EP0537185A1 - Ein enzymatischer prozess für die herstellung von derivaten des wachstumshormon freisehenden faktors und nützlichen peptiden in diesen prozess - Google Patents

Ein enzymatischer prozess für die herstellung von derivaten des wachstumshormon freisehenden faktors und nützlichen peptiden in diesen prozess

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Publication number
EP0537185A1
EP0537185A1 EP91910581A EP91910581A EP0537185A1 EP 0537185 A1 EP0537185 A1 EP 0537185A1 EP 91910581 A EP91910581 A EP 91910581A EP 91910581 A EP91910581 A EP 91910581A EP 0537185 A1 EP0537185 A1 EP 0537185A1
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EP
European Patent Office
Prior art keywords
grf
ser
ala
met
carboxypeptidase
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.)
Withdrawn
Application number
EP91910581A
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English (en)
French (fr)
Inventor
Klaus Breddam
Morten Meldal
Sitg Aasmul-Olsen
Fred Widmer
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CARLBIOTECH Ltd AS
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CARLBIOTECH Ltd AS
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Application filed by CARLBIOTECH Ltd AS filed Critical CARLBIOTECH Ltd AS
Publication of EP0537185A1 publication Critical patent/EP0537185A1/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/16—Serine-type carboxypeptidases (3.4.16)
    • C12Y304/16005—Carboxypeptidase C (3.4.16.5), i.e. carboxypeptidase Y
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/60—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
    • 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)

