PROCESS FOR THE C-TERMINAL MODIFICATION OF PEPTIDES HAVING A C_TERMINAL PENULTIMATE PROLINE RESIDUE

Abstract: C-terminally modified peptides of the formula Peptide - Pro - NH-R, wherein R is selected from hydrogen, hydroxy, C1-6 alkyl, hydroxy C1-6 alkyl and C6-9 aralkyl or R is NHR1 wherein R1 is hydrogen C1-6 alkyl, C6-9 aralkyl or a group CO-R2, wherein R2 is selected from NH2, C1-6 alkyl and C6-9 aralkyl, e.g. calcitonin, are prepared by reacting a substrate component of the formula Peptide - Pro - X, wherein X is an amino acid having a side chain comprising at least two carbon atoms and further comprising at least one hetero atom selected from N, O and S, with a nucleophile component NH2-R, wherein R has the above meaning in the presence of an L-specific serine or thiolcarboxypeptidase enzyme from yeast or of animal, vegetable or other microbial origin, preferably carboxypeptidase Y from yeast, in an aqueous solution or dispersion having a pH of from 7.5 to 10 and optionally containing up to 25 % of an organic solvent. X is preferably selected from Met, Thr, Tyr, Met(o), His, Gln, Asn, Arg, Lys and Trp. Human calcitonin(1-32)Met-OH is a useful intermediate. The process enables a selective C-terminal amidation of peptides in good yields.

Description: Process for the C-terminal modification of peptides having a C-terminal penultimate proline residue The present invention relates to a process for the C- terminal modification of peptides, especially for the preparation of peptides having a C-terminal proline amide or N-substituted amide, e.g. human or salmon calcitonin or their analogs. The use of biologically active peptides for pharmaceutical purposes and in agriculture has increased the importance of being able to synthesize such compounds in bulk-scale. Three methods are available: (a) chemical synthesis, (b) enzymatic synthesis, and (c) fermentation with genetically manipulated microorganisms. While the methods (a) and (b), or a combination of them, are the preferred for short peptides it becomes more and more apparent that long peptides in the future will be produced through exploita- tion of the advances in the methods dealing with recombi- nant DNA. However, these methods do not permit a number of modications such as incorporation of D-amino acids and C- terminal amide or N-substituted amide groups which may be of importance for the biological activity. A subsequent enzymatic modification is therefore highly desirable and such reactions have only been studied to a limited extent. An enzyme has been described which catalyses the hydroxyl- ation of C-terminal glycyl residues which subsequently decomposes leaving the penultimate residue amidated (US Patent No. 4.708.934, EP 308067A, DK Application No. 4489/88). This glycine oxidase enzyme which is dependent on Cu , C" 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 applicab- ility for large scale work is still questionable. The enzyme, as isolated from natural sources like rat medullary thyroid carcinoma is very costly. In the above-mentioned EP 308067 a number of similar a- amidating enzymes of natural origin capable of specifically cleaving C-terminal Gly are described. It is stated that their amidation activity is based upon the conversion of short D-amino acid containing substrates, and that activities vs. any physiologically relevant substrates in L-substrate had not been demonstrated. In application WO 90/08194 (Tanaka et al) an α-amidating enzyme from the skin of Xenopus laevis (a frog) is described. Tanaka et al have produced this enzyme biosynthetically, but its α-amidating activity is also conditioned by the presence of C-terminal Gly. 