CA2069199A1 - Process for the clostripain-catalyzed linkage of arg-pro and arg-b (b=proteinogenous and non-proteinogenous amino acids) containing peptides - Google Patents
Process for the clostripain-catalyzed linkage of arg-pro and arg-b (b=proteinogenous and non-proteinogenous amino acids) containing peptidesInfo
- Publication number
- CA2069199A1 CA2069199A1 CA002069199A CA2069199A CA2069199A1 CA 2069199 A1 CA2069199 A1 CA 2069199A1 CA 002069199 A CA002069199 A CA 002069199A CA 2069199 A CA2069199 A CA 2069199A CA 2069199 A1 CA2069199 A1 CA 2069199A1
- Authority
- CA
- Canada
- Prior art keywords
- clostripain
- arg
- amino
- peptide
- pro
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
A process for the clostripain-catalyzed linkage of Arg-Pro and Arg-B (B = proteinogenous and non-proteinogenous amino acids) containing peptides The clostripain obtained from the culture filtrate from Clostridium histolyticum DSM 627 is employed for the linkage of arginine with amino-terminal protection or of peptides with amino-terminal protection and carboxyl-terminal arginine (acyl donor) to an amino acid or to a peptide with amino donor function. The enzyme can be obtained in high purity by alcohol precipitation and subsequent adsorption of the enzyme obtained from the alcohol-precipitated culture filtrate onto reactive dye and used for the linkage. The specific activity of clostripain is at least 80 U/mg. Clostripain catalyzes not only the abovementioned linkage of proteinogenous amino acids but also the linkage of a proteinogenous to a non-proteinogenous amino acid.
Description
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 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 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.
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 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 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~
catalyzes the linkage of amino acids or peptides which have arginine as acyl donor.
The clostripain according to the :invention is obtained as follows:
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~
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 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 or 5 - 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, or 0 - 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.
- 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 or 5 - 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, or 0 - 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 82~ 90~ 74%
(310 U/mg) (238 U/mg) (160 U/mg) 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~
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 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 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 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 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 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 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:
~ ~ ' 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 82~ 90~ 74%
(310 U/mg) (238 U/mg) (160 U/mg) 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~
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 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 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 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 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 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 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%.
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 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.) 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 2o69l99 2 ~cOOCH3 DL-2-phenyl-glycine ~H2 3 ~ COO L-homophenylalanine H3C ~ COO DL-3-(dimethylphosphinyl)-alanine H
5 ~S ~ COO L-(2-thiazolinyl)-cysteine O O
Il H p 5 6 H3C-P ~ C ~ L,L-phosphinotricinyl-phenylalanine COOCH3 L-3-(2-furyl)-alanine 2069~9 8 ~ COOCH3 L-3-(2-thienyl)-alanine 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 .
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 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.
':
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%.
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 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.) 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 2o69l99 2 ~cOOCH3 DL-2-phenyl-glycine ~H2 3 ~ COO L-homophenylalanine H3C ~ COO DL-3-(dimethylphosphinyl)-alanine H
5 ~S ~ COO L-(2-thiazolinyl)-cysteine O O
Il H p 5 6 H3C-P ~ C ~ L,L-phosphinotricinyl-phenylalanine COOCH3 L-3-(2-furyl)-alanine 2069~9 8 ~ COOCH3 L-3-(2-thienyl)-alanine 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 .
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 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.
':
Claims (9)
1. Clostripain with an enzyme activity of at least 80 U/mg obtainable from Clostridium histolyticum DSM 627 by - fermentation of Clostridium histolyticum DSM 627 - preparation of a culture filtrate - alcohol precipitation of the clostripain from the culture filtrate - adsorption of the alcohol-precipitated 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 process as claimed in claim 2, wherein clostri-pain is prepared from the culture broth from Clostridium histolyticum DSM 627 by filtration, ultrafiltration, ammonium sulfate precipitation, alcohol precipitation, hydrophobic chromatography or affinity chromatography.
4. The process as claimed in claim 2, wherein clostri-pain is obtained from the culture broth from Clostridium histolyticum DSM 627 by alcohol precipi-tation of the clostripain from the culture filtrate and subsequent adsorption of the alcohol-precipi-tated enzyme onto reactive dye.
5. The process as claimed in claim 4, wherein methanol is used for the alcohol precipitation of the clos-tripain.
6. The use of the clostripain as claimed in claim 1 or of the clostripain obtainable as claimed in one or more of claims 2 to 5 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.
7. The use of the clostripain as claimed in claim 1 or of the clostripain obtainable as claimed in one or more of claims 2 to 5 for the linkage of an amino acid or of a peptide to a non-proteinogenous amino acid which has amino-donor function or to a peptide which has an amino acid of this type.
8. The use of the clostripain as claimed in claim 1 or of the clostripain obtainable as claimed in one or more of claims 2 to 5, wherein the clostripain is employed in immobilized form.