Definitions

  • the present invention relates to.a process for the preparation of GRF(1-29)NH 2 , an interesting biologically active derivative of growth hormone releasing factor, and analogs thereof as well as new peptides which are useful as intermediates in this process.
  • Patent No. 4.708.934 EP 308067A, DK Application No.
  • This glycine oxidase enzyme which is dependent on Cu 2+ , O 2 and ascorbate as cofactors is considered to be the enzyme responsible for in vivo formation of peptide amides. It has been utilised for amidation of peptides in small scale but as it exhibits low activity its applicability for large scale work is still questionable.
  • the enzyme as isolated from natural sources like rat medullary thyroid carcinoma is very costly.
  • Amidation may also by achieved by protease-catalysed condensation reactions using an amino acid amide or peptide amide as nucleophile.
  • the yields of condensation reactions are generally low even in the presence of organic solvents unless the product precipitates in the reaction mixture and this is often not the case with long peptides.
  • the precursor peptide may exhibit poor solubility in such media.
  • serine or thiol- protease catalysed transpeptidation reactions may be carried out in high yield but it is a prerequisite that the enzyme exhibits specificity for a peptide bond close to the C-terminus. Endopeptidases are not generally suitable since they usually will cleave at other positions in the peptide chain as well.
  • Serine carboxypeptidases exhibit strict specificity for the C- terminal peptide bond and are able to catalyse the exchange of the C-terminal amino acid with an amino acid amide, added to the reaction medium to compete as nucleophile with water.
  • carboxypeptidase Y (CPD-Y) from yeast.
  • C-terminal amidation of a peptide by means of a serine carboxypeptidase catalysed transpeptidation reaction is dependent on: a) solubility of the peptide in an aqueous medium in which the enzyme is active and relatively stable, b) accessibility of the C-terminus to enzymatic cleavage and c) availability of a serine carboxypeptidase of suitable substrate preference.
  • H-C-OH has, in the case of CPD-Y, been shown to significantly influence this ratio as well (14, 15) albeit with no obvious trend. Since this amino acid residue does not constitute part of the desired amidated product (Tl) it may in principle be chosen freely.
  • a serine carboxypeptidase may also catalyse the formation of an elongated condensation product (C1) in competition with H1 and T1.
  • C1 elongated condensation product
  • the hydrolysis product (H1) may be transformed via the same type of reactions into the products T2, H2 and T1, followed by similar reactions with H2, etc.
  • new products may also arise when the enzyme acts on the C- terminal amide bond of T1, i.e. ammonia functions as leaving group, producing another transpeptidation product (TT1) or hydrolysis product (HT1).
  • TT1 transpeptidation product
  • HT1 hydrolysis product
  • the process is not always sufficiently selective and necessitates purification procedures in order to remove products of various side reactions in particular when longer peptides are used, in which case the optimal reaction conditions for suppressing the side reactions are difficult to establish.
  • Tamaoki obtained human calcitonin amide in a yield of 24.7%, leaving 57% unreacted substrate and 17.2% non- amidated side products (including human calcitonin).
  • Tamaoki patents disclose a process for the preparation of a peptide having a C- terminal proline amide, which comprises reacting in aqueous solution a peptide substrate having C-terminal Pro-Leu, Pro-lle, Pro-Val or Pro-Phe with carboxypeptidase Y in the presence of ammonia.
  • the present invention relates to a process for the preparation of derivatives of growth hormone releasing factor and analogs thereof.
  • GRF Growth hormone releasing factor
  • somatocrinin stimulating the release of growth hormone in the pituitary, is a 44-residue amidated peptide 5 10
  • Gly-Ala-Arg-Ala-Arg-Leu-NH 2 which may be used to stimulate growth of vertebrates (31). Both the amide group as well as the 15 amino acid residues closest to the C-terminus can be removed without abolishing the activity, but each results in a gradual decrease in the biological activity. However, a large part of the activity may be regained by amidation of these truncated peptides.
  • GRF (1-29)NH 2 which can be regarded as the bioactive core of GRF. Since the determination of the activity of GRF(1-29)NH 2 numerous structural linear and cyclic analogs have been made by exchanging, deleting or modifying one or more of the 29 L-amino acids in the native GRF-chain.
  • Val, Leu, Aib and Ala resulted in analogs with increasing biological activity, while replacement by Sar, a helix- breaking residue resulted in a profound loss of biological activity.
  • EP-A-352014 (Salk Inst. for Biol. Studies) showing a number of possible modifications of GRF and GRF(1-29)NH 2 in the 1, 2, 3, 8, 12, 13, 15, 18, 21, 22, 24, 25, 27 and
  • Arg 29 may be replaced by Cys, Abu, Asp, Glu, Orn, Lys, Dab or Dap (all L or D). Ostensibly these peptides are more active with better resistance to enzymatic degradation than native GRF.
