Note: Descriptions are shown in the official language in which they were submitted.
20691~
HOECHST Al~TIENGESELLSCHAFT HO}3 91~F 154 Dr.Sl/St
~escription
A process for the rlostripain-catalyzed linkage of Arg-
Pro and Arg-B (B = proteinogenous and non-proteinogenou
amino acids) containing peptides
S Enzymatic peptide syntheses with a-amino-protected
arginine esters and various amino-unprotected, carboxyl-
protected or unprotected amino-acid derivatives as amino
donors can in principle be carried out with metallopro-
teases, serine proteases or thiol proteases. These
proteases are able to catalyze, depending on the reaction
equilibrium, not only the clea~age of the peptide or of
the protein into amino acids (= hydrolysis) but also the
reverse reaction. The peptide synthesis preferably takes
place under kinetic control.
Suitable and preferred in many cases for the aminolysis
of N-protected arginine esters is trypsin, which is
reasonably priced. However, as investigations by Oka (Oka
et al. J. Biochem. 82, 1055-1062, 1977) have shown, this
does not apply in the case of linkage with proline
derivatives as N components.
Mitchell (Science 162, p. 374, 1968) showed that the Arg-
Pro bond in Met-Lys bradykinins is hydrolyzed not by
trypsin but by clostripain.
Clostripain (EC 3.4.22.8) was isolated by Kochalaty
(Kochalaty et al. Arch. Biochem. 18, 1, 1948) from
Clostridium histolyticum and characterized by Laboues~e
and Gros (Bull. Soc. Chim. Biol. 42, 543, 1969), Xula
(Biochem. Biophys. Res. Comm. 69, 389, 1976) and Mitchell
(J. Biol. Chem. 242, 4683, 1968).
The method of enzymatic peptide linkage of N-protected
arginine esters with proline de~ivatives or di- and
tripeptides containing amino--terminal proline is
described by Andersen (Peptides, Proc. 9th APS, p. 355~
20~9199
-- 2 --
1986, Ed. Deber, Hruby) and Fo:rtier (Arch. of Bioch.
Biophys. 276, 317, 1990). Andersen and Fortier used for
their investigations clostripain which had been highly
purified by many consecutive steps and had a low ~pecific
activity (for example Fortier et al.: 38 U/mg).
The preparation of clostripain with an activity
2 80 U/mg, which is purified from the culture filtrate
from Clostridium histolyticum DSM 627 by alcohol
precipitation of the culture filtrate and subsequent
adsorption onto reactive dye, and its use for the
enzymatic peptide linkage of N-protected arginine esters
to proline derivatives or peptides containing amino-
terminal proline have not yet been described.
Furthermore, the clostripain-catalyzed peptide synthesis
with non-proteinogenous amino acids as amino donor in
place of the amino acid proline, and the use of clostri-
pain immobilizate for the linkage reaction, is unknown.
All the peptide linkages with clostripain described to
date in the literature have furthermore been carried out
only on the analytical scale without preparative isola-
tion of the products.
Analytical scale means that the synthesized peptide can
be detected by thin-layer chromatographic methods or
HPLC.
Preparative isolation is the term used when the syn-
thesized peptide is isolated by extraction processes for
subsequent use of the peptide on the industrial scale,
i.e. in gram or kilogram quantities.
In addition, no process with which Arg-Pro or Arg-B
containing peptide compounds can be prepared using
clostripain on the industrial scale has yet been
disclosed.
20~9199
-- 3 --
Numerous active peptides which have an Arg-Pro fragment
as part of their sequence are known. They are in some
cases used as pharmaceuticals. ThuS, for example, LHRH
(Gly-His-Trp-Ser-Tyr~Gly-Leu-Arg-Pro-Gly-NH2) or LHRH
analogs (gonadoliberin agonists, antagonists) such as,
for example, buserelin (Gly-His-Trp-Ser-Tyr-D-Ser(tBu)
~eu-Arg-Pro-NHEt) and many others. Shorter peptides with
Arg-Pro as part of the sequence likewise ha~e properties
of pharmacological interest.
Enzymatic peptide linkage processes are particularly
distinguished by the absence of interfering racemization
and the possible dispensation with side-chain protection.
Thusl in the specific case of arginine, the enzymatic
process has the advantage that the guanidino group does
not have to be modified with costly and difficult to
introduce and eliminate protective groups (for example
-Mtr).
Most industrially produced active peptides are syn-
thesized by the classical solution method of peptide
synthesis.
Enzymatic peptide linkages can be carried out in aqueous,
aqueous/organic or pure organic solvent systems. If, for
example, aqueous methanol is used as solvent, the pro-
ducts cannot be isolated with simple workin~-up processes
because the Arg-peptides are still sufficiently soluble
in water and, as a rule, do not precipitate and can be
extracted only inadequately with an organic extractant
such as ethyl acetate or methyl isobutyl ketone.
The use of a mixed phase solution for the peptide reac-
tion, which represents reaction and extraction mediumsimultaneously, has not hitherto been described.
It has now been found that enzymatic peptide synthesis
for preparing peptides which contain arginine as acyl
donor and proteinogenous and non-proteinogenous amino
20691~
acids as amino donors is efficiently catalyzed by
clostripain from methanol-precipitated culture filtrate
from Clostridium histolyticum DSM 627 and by enzyme which
has been purified by adsorption onto reactive dye.
The invention thus relates to:
1. Clostripain with an en~yme activity of at least
80 U/mg obtainable from Clostridium histolyticum
DSM 627 by the following preparation process:
- Fermentation of Clostridium histolyticum DSM 627
- preparation of a culture filtrate
- alcohol precipitation of the clostripain from the
culture filtrate
- adsorption of the alcohol-precipitat0d clostripain
onto reactive dye.
2. A process for the preparation of clostripain, which
comprises preparing clostripain with an enzyme
activity of at least 80 U/mg by fermentation of
Clostridium histolyticum DSM 627.
3. The use of clostripain for the linkage of arginine
which has amino-terminal protection or of peptides
which have amino-terminal protection and carboxyl-
terminal arginine (acyl donor) to an amino acid or
a peptide with amino-donor function, which in each
case have a carboxyl-protective group, a carboxamide
or carbohydrazide.
4. The use of clo~tripain for the linkage of an amino
acid or of a peptide to a non-proteinogenou~ amino
acid which has amino-donor function or to a peptide
which has an amino acid of this type.
The invention i5 described in it6 preferred embodiments
hereinafter. The invention i~ furthermore defined by the
contents of the claims.
In the process according to the invention clostripain
20~919~
-- 5 --
catalyzes the linkage of amino acids or peptides which
have arginine as acyl donor.
The clostripain according to the :invention is obtained as
follows:
5 - fermentation of Clostridium histolyticum DSM 627
- alcohol precipitation of the clostripain from the
culture filtratel in particular methanol precipi-
tation, and
- adsorption of the alcohol-precipitated clostripain
onto reactive dye, in particular onto reactive red
120
The clostripain according to the invention can also be
obtained as follows (see scheme 1):
- from sterile-filtered culture filtrate from
Clostridium histolyticum DSM 627 which has pre-
ferably been concentrated 50-fold or
- from ultrafiltered culture filtrate from Clostridium
his~olyticum DSM 627 or
- from culture filtrate from Clostridium histolyticum
DSM 627 after the alcohol precipitation, in particu-
lar after the methanol precipitation, or
- by ammonium sulfate precipitation of the ultra-
filtered culture filtrate from Clostridium histoly-
ticum DSM 627 or5 - by adsorption of clostripain obtained from alcohol-,in particular methanol-, precipitated culture fil-
trate from Clostridium histolyticum DSM 627 and
adsorption onto reactive dye, in particular onto
reactive red 120, or0 - by hydrophobic chromatography of the ammonium
sulfate- or alcohol-precipitated culture broth, in
particular on aminobutyl- or hexylagarose.
Clostripain can likewise be obtained by another combina-
tion of the purification steps indicated in scheme 1.
- 6 - 20~91~ 9
Scheme 1: Obtaining clostripain
Fermentation
(Clostridium histolyticum .DSM 627)
Centrifugation
Sterilization by filtration
Culture filtrate
(100%, about 80-100 U/mg)
Ultrafil lation ~ Treatment with
CL1 (100 U/mg) 5-10 mM CaCl2
max. 97%
~ ~ '
Ammonium sulfate ~ Alcohol precipitation
precipitation 70~ CL1 (10U U0mg)
CL2 CL3 (200 U/mg)
Hydrophobic chromatography Affinity chromatography
~ ~ 1 ~ for e Kample on
aminobutylagarose hexylagarose ti d 120
CL5 CL6 CL4
82~ 90~ 74%
(310 U/mg)
(238 U/mg) (160 U/mg)
2069199
To obtain clostripain, the strain DSM 627 is fermented
with the optimized nutrient medium NL 1581 or the
proteose peptone medium for 12-28 hours. The temperature
of the preculture is 33-39C, pr,eferably 37C, and that
of the main culture is 31-36C, preferably 33C.