9. The use of the clostripain as claimed in claim 1 or of the clostripain obtainable as claimed in one or more of claims 2 to 5, wherein the reaction and extraction medium of the peptide linkage is a mixed phase solution.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4116753 | 1991-05-23 | ||
| DEP4116753.8 | 1991-05-23 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2069199A1 true CA2069199A1 (en) | 1992-11-24 |
Family
ID=6432196
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002069199A Abandoned CA2069199A1 (en) | 1991-05-23 | 1992-05-22 | Process for the clostripain-catalyzed linkage of arg-pro and arg-b (b=proteinogenous and non-proteinogenous amino acids) containing peptides |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP0518088A2 (en) |
| JP (1) | JPH05252948A (en) |
| CA (1) | CA2069199A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6461834B1 (en) | 1998-11-06 | 2002-10-08 | Bionebraska, Inc. | Clostripain catalyzed amidation of peptides |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8236356B2 (en) * | 2008-06-11 | 2012-08-07 | Roche Diagnostics Operations, Inc. | Growth medium for Clostridium histolyticum |
| JP2022529565A (en) * | 2019-02-14 | 2022-06-23 | ノルドマルク ファーマ ゲーエムベーハー | Chromatographic purification of at least one enzyme selected from the group consisting of collagenase type I, collagenase type II, neutral protease, and clostripain. |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2366306A1 (en) * | 1976-02-20 | 1978-04-28 | Anvar | Pure clostripain enzyme prepn. - by affinity chromatography of Clostridium histolyticum culture or crude collagenase |
-
1992
- 1992-05-20 EP EP19920108509 patent/EP0518088A2/en not_active Withdrawn
- 1992-05-22 JP JP4130687A patent/JPH05252948A/en active Pending
- 1992-05-22 CA CA002069199A patent/CA2069199A1/en not_active Abandoned
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6461834B1 (en) | 1998-11-06 | 2002-10-08 | Bionebraska, Inc. | Clostripain catalyzed amidation of peptides |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0518088A3 (en) | 1994-03-23 |
| JPH05252948A (en) | 1993-10-05 |
| EP0518088A2 (en) | 1992-12-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4543329A (en) | Process for the specific cleavage of protein sequences from proteins | |
| FI70597C (en) | FOER FARING FOR ENZYMATIC FRAMSTAELLNING AV PEPTIDER | |
| US5338668A (en) | Opioid peptides derived from wheat proteins | |
| US4645740A (en) | Process for enzymatic replacement of the B-30 amino acid in insulins | |
| EP0278787B1 (en) | A process for enzymatic production of dipeptides | |
| Clapés et al. | Enzymatic peptide synthesis in low water content systems: preparative enzymatic synthesis of [Leu]-and [Met]-enkephalin derivatives | |
| CN111819191A (en) | Chemoenzymatic synthesis of liraglutide, somaglutide and GLP-1 | |
| CN111757891A (en) | Chemical-enzymatic synthesis of somaglutide, liraglutide and GLP-1 | |
| GB2131813A (en) | Peptide substrates for mammalian collagenase | |
| Gaertner et al. | Peptide synthesis catalyzed by modified. alpha.-chymotrypsin in low-water organic media | |
| CA2069199A1 (en) | Process for the clostripain-catalyzed linkage of arg-pro and arg-b (b=proteinogenous and non-proteinogenous amino acids) containing peptides | |
| Shui-Tein et al. | Stable industrial protease catalyzed peptide bond formation in organic solvent. | |
| Stepanov et al. | Subtilisin and α-chymotrypsin catalyzed synthesis of peptides containing arginine and lysine p-nitroanilides as c-terminal moieties | |
| Kase et al. | Hydrolysis of neo-kyotorphin (Thr-Ser-Lys-Tyr-Arg) and [Met] enkephalin-Arg6-Phe7 by angiotensin-converting enzyme from monkey brain | |
| Gaertner et al. | Papain-catalyzed peptide synthesis and oligomerization of amino acid amides in organic solvents | |
| EP0324659A2 (en) | Enzymatic process for producing immunomodulating pentapeptides and intermediates for use in the process | |
| Čeřovský et al. | Peptide synthesis catalyzed by native proteinase K in water-miscible organic solvents with low water content | |
| JP2779171B2 (en) | Vibriolysin binding method | |
| Ricca et al. | Peptide synthesis catalyzed by chymotrypsin in organic solvents | |
| Groeger et al. | Papain‐catalyzed synthesis of dipeptides | |
| Belyaeva et al. | Peptide synthesis in organic media with the use of subtilisin 72 immobilized on a poly (vinyl alcohol) cryogel | |
| Fortier et al. | Substrate-and stereo-specificities in clostripain-catalyzed peptide synthesis | |
| JP2000078971A (en) | New enzyme for synthesizing oligopeptide from d- or l- aminoacid ester and microorganism producing the same | |
| EP0458475A2 (en) | Transpeptidation reagent and method | |
| YASUTAKE et al. | Cyclic Peptides: VIII. Synthesis and Tryptic Hydrolysis of Cyclic Depsidipeptides Containing a Lysine Residue |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |