.
It is seen that very high yields are obtained.
Table VII
Carboxypeptidase-Y catalyzed synthesis of peptides with
depsipeptides as substrate components.
Substrate Amine component Product Yield %
(conc.) (concentration)
.
Bz-Gly- Glycine amide (0.25 M) Bz-Gly-Gly-NH2 90
Odes-NH2- Leucine amide (0.25 M) Bz-Gly-Leu-NH2 95
( 20 mM) Phenylalanine amide Bz-Gly-Phe-NH2 95
) _ . . .
Bz-Gly- Glycine amide (0.25 M) Bz-Gly-Gly-NH2 95
Odes-NH2- Leucine amide ~0.25 M) Bz-Gly-Leu-NH2 95
( 20 m~S) ( 0. 25 M) --- Bz-Gly-Phe-NH2 95
Bz-Phe- Glycine amide (0.25 M) Bz-phe-Gly-NH2 90
Odes-NH2- Leucine amide (0.25 M) Bz-Phe-Leu-NH2 80
20 mM) Phenylalanine amide Bz-Phe-Phe-NH2 80
(0.25 M~ _ _
39
~XAMPLE 10
. ,
Peptides as su~strate comp~nen-ts
Analogously with examples 1, 2, 3, and 4 the peptides
stated in Table VIII were pl~oduced. The experimen`ts were
carried out in a Ra~iometer pH-stat and -the yields dc-
termined by IIPLC. The condi~ions were 5 /uM CPD-Y,
pH = 7.6 and 25C.
Table VIII
Carboxypeptidase-Y catalyzed synthesis of peptidcs ~Jith
peptides as substra-tes.
Substrate Amine component Product Yleld %
(conc.) (concentration)
.. _,
(20_m ~ Leucine amide (0.25 M) Bz-Phe-Leu-NH2 90
Bz-Gly-Phe Leucine amide (0.25 M) Bz-Gly-Leu-NH2 10 .
(20 mM Leucine amide (0.25 M) Z-Ala-Leu-NH2 74
_ ~ __ _
Z-Phe-Ala Leucine amide (0.25 M3 Z-Phc-I.eu-NH2 60
(20 mM)
. _ _ _
Z-Phe-Ser ¦Leucine amide (0.25 M) Z-Phc-Leu-NH2 7o
(20 mM3 l _ _
.
. . .
.
~)7~
.
= . . . . 40
Pcptide synthesis as a function of pH .. .
In cases where the synthesized peptide is a poor substrate
for CPD-Y the synthesis may be carried out at pH-v~lues
bclow the preferred 9 - 10.5.
EXAMPLE 11
Analogously with e~arnples 1 and 3 the peptides statcd in
Table IX were produced in a pl~-scat at the listed pH-
values.
The conditions wcre 15 /uM CPD-Y and 25C.
Table IY
Carboxypcptidase-Y catalyzed synth~sis of peptidcs as
function o~ pH.
..
Substrate Amine componcnt Product pH Yicld %
(conc.) (concentration)
. . .
Bz-~la-OMe Glycine (1.3 M) Bz-Ala-Gly 99 05 57
8 0 60
_ .. 6 o 60
Glycine amide Bz-Ala-Cly- 9.5 77
(1-3 M) NH2 9.0 95
8 0 1~7
_ . 6.0 _ 4/~
- ~7~
_ .
.
.
- - 41
~ . . . .
Various other amine components
--. .
_ AMPLE 1
Analogously with examples 3 and 4 the peptides stated in
Table X were produced. The experiments wcre carried out
in a pl~-stat and the yields determined by ]~PJ,C. The
conditions were 4.5 /uM CPD-Y, pH 9.7 and 25C.
Table X
Carboxypeptidase-Y catalyzed synthesis of peptides wi.th
diflerent amino acid derivatives as amine components.
Substrate ¦ Amine component ¦ Product ¦Yield
(conc.) ¦ (concentration) I l
._.............. _.__ _ ___ _
Bz-Al~-OMe ~-alanine amide (0.2 M) Bz-Ala-~Ala- 80
(50 mM) NH2 .
Glycine hydroxamic Bz-Ala-Gly- . 45
acid (0.2 M) NH-OH
D,L-Alanine hydroxamic Bz-A].a-Ala- 40
acid (O.2 M) NH-OH
. .