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. In addition, the precursor peptide may exhibit poor solubility in such media. However, 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, on the other hand, 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 nucleo- phile with water. This property of serine carboxypeptidases was realized by a group of researchers at Carlsberg Research Center and lead to a large family of patents assigned to the present assignee based on DK application No. 1443/79 represented by EP Patent No. 17485, US Patent No. 4.806.534 and its parent US patent No. 4.339.534 and International Applica¬ tion WO 80/02151, which lead to a number of patents i.a. DK Patent No. 155613 and JP Patent No. 1.489.494. These patents were based on the at that time surprising finding that exopeptidases were suitable as catalysts for enzymatic peptide synthesis, while the prior art dealt exclusively with endopeptidases. Dependent on the nature of the reactants (substrate and nucleophile components), and the reaction conditions, particularly the pH, serine and thiol carboxypeptidases may catalyze peptide synthesis by chain elongation or by transpeptidation. The preferred enzyme is carboxypeptidase Y (CPD-Y) from yeast. The underlying and subsequent research has been further described in a number of articles (Ref. 1-8), which together with the above-mentioned patents are all incorporated by reference. The general principle of enzymatic peptide synthesis by transpeptidation in the presence of serine or thiol carboxypeptidases is disclosed in US Patent No. 4.806.473 and its parallel Danish Patent No. 155613. With particular reference to the production of peptide amides these patents generally disclose and claim the production of peptide amides A-B-NH- where A represent an N-terminal protected amino acid residue or an optionally N-terminal protected peptide residue and B-represents an L-amino acid residue, by reacting as substrate component an optionally N-terminal protected peptide A-X-OH, where A is as defined above and X represents an amino acid, with a nucleophile (amine) component H-B-NH^ in the presence of an L-specific serine or thiol carboxypeptidase enzyme from yeast, or of animal, vegetable or microbial origin in an aqueous solu- tion of dispersion being a pH from 5 to 10.5. As further explained in Ref. 1 the preferred pH is about neutral if the formation of a peptide amide is desired. Further experiments are disclosed in Ref. 1-5 which support the pioneer character of these early patents and the general applicability of serine carboxypetidases as catalysts for C-terminal modification of peptides. In particular, Ref. 3 compared the reactivity of various nucleophiles in the exchanges of C-terminal amino acid residues in peptides, and i.a. concluded that ammonia was an applicable nucleophile in transacylation reactions in a manner equivalent to the above-mentioned use of amino acids H-B-NH-. Thus Z-Ala-Ala-OH was reacted with H-Gly- NH 2 and with NH« resulting in the formation of Z-Ala-Gly- H 2 and Z-Ala-NH 2 in coupling yields of 100% and 75%, respectively. In comparison the reaction with H-Gly-OMe also lead to the formation of Z-Ala-Gly-OMe in a yield of 75%. So already before the publication of the above patent family it was obvious to a person skilled in the art that ammonia was an applicable nucleophile in C-terminal modification of peptides along with amino acid amides and amino acid esters. In ref. 2 was described the use of a series of primary amines other than α-amino carboxylic acids as nucleophiles in carboxypeptidase catalyzed coupling to amino acid esters. The nucleophiles included ammonia, hydrazine and N-alkyl or other substituted derivatives of these. However, the only substrates used were particular N-α protected amino acid esters, viz. BzAlaO e, and no transpeptidation reactions were attempted. Application WO 91/18998 assigned to the present assignee describes a process for the preparation of derivatives of growth hormone releasing factor GRF(1-29)NH 2 and analogs thereof by serine carboxypeptidase catalyzed trans¬ peptidation. In this process 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 uncharged hydrophilic acyclic a- amino carboxylic acid residue having the side chain of at least the size of a methyl group, is reacted 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 the desired N-terminal (l-(n-l)) fragment is coupled chemically or enzymatically. Preferred amino acids X are Ala, Thr, Ser, Asn or Gin. In this application, which is incorporated herein by reference, a general discussion of the transpeptidation principles and the competing reactions are given. To recapitulate the essence of the above observations, incorporation of C-terminal amide groups in peptides by transpeptidation in the presence of a serine carboxy¬ peptidase using the proper amino acid amide as the nucleophile as broadly described and claimed in US patent no. 4,806,473 and the other family members is a very appropriate method virtually applicable for any peptide. Also as shown already in Ref. 3, it could be expected that ammonia would be a suitable nucleophile in such amidation reactions. However, 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. In the early articles by the original inventors published shortly after filing of the above patent applications, attempts were made to analyze the influence of the C- terminal (leaving group) amino acid and the penultimate amino acid. Thus in Ref. 1 Breddam et al using Leu-NH 2 as the nucleophile and Gly, Ala, Ser, Val, Leu and Phe as the leaving groups suggested on the basis of the obtained yields that only in cases where the leaving group is one of the smallest amino acids, i.e. Gly, Ala or Ser is the reaction successful, and at least for the simple sub¬ strates tested there was no dependence on the penultimate residue (being Ala, Phe and Gly). In Ref. 3 Breddam et al using Gly-NH^ as the nucleophile and Z-Ala-X as the substrate, where X was Gly, Ala, Ser, Arg, Pro, Lys, Asn, His, Val, Met, Phe and Asp modified the earlier statement to the effect that when using Gly- NH.~ as the nucleophile, the yield is strongly dependent on the nature of the C-terminal (leaving group) amino acid. The yields varied from 10 to 100% with the lowest yields obtained with substrates where a hydrophσbic acid (Val, Met, Phe) serves as leaving group. It should be noted that the yields with basic acids (Arg, Lys) are comparable to the yields with the hydrophilic acids (Ala, Ser), Lys being even better than Ser. As for the penultimate amino acid residue of the peptide substrates the influence was investigated using a series of N-blocked dipeptides with different penultimate amino acids (Ala, Val, Leu, lie, Phe and Val) as the leaving group. Using Gly-NH 2 as the nucleophile, it was apparent that the coupling yield which varied from 45% for lie to 5% for Phe is dependent on the penultimate amino acid residue, but no obvious trend could be found. In Ref. 4 some of the experiments underlying US patent no. 4,645,740 and its family members were discussed. Here porcine insulin Ins-Pro-Lys-Ala was reacted with i.a. Thr- NH 2 and it was concluded that Ins-Pro-Lys-OH was a better substrate that Ins-Pro-Lys-Ala-OH, since Ins-Pro-Thr-NH 2 was formed in greater yields than Ins-Pro-Lys-Thr-NH 2 . By inference Lys in this reaction was a better leaving group than Ala. Also a significant oligomerization under formation of Ins-Pro-Lys-Thr-Thr-NH 2 occurred. These results were further confirmed in Ref. 5 using Bz- Lys-Ala-OH as a model peptide alongside with porcine insulin. The conclusive message was that for the future use of CPD-Y (the serine carboxypeptidase used in the experiments) in transpeptidation reactions it is important to be aware of the possibility that side products may be formed. Besides the above investigations of the applicability of serine carboxypeptidase in C-terminal modifications of insulin, a further experiment with amidation of longer peptides using CPD-Y as a catalyst has been reported. Thus in EP-B2-197794 and the parallel US patent no. 4,709,014 (Tamaoki) human calcitonin-Leu peptide was reacted with ammonia as the nucleophile using CPD-Y as the catalyst under conditions otherwise similar to those used by Breddam et al in Ref. 3. Tamaoki obtained S-sulfonated human calcitonin amide in a yield of 24.7%, leaving 57% unreacted substrate and 17.2% non-amidated side products. The S-sulfonated calcitonin was reduced with glutathione to give a mature human calcitonin, but no yield is stated. In its more general aspects the Tamaoki patents which are incorporated by reference disclose a process for the pre¬ paration of a peptide having a C-terminal proline amide, which comprises reacting in aqueous solution a peptide substrate having C-terminal Pro-Leu, Pro-lie, Pro-Val or Pro-Phe with carboxypeptidase Y in the presence of ammonia. Without in any way wanting to endorse the statements made by Tamaoki, it should be mentioned that he claims that contrary to the findings of Breddam et al in Ref. 3, where a preference for hydrophilic C-terminal amino acids as leaving groups is expressed, the use of hydrophobic amino acids (Leu, lie, Val and Phe) gives better yields than Gly, when Pro is the penultimate amino acid. Nevertheless the yields of the amidation products in Tamaoki's examples using Cbz-Ala-Pro-X-OH as the sub¬ strate, where X is Leu, Leu, Val, Phe and lie, were only 35,1%, 43%, 15,4%, 13,4%, and 22,6%, respectively. The remainder was - to the extent reported - unreacted starting material and non-amidated side-products Cbz-Ala- Pro-OH. Summing up what has been said above, previous literature on carboxypeptidase catalyzed transpeptidations using amino acid amides as nucleophiles has shown no obvious trend in the influence of the penultimate amino acid residue, as stated in Ref. 3. As for the influence of the leaving group likewise varying results have been found. Thus, in the above-mentioned reference, small hydrophilic leaving groups, or even larger positively charged ones, gave the best yields on Z-Ala-X models, while in WO 91/18998 large uncharged hydrophilics were preferable for transpeptidation on R-Met-Ser-X substrates. Likewise, using ammonia as a nucleophile, Ref. 3 indicates superior yields for Z-Ala-X substrates using Ala as leaving group. Using peptides of type R-Pro-X Tamaoki states a particular group of hydrophobic leaving groups to be unique for the reaction with ammonia, viz. Leu, Phe, lie and Val, for which reasonable, albeit modest yields were obtained, in contrast to using Gly as a leaving group or Ala as apparent from Tamaoki's JP priority application no. 72705/1985, where no reaction product was formed. The present invention is based on the surprising finding that in peptides of the above type, which may serve as models for i.a. Calcitonins, a different group of amino acid residues is able to act as good leaving groups in carboxypeptidase catalyzed reactions with ammonia, hydrazine or substituted derivatives thereof, ensuring both speediness and high yields, usually far superior to the ones reported in the above-mentioned Tamaoki patent. Consequently, the invention relates to a process for the preparation of C-terminally modified peptides of the general formula Peptide-Pro-NH-R wherein R is selected from hydrogen, hydroxy, C.,-Cg alkyl, hydroxy C-_, alkyl, C fi _ q aralkyl or R is a group NHR.., wherein R. is hydrogen, C._ 6 aralkyl, Cg_ g aralkyl or a group C0-R 2 , wherein R 2 is selected from H 2 >* ._g alkyl and C ft g aralkyl. C, ft alkyl encompasses straight chain or branched alkyl, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl tert.butyl, pentyl and hexyl. A preferred alkyl group is ethyl. C fi _ q aralkyl encompasses e.g. phenyl-C., alkyl, e.g. benzyl, phenylethyl and phenyl rop l. The alkyl and aralkyl groups may be substituted with one or more inert substituents, e.g. halogen (F, Cl, Br, I), hydroxy or nitro. The process according to the invention is characterized by reacting a substrate component of the general formula Peptide - Pro - X wherein X is an amino acid having an uncharged or positively charged side chain comprising at least two carbon atoms and further comprising at least one hetero atom selected from N, 0 and S, with a nucleophile component NH ^ -R, wherein R has the above meaning 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 7.5 to 10, and if desired converting a reaction product wherein R is different from hydrogen into a peptide amide. For reasons of record it is noted that among the applicable nucleophiles R = H is ammonia, R = NHR- , wherein R. = H is hydrazine, R = NHC0R 2 , wherein R 2 is NH 2 is semicarbazide. The applicable group of amino acid leaving groups X spans a wide range of hydrophilicity - from the hydrophobic tryptophan and tyrosine over methionine and its protected derivatives, e.g. sulphone, Met(o), histidine and threonine to the hydrophilic glutamine, asparagine, arginine and lysine using the scale of Hopp & Woods (Ref 13) . They share the common structural property of having large (at least C ) side chains, which carry at least one hetero atom chosen among N, 0 and S, said side chains being uncharged or positively charged. Using ammonia as the nucleophile, the process of the invention is suitable for the production