The strain DSM 627 is freely available from the Deutsche
Gesellschaft fur Mikroorganismen und Zellkulturen,
Mascheroder Weg lB, 3300 Braunschweig~
Nutrient medium NL 1581:
(the figures relate to 1 1 of nutrient medium)
- 30 g of meat extract
- 30 g of casein peptone
- 5 g of yeast extract
- 1.74 g of K2HPO4
- 0.5 g of cysteine
pH 7.4
Proteose peptone medium:
(the figures relate to 1 1 of medium)
- 50 g of proteose peptone
- 1.74 g of K2HPO4
- 0.5 g of cysteine
pH 7.2
The fermentation with the optimized nutrient medium
NL 1581 or with the proteose peptone medium yields after
centrifugation (10,000 g for 10 min) and sterilization by
filtration (pore size of the membrane 0.2-0.4 ~m, pre-
ferably 0.3 ~m, such as, for example, Omega 0.3 ~m
filter supplied by Filtron) a culture filtrate with up to
50,000 U/L (corresponds to 60-120, in particular 80-
100 U/mg clostripain), which is concentrated by
ultrafiltration without loss of protein. This enzyme
quality is called C~l.
The ultrafiltration is used to concentrate $he culture
broth.
- 8 - - ~691 99
Suitable as ultrafiltration membrane are all membranes
with a pore size 5-50, preferably 10 RD (KD = kilodalton)
(for example Omega 10 K supplied by Filtron). Larger
batches (> 5 1 - 1 m3) are, however, concentrated by
means of a tangential flow (crossflow) cassette system.
Linkages can be carried out even with the clostripain
obtained from the ultrafiltration. The enzyme has an
acti~ity of 80-100, in particular 90 U/mg.
An alternative working up via ~mmonium sulfate precipita-
tion (40 or 70~ (NH4)2SO4) in analogy to Labouesse (Bull.
Soc. Chim. Biol. 42, 543-68, 1960) resulted in the
quality CL2 in 32~ yield, while alcohol-, in particular
methanol-, precipitation in analogy to Ogle (Arch.
Biochem. Biophys 42, 327-36, 1953) provides CL3 in about
70% yield of enzyme with a specific activity of up to
250 U/mg of protein.
The clostripain obtained by ammonium sulfate
precipitation can be further purified by hydrophobic
chromatography, in particular on aminobutyl- or
hexylagarose. The enzyme then has a quality of 150~250,
in particular 150-170 (hexylagarose) or 220-250 U/mg
~aminobutylagarose).
Before the methanol precipitation, to stabilize the
enzyme it is absolutely necessary to check the Ca2~ ion
content, otherwise CaCl2 or calcium acetate must be added
(requirement: 5-10 mM, 60C, 15 min). The procedure for
this is such that sufficient CaCl2 or calcium acetate is
added to the culture broth until the final concentration
in the culture broth reaches 5-10 mM.
The methanol precipitation makes it possible to obtain
enzyme of quality CL3 at reasonable cost and on the gram
scale.
2~9199
g
The clostripain obtained by alcohol precipitation can be
purified again by adsorption onto reactive dye, in
particular reactive red 120 [Sigma], and subsequent
elution with salts, preferably NaCl or KCl (0.4 M). This
procedure makes it possible to obtain highly pure clos-
tripain in only a two-stage process (1. methanol precipi-
tation of the culture filtrate and 2. adsorption of the
enzyme obtained in the first step onto a reactive dye).
In addition, high yields are obtained. The enzyme
activity is 250-350, in particular 280-330 U/mg.
Clostripain can be employed in immobilized and non-
immobilized form for the linkage reaction.
Carrier materials which are preferably employed are
carriers which act by adsorption, such as, for example,
silica gel or CNBr-Sepharose. The carriers ar~ commer-
cially available.
The enzyme is immobilized by mixing it with the carrier.
The duration of the coupling reaction, the reaction
medium and the mixing ratio of enzyme and carrier depend
on the nature of the chosen carrier and can easily be
determined by the person skilled in the art by pre-
liminary tests for each individual case.