.1
- . -
;~c~ ~ . . 42
~ Y~PLE 13 ~~
. . .
Synth sis of ~ ides w th c~rboxypeptidases from bar].~y
Gerlninating barley, e.~. in the form of rnalt, contains two
dif~erent peptidascs denominated CP-l-l and CP-2-1 (see
~ 5 Lee E. Ray, Carlsberg Res. Cornm. 41, 169-182 (1976)).
CP-l-l and CP-2-1 ~ere isola-ted as described by L.E. Ray
and the peptides listed in Table XI were produced
analo{~ously with e~amples 1, 2~ 3, and 4 from the listed
startjng materials. ~he conditions were 6 /uM CP-l-l or
CP-2-1, pl1 8.0 and 25C.
Table XI
Synthesis of peptides usin~ carboxypcptida.ses from
~erillinating barley CP-l-l and CP-2-].
. . .
S~bstrate Amine component Product Yield i/o ~nzyme
(conc.) (concentration)
. ._ _ _ . ._ ,
Bz-Ala-OMe Valine amide Bz-Ala-Val- 43 CP-l-l
(50 m~) (0.25 M) NH2
Alanine amide Bz-Ala-Ala- 58 CP-l-l
(0.25 M) NH2
Lysine (1.7 M) Bz-Ala-Lys 11 CP-l-l
Valine amide Bz-Ala-Val- 57 CP-2-1
(0.25 M) '~H2
Alanine amide Bz-~la-Ala- 64 CP-2-1
~0.25 M) NH2
Lysine (1.7 M) Bz-Ala-Bys 11 P-2-1
~7'7~
_3 ~ ~ L~ 3
EXAI~PLE 14
Carboxypeptidase-Y catalyzed deamidation o~ peptide
amides
To a solution of 2 ml 15 rnM Bz-Ala-Leu NH2 in 0.1 M KCl -
5 1 mM EDTA, pH 9.7, 25C and 10/o dimethyl formamide Jas
added 2 mg CPD-Y. The reaction course as shown on fig. 5
was followed by HPLC as described in example 1. It is scen
that Bz-Ala-Leu-NH2 after 20 minutes was completely
converted to about 68% Bz-Ala-Leu~OH and 32% Bz-Ala-OH.
Amino acid analysis on the reaction mixture showed that
the Bz-Ala-OH was mainly formed by cleavage of Leu-NH2
from Bz-Ala-Leu-NH2.
EXAMPLE 1 5
Com~arison of ~e~tide ester substrates with and without
N-terminal ~rotective ~roups
Analogously with examples ~ and 4 the peptides stated in
Table XII were produced under the following conditions:
50 mM substrate, 5 ~uM CPD-Y 9 pH 9.5 and 25C.
Table XII
Comparison of carboxypeptidase-Y catalyzed syn-thesis of
peptides using peptide esters with and without N-terminal
protective groups as substrate.
. __ __ .. _ __
Substrate Amine component P Product Yield %
(conc.) (concentration~
, __ . _ . _. .. _ .
Ac-Al~-Ala- Leucine amide (0.15 M) Ac-Ala-Ala 90
Ala-OI-ie Ala-Leu-NH
(50-~'~) 2
Ala-Ala- Lcucine amide (0.15 M) Ala-Ala-Ala- 75
Ala-Oi;e Leu-NH2
(50 ~1) _
a. Y ~ ~,'d~J
,, . . __ . , .
_ 44
_. ~ __ . ... .
EX~MPLE 16
--~ . _
Svnthesis in the resence of or anic solvents
P
Analogously with examples 1, 2, 3, 4, and 6 -the peptides
stated in the below Tables XIII, XIV and XV were produced
from the listed starting materials and with the listed
concentrations of organic solvents.
The conditions were: 50 mM substrate, 5 /uM CPD-Y, pH 9.6
and 30 C.
Table XIII
Carboxypeptidase-Y catalyzed synthesis of peptides with
50 mM Bz-Ala-OMe as substrate and free amino acids as
amine component with and without 20% methanol (MeOH) in
0.1 M KCl, 1 mM EDTA. ,.
_ . _ ...