of various peptide hormones of the calcitonin type, e.g. human calcitonin having the amino acid sequence: I S S 1 Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln- 5 10 Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly- 15 20 25 Val-Gly-Ala-Pro-NH 2 30 32 A particularly interesting calcitonin having a 20 times greater potency is salmon calcitonin having the amino acid sequence: |—S S 1 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln- 5 10 Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly- 15 20 25 Ser-Gly-Thr-Pro-NH„ 30 32 Also calcitonin from other natural species, e.g. eel, chicken, ovine, bovine, porcine and murine calcitonin may be prepared by the process according to the invention. The structures of these calcitonins are described in US patent No. 4,652,627 (Kempe et al. ) which is incorporated by reference. Kempe also discloses such calcitonins having a D-amino acid substituent in position 31. They can also be made by the process according to the present invention. If hydrazine is used as nucleophile, the process may be used to produce the corresponding hydrazides, e.g. calcitonin hydrazides. If it is desired to convert these further by chemical methods, e.g. the azide method, it is necessary first to protect N-terminal and possible side chain amino groups in the substrate, by e.g. Boc, before the reaction. The protected azide may then be reacted with ammonia to form a protected peptide amide, from which the protective groups may be removed in a manner known per se to form the desired peptide amide, e.g. calcitonin. It has also been found that in contrast to the statements made by Tamaoki better yields are obtainable using as the nucleophile elevated concentrations of ammonia in free base form, particularly in the presence of organic acids, preferably acetic acid or formic acid, but not equal yields using lithium hydroxide as base in adjustment of NH.C1. Without wishing to be bound by any particular theory, it may be envisaged that the sterical effects of the large residues in combination with hydrogen bonding or salt or electronic effects often possible from hetero atoms may cause the action, but the magnitude, influence or exact nature of these interactions are not yet clear. Finally, it apparently was not possible to keep the substrates preferred by Tamaoki soluble in the human Calcitonin precursor, necessitating an oxidation of the disulfide bridge before the transpeptidation and a subsequent reduction before the desired Calcitonin could be obtained. Using the analogous substrates in the preferred embodiment of the present invention, it has been possible in many cases to keep the substrates in solution and thus performing directly a one-step reaction instead of Tamaoki's three-step reaction provided that guanidinium hydrochloride is added. However, if for technical reasons, it is desirable to use an open chain or solubilized intermediate, this is also possible in the process according to the invention. Using ethyl amine or semicarbazide as nucleophiles, compounds containing a C-terminal proline N-ethyl amide or proline semicarbazide may be prepared according to the process. As examples of such peptides containing these groups of biological interest are e.g. the nonapeptide Luteinizing Hormone Releasing Hormone analogue drugs, Leuprolide®, see GB patent No. 1.434.694, and Buserelin, see US patent No. 4.263.282, having C-terminal Pro-NEt and Goserelin, see US patent No. 4.100.274, having C-terminal Pro-SEM. The applicable carboxypeptidases in the process of the invention are L-specific serine or thiol carboxy- peptidases. 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. 10) who developed a particularly expedient purifica¬ tion method by affinity chromatography on an affinity resin comprising a polymeric resin matrix with coupled benzylsuccinyl grups. CPD-Y, which is a serine enzyme is available in large amounts and displays relatively great stability. Further details are given in Ref. 1. The native CPD-Y is a well characterized serine carboxypeptidase. A comparison with other such carboxypeptidases is given in Ref. 7. These were also from other sources than yeast or genetically or chemically modified types. Another CPD-Y homologous serine carboxypeptidase from yeast, KEX 1, is described in Ref. 14 and further characterized in Ref. 15. A combination of chemical and genetic modification of a yeast carboxypeptidase is described in Ref. 16. CPD-Y is easily isolated from baker's yeast after autolysis (Ref. 10) or from the medium of genetically manipulated yeast cells (Ref. 11) as applied in example 18. This enzyme has a different glycosylation and molecular weight, but has proved to be equally useful in the native form. 