For the peptide coupling it is necessary for an amino
component (= amino donor~ and a carboxyl component
(= acyl donor) to be present.
Used as carboxyl component are N~protected arginine alkyl
esters or peptide fragments which have at the carboxyl
terminus an arginine alkyl ester group, but preferably Z-
Arg-OMe, Z-Leu-Arg-OMe, Z-Gly-Pro-Arg-OMe, Boc-D-Arg-Arg-
OMe.
Used as amino component are amino-acid derivatives or
peptide fragments which have as amino terminus a pro-
teinogenous amino acid, with the exception of the amino
2069~9~
-- 10 --
acids Asn, Gln or Thr, or proline esters and amides,
preferably Pro-NH2, Pro-NHEt, Pro-Gly-NH2, Pro-Ala-NH2,
Pro-AzaEly-NH2, D-amino acids or non-proteinogenous amino
acids such as, for example, halogen-substituted phenyl-
S alanines, furyl-, thienylalanines, phenylglycine, homo-
phenylalanine, 2-aminonorbornane-2-carboxylic acid or
phosphorus-containing amino acids.
The ami.no acids or peptides to be coupled must be amino-
~carboxyl component) or carboxyl-protected (amino com-
ponent) by methods known to the person skilled in the artbefore the coupiing reaction.
Suitable for this are the amino-protective group~: Z,
Boc, Ac, For, Bz and the carboxyl-protective groups OR
(with R = C1-Cs, Bzl, tBux), NH2 and NHR' (R' = Cl-Cs,
NH-CONHz). A carboxamide or carbohydrazide can also be
used in place of the carboxyl-protective qroup.
The reaction conditions for the coupling reaction depend
on the amino acids or peptides to be coupled and are
detailed in the examples. The nucleophile iB added in
2-10-fold excess. Prolinamide or proline-containing
nucleophiles with N-terminal proline are preferably
employed in 2-6-fold excess compared with the acyl donor.
The peptide coupling can be carried out successfully with
this low excess of nucleophile only when
a) the methanol concentration and, at the same time,
b) the absolute concentration of the reactants is
increased.
The methanol concentration when a 2-6-fold excess is used
is in the range 40-90%. The concentration of the reac-
tants in the case of a 2-6-fold excess is increased by a
factor of up to 3 compared with the 10-fold excess.
Since, despite the high methanol content, the solubility
limit of the arginine derivatives is exceeded, the amino
acids must first be dissolved by heatiny, and the enzyme
2069199
11
added only after the solution has conled.
Apart from methanol, which is preferably employed for the
peptide coupling with the condition of a small excess of
nucleophile, it is also possible to use in addition: THF
(tetrahydrofuran), dioxane, dimethoxyethane, ethylene
glycol, diethylene glycol diethyl ether or ethanol.
The reaction conditions indicated for methanol likewise
apply to these solvents.
When butanol is used as solvent the excess of nucleophile
must be 10-fold.
The peptide coupling can also be carried out in a mixed
phase system which represents reaction and extraction
medium simultaneously. This mixed phase system is pre-
ferably composed of butanol and aqueous NaCl solution.
lS The reaction is carried out in a continuous or batchwise
process.
As described above, the linkage preferably takes place in
a mixed phase system which is reaction and extraction
medium simultaneously. Extraction results in transfer of
the peptide into the butanol phase, which is removed
after the phase separation. Extraction of the sodium
chloride phase several times by shaking with butanol
increases ~he peptide yield.
S
1. Immobilization of clostripain:
Clostripain can be ionically bonded to DEAE-
Sephadex A 50 (Pharmacia). However, covalent
immobilization is more advantageous for clostripain.
Thus, very high binding yields are achieved with
some of the materials listed in Table 1.
206~199
- 12 -
Table 1: Immobilization of clostripain on various
carriers
Carrier material D~T-pre- BindLing Activity Long-
activa- yield ~ U/ml term
5tion stability
DEAE-Sepharose A 50 no 32 13 --
" " yes 24 103
Silica gel yes 95 59 ~+
" no 95 46
Eupergit C no 90 432 +
" yes 99 166
VA epoxy yes 99 66 +
" no 92 16
15Tresyl Sepharose 4B yes 13 17 n.d.
" no 36 19
Cyanogen bromide
Sepharose 4B yes 34 45
" no 92 500
DEAE-Sepharose A 50-clostripain:
The immobilization was carried out in analogy to H.