  • EP-A-307860 (Roche) describes a number of linear and cyclic GRF analogs related to the ones earlier discussed.
  • EP-A-292334 (Salk) describes a number of possible modifications of GRF and GRF(1-29)NH 2 in the 1, 2, 3, 8, 10, 12, 13, 15, 18, 21, 22, 24, 25, 27 and 28 positions.
  • FR-A-2594832 (Sanofi) describes modified GRF(1-29)NH 2 , where Val 19 may be exchanged with lle, Met 27 may be exchanged with Nle and/or Ser 28 may be exchanged with Asn and in which at least 1 and up to 6 amino acids, preferably in positions 2, 5, 12, 17, 21 or 27, are replaced by an ⁇ -amino alkynoic acid, preferably ⁇ -amino- 4-pentynoic acid, ⁇ -amino-4-hexynoic acid and 2,6-diamino-
  • EP-A-216517 (Salk) corresponding to US-A-4689318 describes a number of GRF(1-29)NH 2 analogs where at least 4 of amino acids 5-13, 15, 18-21 and 26 are different from the native sequence and preferably containing Ala 28 .
  • the invention is based on the surprising finding that by selecting as the leaving group X in the starting material GRF(1-28)X a number of amino acids which does not logically fit into any of the preference patterns suggested in the earlier works by Breddam et al and Tamaoki discussed above, viz. the acyclic ⁇ -aminocarboxylic acids having an uncharged hydrophilic side chain of at least the size of a methyl group, it is possible to obtain GRF(1-29)NH 2 in a surprisingly high yield.
  • Preferred amino acids are Thr, Ala, Ser, Gln and Asn, in particular Thr and Ala. Very small amounts of unreacted substrate and byproducts resulting from the competing reactions are observed, in particular when the experiences with human insulin amide described above and Tamaoki's results with calcitonin are taken into account.
  • the present invention relates to a process for the preparation of derivatives of Growth Hormone Releasing Factor, viz. GRF(1-29)NH 2 and analogs thereof, characterized by reacting a substrate component of the formula GRF'-Met-Ser-X wherein GRF' denominates the native GRF(1-26) sequence or analogs thereof including GRF(n-26) fragments, where n is from 1 to 8, and X is an acyclic ⁇ -amino carboxylic acid residue having an uncharged hydrophilic side chain of at least the size of a methyl group, with H-Arg-NH 2 as nucleophile component in the presence of an L-specific serine or thiolcarboxypeptidase enzyme from yeast or of animal, vegetable or other microbial origin in an aqueous solution or dispersion having a pH of from 6 to 9, and if necessary coupling the desired N-terminal (1-(n-1)) fragment chemically or enzymatically.
  • an endopeptidase enzyme may be used having the proper specificity with regard to the C-terminal of the (1-(n-1)) fragment, e.g. papain or chymotrypsin.
  • a chemical fragment coupling may be carried out following introduction of suitable protective groups in solution phase in the presence of suitable catalysts, e.g. DCC together with additives, e.g. HOBt.
  • suitable catalysts e.g. DCC together with additives, e.g. HOBt.
  • a preferred group of substrate components are those of the formula GRF"-Met-Ser-X, wherein GRF" denominates the native GRF(1-26) sequence, wherein from 1 to 4 of the amino acid residues may be replaced or deleted or the des- ⁇ -NH 2 derivatives thereof and X has the above meaning.
  • the applicable carboxypeptidases in the process of the invention are L-specific serine or thiol carboxypeptidases.
  • Such enzymes can be produced by yeast fungi, or they may be of animal, vegetable or other microbial origin.
  • a particularly expedient enzyme is carboxypeptidase Y from yeast fungi (CPD-Y). This enzyme is described in the earlier patents i.a. with reference to Johansen et al (Ref. 28) who developed a particularly expedient purification method by affinity chromatography on an affinity resin comprising a polymeric resin matrix with coupled benzylsuccinyl s. CPD-Y, which is a serine enzyme is available in large amounts and displays relatively great stability. Further details are given in Ref. 14.
  • the pH-control may be provided for by incorporating a suitable buffer for the selected pH-range in the reaction medium.
  • the pH-value may also be maintained by adding an acid, such as HCl, or a base, such as NaOH, during the reaction. This may conveniently be done by using a pH-stat.
  • an acid such as HCl
  • a base such as NaOH
  • the conditions may also be influenced upon by varying the enzyme concentration, reaction time, etc.
  • the reaction is carried out in an aqueous reaction medium which, if desired, may contain up to 80% by volume of an organic solvent.
  • organic solvents are alkanols, e.g. methanol and ethanol, glycols, e.g. ethylene glycol or polyethylene glycols, glycerol, alkanoic acids, e.g. acetic acid, dimethyl formamide, dimethyl sulfoxide, tetrahydrofurane, dioxane and dimethoxyethane.
  • composition of the reaction medium depends particularly upon the solubility, temperature and pH of the reaction components and the reaction products involved and upon the stability of the enzyme.
  • the reaction medium may also comprise a component that renders the enzyme insoluble, but retains a considerable part of the enzyme activity, such as an ion exchanger resin.
  • the enzyme may be immobilized in known manner, e.g. by bonding to a matrix, such as a cross-linked dextran or agarose, or to a silica, polyamide or cellulose, or by encapsulating in polyacrylamide, alginates or fibres.
  • the enzyme may be modified by chemical means to improve its stability or enzymatic properties.
  • a chelating agent e.g. EDTA is included in the reaction medium which may also comprise salts.
  • concentration of the two participants in the reaction may vary within wide limits, as explained below.
  • a preferred starting concentration for the substrate component is 0,1-20 mM, preferably 1-10 rnM, and for the nucleophile component 0.02 to 2 M, preferably 0.2 - 1.5 M.
  • the enzyme activity may vary as well, but the concentration is preferably 10 -8 to 10 -4 M.
  • concentration is preferably 10 -8 to 10 -4 M.
  • the most advantageous activity depends i.a. on the substrate chain and concentration, the nucleophile concentration, the reaction time, the reaction temperature, the pH, and the presence of organic solvents and/or salts.
  • the amount of enzyme should in each case be adjusted to give the necessary degree of conversion for formation of an optimal absolute amount of product.
  • the reaction temperature is 10° to 50°C, preferably 20° to 40°C.
  • the most appropriate reaction temperature for a given synthesis can be determined by experiments, but depends particularly upon the concentration of the nucleophile component and the enzyme concentration.
  • An appropriate temperature will usually be about 20° to 30°C, preferably about 25°C, taking into account due consideration for enzyme activity and stability.
  • the standard reaction time in the process of the invention is about 1 - 3 hours.
  • the abbreviations of- amino acids, amino acid derivatives and peptides are according to Guidelines of the IUPAC-IUB Commission on Biochemical Nomenclature and the amino acids are on L-form unless otherwise stipulated.
  • the substrate positions are all denoted P 1 ', P 1 , P 2 7-8 P i in correspondence with the binding sites (Ref. 1).
  • DMF was purified by fractional distillation in vacuo. THF was passed through active Alumina prior to use. Fmoc-amino acids were purchased from Milligen and were converted into Dhbt esters by reaction with carbodiimides and Dhbt-OH (Fluka) in THF (20). The peptide synthesis resin (Macrosorb SPR) was purchased from Sterling Organics. Sequence analysis was carried out on an ABI gas phase sequencer and d Durrum D-500 instrument was used for amino acid analysis. The HPLC equipment was from Waters Associates. Samples were hydrolyzed for 24 h in 6 N hydrochloric acid and evaporated, prior to amino acid analysis.
  • H-Arg-NH 2 -2HCl, Bz-Met-OH, other amino acids and amino acid derivatives and GRF (1-29)-NH 2 were obtained from Bachem, Switzerland.
  • CPD-Y is commercially available from the applicants.
  • the substrates Bz-Met-Ser-Ala-OH, Bz-Met- Ser-Leu-OH, Bz-Met-Ser-Thr-OH, Bz-Met-Ser-Arg-OH, Bz-Met- Ser-Gly-OH and Bz-Met-Ser-Ser-OH were synthesized as described below.. All other reagents and solvents were from Merck, W. Germany.
  • CPD-W-II and CPD-S-1 were prepared as previously described (18, 21).
  • BzMetSerAla, BzMetSerSer, BzMetSerGly, BzMetSerLeu, BzMetSerArg, BzMetSerAsn, BzMetSerGln and BzMetSerMet were prepared in an analogous manner, using the free amino acids Ala, Ser, Gly, Leu, Arg, Asn, Gin and Met as nucleophiles instead of Thr, respectively, and similarly pure products were obtained.
  • a standard synthesis cycle consisted of a sequence of 10 min deprotection with piperidine in DMF (20%), a wash with
  • the peptide was dissolved in 10 ml DMF/1% AcOH (2/3, 10 ml) and chromatographed on a G15 column with 1% AcOH. The fractions containing the major component was collected and lyophilized. This was separated by preparative HPLC on a 25 mm x 300 mm 15 ⁇ reversed phase column (Waters deltaprep.). The sample was divided into two portions each of which were dissolved in 20 ml 15% DMF in water. The sample was applied to the column and eluted at 10 ml/min first for 10 minutes with 30% B and then with a gradient (20% -70% B) over 30 min.
  • the main peak eluted after 32 min and was collected and lyophilized to yield a total of 44 mg of peptide.
  • the amino acid analysis showed: Asp 2.93; Thr 0.94; Ser 2.82; Glu 2.23; Gly 1.08; Ala 3.91; Val 1.05; Met 1.00; lle 2.05; Leu 4.07; Tyr 1.67; Phe 0.90; Lys 2.16; Arg 2.31.
  • the structure of the peptide was confirmed by gas phase sequence analysis.
  • Fig. 2 Separation of the products from GRF(1-28)-Ala-OH after treatment with CPD-Y for 147 minutes in the presence of H-Arg-NH 2 .
  • the separation was carried out on a Vydac C-18,5 ⁇ column using the TEAP buffer system described above and a 30 to 45% B gradient over 30 minutes.
  • 3 GRF(1-28)-OH
  • 4 GRF(1-
  • hydrophilic amino acids are defined as amino acids having side chains more hydrophilic than cysteine, indicated by the hydrophilicity values assigned by Hopp and Woods (Proc. Nat. Acad. Sci. USA, 78 (6), pp. 3824-3828 (1981) (Ref. 34).
  • Hopp and Woods Proc. Nat. Acad. Sci. USA, 78 (6), pp. 3824-3828 (1981) (Ref. 34).
  • the good initial results with Ala versus Leu in this model warranted further studies using different sizes of hydrophilic amino acid residues (i.e. Gly, Ser, Thr, Asn and Gin) to establish the size requirements for possible successful product formation, and also an establishment of the degree of hydrophilicity needed by testing Met as leaving group, which has a hydrophilicity value between Leu and Ala.
  • the data are from Refs. 17, 18, 21, 26, 30.
  • the optimal conditions for serine carboxypeptidase catalysed transpeptidation reactions can be established by experiments in each case.
  • the yield of transpeptidation is generally beneficially influenced by an increase in pH due to deprotonation of the amino group of the amino acid amide nucleophile but simultaneously the peptidase activity decreases, leading to lower rates of conversion, and the amidase activity increases, leading to degradation of the amidated product.
  • An increase in pH can be com- pensated by an increase in the concentration of nucleophile but this also reduces the rate of conversion since the nucleophile acts as an inhibitor (25). With respect to pH the compromise usually is between 7.5 and 8.0 with the exception of CPD-S-1 which is unstable above pH 7.0.
  • the transpeptidation reaction with CPD-W-II and Bz-Met- Ser-Arg-OH was initially carried out at pH 7.6 and 10 mM H-Arg-NH 2 , the low concentration of nucleophile being motivated by the efficient binding of nucleophiles with positively charged side chains to this enzyme. After 47 minutes, 91% of the substrate had been converted into the following products (see Table II): 64% Bz-Met-Ser-Arg-NH 2 (Tl), 20% Bz-Met-Ser-OH- (H1) and 6% Bz-Met-OH (H2). Bz- Met-Arg-NH 2 (T2) as well as other potential products (see Scheme 1) were absent. The fraction of transpeptidation was 0.71 and this value was also obtained at pH 8.0.
  • CPD-S-1 was also tested in transpeptidation reactions with Bz-Met-Ser-Arg-OH and Bz-Met-Ser-Leu-OH. With 0.2 M H-Arg- NH 2 and pH 6.5 the fractions of transpeptidation were 0.38 and 0.48, respectively. Higher concentrations of nucleophile had no beneficial effect.
  • the initial results of the transpeptidation reaction in the screening tests with the N-benzoylated tripeptides seemed to indicate that the most efficient amidation leading to a good yield of T1 and suppression of the formation of undesirable peptides such as T2, could be achieved with CPD-Y and with -Ala-OH as leaving group.
  • GRF( 1-28)-Ala-OH was selected as test intermediate and was prepared by continuous flow peptide synthesis.
  • the assembly of GRF(1-28)-Ala-OH (2) was carried out by the solid phase methodology described above and cleaved from the resin by a 20 h treatment with TFA and phenol.
  • the reaction mixture contained 84% GRF(1- 28)-Arg-NH 2 , i.e. GRF(1-29)-NH 2 .
  • GRF(1-29)-NH 2 i.e. GRF(1-29)-NH 2 .
  • CPD-Y is quite stable at pH 8.0 and 22°C and it is therefore possible to employ a much longer reaction time and correspondingly reduced enzyme concentration. Since CPD-Y is easily isolated from baker's yeast after autolysis (Ref. 28) or from the medium of genetically manipulated yeast cells (Ref. 29) the cost of the enzyme is rather low and the procedure described here therefore seems to be a valuable alternative to the use of the much more rare glycine oxidase.
  • the absolute amount of product formed depends on the degree of conversion and passes through an optimum, which would justify stopping the reaction and recycling the remaining substrate, while in others, running the reaction to virtual completion is fully justified.
  • Ala and Thr as the leaving groups X appear to be in the latter category, while Asn and Gin as leaving groups would appear to be in the former.
  • Table IV below some results at incomplete conversion are listed using the large, but hydrophobic Met as a reference example:
  • a logical standard for evaluation of the applicability of the various possible leaving groups would be the native GRF sequence, in which the C-terminal sequence is -Met- Ser-Arg-OH.

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EP91910581A 1990-05-30 1991-05-29 Ein enzymatischer prozess für die herstellung von derivaten des wachstumshormon freisehenden faktors und nützlichen peptiden in diesen prozess Withdrawn EP0537185A1 (de)

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DK1339/90 1990-05-30
DK133990A DK133990D0 (da) 1990-05-30 1990-05-30 Fremgangsmaade til fremstilling af derivater af growth hormone releasing factor (grf) og peptider anvendelige som mellemprodukter ved fremgangsmaaden

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EP (1) EP0537185A1 (de)
AU (1) AU647796B2 (de)
CS (1) CS163191A3 (de)
DK (1) DK133990D0 (de)
IE (1) IE911831A1 (de)
IL (1) IL98313A0 (de)
WO (1) WO1991018998A1 (de)

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AU7909291A (en) 1991-12-31
IE911831A1 (en) 1991-12-04
IL98313A0 (en) 1992-06-21
CS163191A3 (en) 1992-02-19
DK133990D0 (da) 1990-05-30
AU647796B2 (en) 1994-03-31
WO1991018998A1 (en) 1991-12-12

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