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. In addition to CPD-Y, which is the preferred enzyme at present, the process of the invention is feasible with other carboxypeptidases from other sources than yeast, such as those listed in the following survey: Enzyme Origin Fungi Carboxypeptidase(s) from Penicillium janthinellum Carboxypeptidase(s) from Aspergillus saitoi Carboxypeptidase(s) from Aspergillus oryzae Plants Carboxypeptidase(s) C Orange leaves Orange peels Carboxypeptidase C N Citrus natsudaidai Hayata Phaseolain French bean leaves Carboxypeptidases M from Germinating berlay Carboxypeptidases W from Wheat bran Carboxypeptidases from Germinating cotton plants Tomatoes Watermelons Bromelain(pineapple)powder The close relationship between a number of the above carboxypeptidases is discussed by Kubota et al (Ref. 12) and further expanded in Ref. 7. As stated above the process of the invention may be carried out at pH 7.5 to 10.0, preferably at pH 8.5 to 9.5, most preferably from 9.0 to 9.5. Accordingly, it is necessary for the enzyme to have sufficient stability in alkaline media during the reaction period. The preferred pH-value, which is often within a very narrow range, depends upon the enzyme used and the substrate employed. For CPD-Y, a favourable pH for most substrates is about 9.2. The preferred agents for pH-adjustment in starting solutions containing NH~ are low molecular carboxylic acid, preferably acetic acid or formic acid and good results have also been obtained using some ammonium salts, e.g. H 4 N0 3 . The reaction is carried out in an aqueous reaction medium which, if desired, may contain up to 25% by volume of an organic solvent not including possible organic pH-adjust- ment agents. Preferred organic solvents are dimethyl formamide and dimethyl sulfoxide, but also alkanols, e.g. methanol and ethanol, glycols, e.g. ethylene glycol or polyethylene glycols, triethylene glycol dimethyl ether, glycerol, alkanoic acids, e.g. acetic acid, tetra- hydrofurane, dioxane and dimethoxyethane may be used. Preferably only small amounts, e.g. 2-12%, of organic solvent are used. The selection of the composition of the reaction medium depends particularly upon the solubility of the reaction components and the reaction products involved and upon the stability of the enzyme. These can be affected by addition of urea and/or detergents. Examples are anionic, e.g. pentanesulphonic acid, zwitterionic, e.g CHAPSO, nonionic, e.g. Brij 35 or Tween 20 and cationic, e.g. guanidinium hydrochloride. Stabilization of the enzyme might also be brought about by addition of carbohydrates, e.g. mannitol or proteins, e.g. BSA. Naturally this variety of additives may also affect the course and synthetic ratio of the reaction. The reaction medium may also comprise a component that renders the enzyme insoluble, but retains a considerable part of the enzyme activity, such as an ion exchanger resin. Alternatively, the 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. Besides, the enzyme may be modified by chemical means to improve its stability or enzymatic properties. The addition of a chelating agent e.g. EDTA to the reaction medium is often not necessary. However, the medium preferably contains a gelation inhibiting agent, e.g. guanidium hydrochloride. The concentration of the two participants in the reaction may vary within wide limits, as explained below. A preferred starting concentration for the peptide substrate is 0.1-5.0 mM, preferably 0.2-1.0 mM, in particular about 0.5 mM, and when the nucleophile is ammonia, it is preferably added as a saturated solution or on liquid form, the concentration is 4.0 to 12.0 M, preferably 4.3 - 9.7 M, in particular 5 - 8 M. This is in contrast to Tamaoki, who only used 4.5 M solution