Woodward, Imm. Cells and Enzymes, IRL Press, Oxford 1985.
Silica gel-clostripain:
The immobilization was carried out in analogy to
EP 3 438 189.
50 ml of silica gel (Grace) are mixed with 100 ml of
water and 3 g of aminopropylethoxysilane, stirred at 65C
for 2 h, filtered hot and dried at 90C for 24 h.
Then one part by volume of aminated carrier is mixed with
2 parts by volume of 0.25 M potassium phosphate buffer
pH 8 containing 2.5~ glutaraldehyde, and shaken gently
for 2 h.
The yield of immobilized en2yme after the carrier has
206919g
- 13 -
been filtered with suction ~nd washed several times i8
90-95%. The loading density is 45-70 U/ml of wet carrier.
The silica gel immobilizate is particularly suitable for
preparative purposes hecause of its favorable cost.
Eupergi~ C (Rohm):
Immobilization in O.5 M potassium phosphate buffer
pH 7.5t4C/48 h.
VA-epoxy ~Riedel de Haën)
Immobilization in l M potassium phosphate buffer
pH 7.5/20C/24 h.
Tresyl Sepharose 4B (Pharmacia):
Procedure analogous to Nilsson, Mosbach Biotech. Bioeng.
26, 1146-54, 1984.
Cyanogen bromide Sepharose B (Pharmacia):
Procedure analogous to Cobbs, Biotechnol. Techn. 5, 5-10,
1990 .
2. Assay of various clostripain purification fractions
for peptide synthesis activity and competing ester
hydrolysis during reaction of Z-Arg-OMe with Pro-NHz
in 30~ strength methanol solution (Z-Arg-OMe:Pro-
NH2 = 1:10)
These investigations were intended to establi~h how far
the enzyme must be purified in order to obtain Arg-Pro
linkage with high yield and minimum hydrolysis of the
arginine ester.
The formation of Z-Arg-Pro-N~2 and Z-Arg-OH as a function
of the enzyme quality employed was determined by HPLC
after 10 and 30 min.
The relation between product formation (Z Arg-Pro-NH2)
and the competing hydrolysis of Z-Arg-OMe to 2-Arg-OH
20~9199
- 14 -
indicates the quality of the course of the peptide
synthesis reaction.
Z-Arg-OH is a byproduct r~sulting from the competing
hydrolysis of Z-Arg-OMe. The amount of this byproduct
usually increases as the donor properties of the
nucleophile diminish or impurities in the catalyzing
enzyme interfere with the course of the reaction. In
addition, Z-Arg-O~I is very difficult to separate from the
resulting peptide. The success of the peptide synthesis
increases as the amount of Z-Arg-OH produced falls.
The amounts of Z-Arg-OH and Z-Arg-Pro-NH2 produced after
reaction times of 10 and 30 min were as follows:
Using clostripain from
2-Arg-OH Z-Arg-Pro-NH2
10 min 30 min 10 min 30 min
- ultrafiltered culture
filtrate (CLl): ~ 2.2 53 85
- methanol precipitation
(CL3): 2.5 3.9 67 91
- 70% ammonium sulfate
precipitation ~CL2): 0 0.7 64 88 %
- 40~ ammonium sulfate
precipitation (CL2): 1.8 2.4 78 97 %
- aminobutylagarose
purification (CL5): 1.8 2.5 61 88
- clostripain lyophilizate
after reactive red
treatment (CL4): 2.7 1.8 60 90 %
The amount of Z-Arg-OH or Z-Arg-Pro-NH2 indicated in
percentages is related to the total amount of Z-Axg-Pro-
NH2, Z-Arg-OH and Z-Arg-OMe.
III. Peptide syntheses with clostripain: peptide linkage:
A). ...Arg-Pro-... peptide linkage
20&~1~9
- 15 -
Example la:
Synthesis of Z-Arg-Pro-NH2 with soluble clostripain of
quali~y CL1:
358.8 mg (1 mmol) of 2-Arg-OMe x HCl and 1.5 g (10 mmol)
of Pro-NH2 x HCl are dissolved in 3 ml of 30% methanol,
the pH is adjusted to 7.8, and the solution is incubated
with 26 U of clostripai~ (CL1) at 40C. 1 U of clostri-
pain is defined as the amount of enzyme which hydrolyzes
l mol of N-benzoyl-L-arginine ethyl ester/min at 25C,
pH 7.6 in the presence of 2.5 mM dithiothreitol.