Amine component Product _ Yield %
(concentration) without with
MeOH MeOH
,, . _ _. _ _ .. _
Valine (0.6 M) Bz-Ala-Val-OH 42 53
Threonine (1.2 M) Bz-Ala-Thr--OH 52 61
Phenylalanine Bz-Ala-Phe-OH 16 18
(0.16 M) .
Leucine (0.16 M) Bz-Ala-Leu-OH 33 39
Methionine (0.6 M~ Bz-Ala-Met-OH 42 64
Glycine (3.0 M) Bz-Ala-Gly-OH 55 50
Serine (3.2 M) Bz-Ala-Ser-OH 50 52
Lysine (1.5 M) Bz-Ala-Lys-OH 53 41
Clutamic acid ~-t- Bz-Ala-Glu 54 55
butylester (1,0 M) (~-OBut)-OH
Asparagin (0.6 M) Bz-Ala-Asn-OH 18
.
r _ 4 5
__ = _~ ~
. Ta_le XIV
,_ . _ ...
Carboxypeptj.dasc-Y catalyzed synthesis of Bz-Ala-Thr-OH
from Bz-Ala-OMe (50 mM) and th~eoni.ne with varying
conccntrati.ons of polyethylene ~lycol-~OO (PEG-300).
- Conditions: CPD-Y 4 /uM, pH 9.6 and 2~C.
.~
So PEG-300 in Threonine % Yield of
0.1 M KOl concentration Bz-Ala-Thr-OH
0 0~7 M 36
0.7 M 34
0.7 M 31
~~ 30 0.7 M 29
0.35 M 28
.. . ..
Table XV
Carboxypeptidase-Y catalyzed synthesis of pcptides Irorn
50 mM Bz-Ala-OMe and the listed amino acid csters as arnine
component in 30~0 polyethylene glycol-O.l M KCl, 1 mM ~DTA.
The other conditions were: CPD-Y = 8 /uM, pH 9.6 and 25C. ..
__. . . .. ... _ ,
Substrate Aminc component Product Yield ~0
(conc.) (concentration)
. _ _ __ .. _
Bz-Ala-OMe Vali.nc-OMe (0.5 M~ Bz-Ala-Val_ 3
V~l-O~
Serinc-O~c (0,5 M) Bz-A].a~Scr OH 3o
Clutarnic ~cid (y-OBut) B~-Al.a-Glu- ~8
-OM~ (0.5 M) (Y-OBut)_o~ ~
Bz-Ala-Phc-OlI
Phcnylal.1ninc-OMe ~ Bz-Ala-P}le- ~2
- 46
. ~ Table XV (continued)
,. . ~ . . .
Substrate Amine cornponent Product Yield %
(conc.) (concentration)
Bz-Ala-OMe ~Bz-Ala-Met-OH ~
(50 m~) L-Methionine-OMe ~ Bz-Ala-Met- ~ 85
Bz-Ala-Met-
Me-t-Met-OH J
D-Methionine-OMe Bz-Ala-D-Met-OH O
(0.5 M)
_
EXAMPLE 1 7
Insoluble (immobilized) carboxy~e~tidase-Y
CPD-Y was bonded to Concanavalin A-Sepharose 4B (Pharmacia
Fine Chemicals) followed by cross-lirking between CPD-Y and.
Concanavalin A with glutaraldehyde as described by Hsiao
and Royer (Archives of Biochemistry and Biophysics, Vol.
198, 379-385 (1979). The CPD-Y concentration was 3 mg
per ml packed Sepharose. ..
Analogously with examples 1 and 3 the peptides stated in
Table XVI were produced under the following condi-tions:
50 mM substrate, 0.25 ml CPD-Y gel (5 /uM CPD-Y), pH 9.7
and 35C. After removal of the enzyme by fil-tration the
products and yields were determined by HPLC.
. : .
7~7g,~9
.
_ ~ _ . 47
_ _ . Table XVI
Insoluble car~oxypeptidasc-Y c~talyzed synthesis of
peptides.
__ . __ _ _ __ _
Substrate Amine component Product Yield ~'
(conc.) (concentration) _
___ _ .__ _ ____
Bz-Ala-OMe Phcnylalanine (0.15 M) Bz-Ala-Phe ~1
(50 !nM) --cin~ n z 11) gZ-Al L-Lcu-NH2 91
_ _