The pH is kept constant during the reaction. After 90 min
94% peptide has been obtained according to HPLCo For the
isolation, methanol is removed, and the aqueous solution
is saturated with sodium chloride and extracted by
shakin~ several times with n-butanol. The concentrated
butanol phase contains analytically pure peptide in
85-90% yield.
Example lb:
The mixture is made up as in Example la but the stated
amount of Z-Arg-OMe is dissolved together with 450 mg
(2.5 eq) of H-Pro-NH2 x HCl in 1 ml of 50% strength MeOH
by heating and, immediately after cooling to 40~C, the
enzyme is added. After a reaction time of 1 h, HPLC shows
that the conversion has reached 93~. Working up and
product isolation are carried out as described in la.
Example 2:
Synthesis of Z-Arg-Pro-NH2 with immobilized CL1 in a
methanol/water system
Mixture as in Example l but addition of 100 U of
clostripain-silica gel Lmmobili2ate. The mixture is
slowly stirred under pH-static conditions. After 40 min
there is 95~ peptide present, and the catalyst can be
removed and washed with buffer.
2~6~199
- 16 -
Example 3:
Synthesis of Z-Arg-Pro-NH2 with i~mobilized clostripain
in NaCl solution (6.1 M)/n-butanol (= 50:50) in batch
operation.
1 mmol of Z-Arg-OMe x HCl and 10 mmol of Pro-NH2 x HCl
are dissolved in 1.5 ml of 6.1 M NaCl solution and 1.5 ml
of n-butanol at pH 7.8 and 40~C.
48 U of silica gel Lmmobilizate are added. After 40 min
89% peptide has formed. After the phase separation the
butanol phase is removed and the ssdium chloride phase is
extracted by shaking three times more with n-butanol. 78%
~-Arg-Pro-NH2 is isolated.
Example 4:
Synthesis of Z-Arg-Pro-Gly-NH2 with column bed
reactor/NaClfbutanol:
7.18 g (20 mmol) of Z-Arg-OMe x HCl and 41.5 g (200 mmol)
of H-Pro-Gly-NH2 x HCl are dissolved in 30 ml of satu-
rated NaCl solution and 30 ml of butanol, and the pH is
ad~usted to 7.8 with 16.5 ml of 10 N NaOH, the reaction
temperature being 40C.
The mixture is passed over a 25 ml clostripain immobili-
zate column at a pumping rate of about 14 ml~min.
The reaction is stopped after 80 min, and the phases are
separated. The tripeptide was isolated in 86% yield from
the butanol phase. The remaining brine phase can now be
replenished again with the equivalents which have reacted
and again be pumped over the enzyme column.
After the brine phase had been used 4-5 times it was
possible to detect the diketopiperazine derivative cyclo-
(Pro-Gly), which led to an overall slowing down of the
reaction rate.
Example 5:
Further Arg-Pro linkages
- 17 - 2~9199
Clostripain-catalyzed peptide syntheses were carried out
with Z-Arg-OMe as acyl donor (that is to say Arg in
position P1) and proline (as amino-terminal amino acid of
the nucleophile) in Pl and occupied P2', P3' position.
for example: P2 Pl Pl~ P2' P3'
Z---Arg---Pro---Ala---NHz
Table 2
Nucleophile Peptide ~% yield range)
Pro-NHEt Z-Arg-Pro-NHEt 80-96
Pro-Gly-NH2xHCl Z-Arg-pro-Gly-NH2 70 9
Pro-Azagly-NH2xHCl Z-Arg-Pro-AzaGly-NH2 B0-90
Pro-Ala-NH2xHCl Z Arg-PrO-Ala-NH2 85-95
a) Z-Arg-Pro-NHEt:
Reaction mixture:
Ester:amine = 1:10 in 10% MeOH, pH 7.8; 0.25 ml of
CL3 (230 U/ml) per 1 mmol of ester; reaction time:
30 min;
HPLC yield: 80~.
b) Z-Arg-Pro-NHEt:
Reaction mixture:
Ester:amine = 1:10 in NaCl/BuOH = 1:1, pH 8.3;
0.5 ml of CL3 ~230 U/ml) per 1 mmol of ester;
reaction time: 50 min;
HPLC yield: 96%.
c) Z-Arg-Pro-Gly-NH2:
Reaction mixture:
Ester:amine = 1:10 in NaCl/Bu~H (lol) ~ 2 ml of C~3-
silica gel per 1 mmol of ester; reaction time 1 h;
HPLC yield: ~ 95%.
~he butanol phase contains peptide with a purity of
94%.
2069199
- 18
d~ Z-Arg-Pro-Ala-NH2:
Reaction mixture:
Ester:amine = 1:10, in 10% MeOH; 0.25 ml of CL3
` solution (230 U/ml) per 0.05 mmol of ester. Ester is
no longer detectable after 45 min. The products
according to HPLC were about 94% peptide, 5% %-Arg-
OH.
e) Z-Arg-Pro-OCAM:
C~ ~ C ~ C ~ 2
a
Reaction mixture:
Ester:amine = 1:10 in 10% MeOH,
0.15 ml of CL3 solution (~00 U/ml) per 1 mmol of
ester; reaction time: 2 h.
HPLC yield: 30%
f) Z-Arg-Pro-N2H3
Reaction mi~ture:
Ester:amine = 1:10 in 10% MeOH;
0.1 ml of CL3 solution (600 U/ml) per 1 mmol of
ester; reaction time: 3/4 h
HPLC yield: 56
g ) Z -Arg-Hyp-NH2
Reaction mixture:
Ester:amine = 1:10 in 10% MeOH
O.15 ml of CL3 solution (660 U/ml) per 1 mmol of
ester; reaction time: 1.5 h
HPLC yield: 81~
B) Peptide linkage of non-proteinogenous amino acids as
amino donor
2~69199
-- 19 --
1 mmol of Z Arg-OMe x HC1 and 1-2 mmol of nucleophile are
dissolved in 2-4 ml of 10% strength aqueous methanol and
adjusted to pH 7.8-8 with 5 N NaOH. This is followed by
addition of O.25-0.5 ml of CL3 enzyme solution (400-
500 U/ml). The enzyme has previously been treated withactivation buffer (2.5 mM CaCl2, :LO mM DTT) for at least
1 h. The reaction solution is run under pH-stat condi-
tions (titration with 1 N NaOH).
Some products precipitate directly out of the reaction
solution (for example Z-Arg-4F-Phe-NH2) and are isolated
by filtration/subsequent washing with 5%
methanol/distilled water. No Z-Arg-OMe is detectable by
HPLC after 0.75-2 h. Besides the peptide, 2-50% of Z-Arg-
OH was produced by competing hydrolysis of the ester
employed.
The reaction is stopped by adding a few drops of HCl04
~pH about 2-3).
The solution is concentrated to about 1/3 of the volume
to remove the methanol, saturated with NaC1 and extracted
three times with butanol. The peptide is often still
contaminated with a few percent of Z-Arg-OH. ~The pre-
parative yields are often considerably lower than the
HPLC figures because of incomplete extraction.)
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Table 3:
Nucleophile (formula) Peptide HPLC,
prep.
yield
(%)
4F-Phe-NH2 Z-Arg-4F-Phe-NH2 97,73
4F-Phe-ONe Z-Arg-4F-Phe-OMe 91,70
DL-4F-Phe~OProp Z-Arg-D,L-4F-Phe-OProp 90,69
4F-Phe-4F-Phe-NH2 Z-Arg-4F-Phe-4F-Phe-NH2 85,80
4Cl-Phe-OProp Z-Arg-4Cl-Phe-OProp ~95,76
Nal-OProp 1 Z-Arg-Nal-OProp ~95,83
DL-Phg-OMe 2 Z-Arg-D,L-Phg-OMe ~95,78
Phgly-Ot~u Z-Arg-Phgly-OtBu 90,70
HPhe-OEt 3 Z-Arg-HPhe-OEt 85,72
DL-DMPO-Ala-OProp 4 Z-Arg-D,L-DMPO-Ala-OProp 40,10
Thiaz-Cys-OProp 5 Z-Arg-Thiaz-Cys-OProp 70,50
PTC-Phe-NH2 6 Z-Arg-pTc-phe-NH2 >95,63
Fur-Ala-OMe 7 Z-Arg-Fur-Ala-OMe 75,45
Thi-ONe 8 Z-Arg-Thi-OMe 73,59
H-Sar(*)-NH2 Z-Arg-Sar-NH2 66
H-Gly-N2H4 Z-Arg-Gly-N2H4 92
H-Leu-N2H4 Z-Arg-Leu-N2H4 66
~)Sar = N-methylglycine
(Amino acid hydrazides must be regarded more as non-
proteinogenous amino acids than, for example, protected
derivatives of proteinogenous amino acids since, in
contrast to the latter, carbohydrazides of peptides or
amino acids cannot be cleaved back to the latter).
Formula: Amino-acid parent compound:
1 ~ ~ L-3-(2-naphthyl~-alanine
NX2
2o69l99
-- 21 --
2 ~cOOCH3 DL-2-phenyl-glycine
~H2
3 ~ COO L-homophenylalanine
NH2
H3C ~ COO DL-3-(dimethylphosphinyl)-alanine
H3C NH2
H
5 ~S ~ COO L-(2-thiazolinyl)-cysteine
NH2
O O
Il H p
5 6 H3C-P ~ C ~ L,L-phosphinotricinyl-phenylalanine
OH NH2
COOCH3 L-3-(2-furyl)-alanine
NH2
2069~9
8 ~ COOCH3 L-3-(2-thienyl)-alanine
NH2
3. Two-phase process with immobilizate in stirred
reactor with accumulation of the product
Four consecutive batch runs (each 1 mmol) with silica
gel/CL3 were carried out in an NaCl/butanol solution (50
butanol).
The reaction is slower in this system, the yield of
peptide is lower, and the percentage content of acid
produced is higher than in a comparable single phase
system. Furthermore, the activity of the catalyst
decreases more quickly than with the single phase
methanol/water system.
After the activation before the 4th run, the immobilizate
had only 50~ of its initial activity remaining.
Example 6:
Z-Arg-Pro-AzaGly-NH2
Cycle Ester Amine 10% MeOH CL3 React. Y~d
No. solution* time HPLC
mmol mmol ml ml min
1 5 50 15 3 2~ 78
2 +5 +5 15 - 45 81
3 +5 +5 20 2.5 40 80
4 +5 ~5 25 - 30 79
+5 +5 15 3 20 76
6 +5 +S 20 1 30 84
~CL3: 80 U/ml
After 6 cycleæ and extraction with n-butanol .
2069199
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4. Two-phase process with Lmmobilizate in column reac-
tor with accumulation of product
As preliminary experLments showed, precursors dissolved
in the butanol/NaCl solution can be circulated by pumping
without phase separation through a catalyst bed without
difficulty.
Then 2 batch runs (each 15 mmol) were carried out in each
case with a silica gel and a Eupergit column.
The reaction rate is somewhat decreased, and the yield i8
somewhat less. In the second batch run the column shows
a distinct loss of activity. Furthermore, the acyl donor
does not react quantitatively.
Nevertheless, preparative amounts can be conveniently
prepared with this method, because an important advantage
of the two-phase system is regarded as being that the
product can be removed directly from the butanol phase
after the phase separation.
Example 7:
Z-Arg-pro-Gly-NH2:
3 cycles were run, selecting a 10-fold nucleophile excess
only for the first mixture. In the following cycles only
the equivalents which had been removed by reaction were
replaced, and the reaction rate was corrected by addi-
tional enzyme addition.
20~9199
- 24 -
Cycle Ester Amine ~uQH NaCl CL3 React. Vi~ld
No. solution time HPLC
mmol mmol ml ml ml min %
1 5 50 12.5 12.5 0.25 60 >95
After the 1st cycle, the butanol phase is separated off,
and the NaCl phase replenished with the equivalents
cycle 2
2 +5 +5 +12.5 - 1.0 120 >95
After the 2nd cycle, the butanol phase is separated off,
and the NaCl phase replenished with the equivalents - 3
3 +5 ~5 +12.5 - 0.7 75 ~90
The combined butanol phases are dried and concentrated.
According to TLC, HPLC, there are still 2-5% Z-Arg-OH
present, which can be removed by recrystallization
several times.
Overall preparative yield 72~ (about 95~ pure).
The yield decreases at an increasing rate as the number
of cycles increases. As the reaction time increases under
these reaction conditions there is formation of relevant
small amounts of the diketopiperazine:cyclo-(Pro-Gly)
which initially reduces the effective amino concentration
and possibly also might have additional inhibiting
effects on the reaction. Thus, it is worthwhile to use
this reaction only for a limited number of batch runs.
A continuous process continued for a long time i8 not
advisable because of the instability of the amine.
':