and claimed this to be optimal. For many of the other nucleophiles, e.g. benzyl amine, a much lower concentration, viz. 0.1 M may be used. Generally concentrations of 1.0 to 4.0 M, preferably 2.0 to 3.0 M are used. It is not necessary to protect the N-terminal or possible side chain amino acids or carboxylic groups in the substrate during the reaction with the nucleophile. However, it may be necessary if the reaction product, e.g. a hydrazide, is to be used for further reactions. By the same token it may be desirable to protect one of the amino groups in hydrazine e.g. with Boc. The enzyme activity may vary as well, but for CPD-Y the concentration is 5 - 50 urn, preferably 5 - 20 um. 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. According to the invention the reaction temperature is 20 β to 40°C. An appropriate temperature will usually be about 33° to 39°C, preferably about 37°C, taking into account due consideration for enzyme activity and stability. Similar variations occur for the reaction time which depends very much upon the above-mentioned reaction parameters, especially the enzyme concentration. The standard reaction time in the process of the invention is about 1 - 5 hours. As for the pressure, the reaction is preferably carried out in closed vessels at a pressure of 1 - 3 bar, preferably 1 - 2 bar. 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 following additional abbreviations are used: HOAc, acetic acid; Bz, N-benzoyl; Boc, tert.butyloxycarbonyl; DMF, N,N-dimethylformamide; EDTA, ethylene diamine tetraacetic acid; GRF, growth hormone releasing factor; HPLC, high performance liquid chromatography; SEM, semicarbazide, TFA, trifluoroacetic acid; TGME, triethylene glycol dimethyl ether; THF, tetrahydrofuran; Z, carbobenzoxy; CHAPSO, 3-[(3-cholamidopropyl)- dimethylammonio]-2-hydroxy-l-propane-sulfonate. Before the process of the invention will be illustrated by examples, starting materials, methods of measurement, etc. will be explained in general terms. General procedure for examples 1 - 19 The reactions were performed on four different groups of test substrates, wherein X designates the amino acid leaving group: Tripeptides of formula Z-Thr-Pro-X-OH and Z-Ala-Pro-X-OH served as short model substrates for salmon and human calcitonin, respectively. Substrates of formula: H-Leu- His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly- Thr-Pro-X-OH served as longer model substrates for salmon calcitonin, corresponding to residues 16 - 32 elongated by X, and henceforth referred to as SAL (16-32)-X. These substrates were obtained by chemical synthesis, by employing liquid and solid phase methods at conventional state of the art level, e.g. as described in appln. WO 91/18998. Finally, human calcitonin elongated by one amino acid residue can be obtained by an enzymatic coupling of the free amino acid to the corresponding calcitonin methyl ester using CPD-Y catalysis in aqueous medium (see example 13 below) or by standard solid phase methods followed by formation of the disulfide bridge by cyclization. Following HPLC purification all shortened substrates were found to be more than 95% pure by HPLC in systems similar to the ones used for monitoring the reaction, and for selected specimens including the longest peptides, amino acid analysis or gas sequence analyses were performed and proved the correct identity. Met-sulfone substrates were obtained by oxidizing the corresponding methionine substrates e.g. by treatment with hydrogen peroxide. By synthetical methods similar to the ones described above the corresponding amide products, byproducts and hydro¬ lysis byproducts were synthesized as HPLC reference substances. Reaction monitoring, product identification and determina¬ tion of product yield were performed by means of reverse phase HPLC (Waters 6000 A pumps, automated gradient controller, UK 6 injector) on a C lg NOVA PAK column (Waters, RCM) using suitable gradients of elution systems containing 50 mM triethylammonium phosphate, pH 3.0 or 7.0 from 0% to 80% acetonitrile with a flow of 2 ml/min. Elution was monitored by means of a UV detector (Waters 480) at 230 nm, 254 nm or 278 nm. The products were identified by amino acid analysis of fractions from the HPLC analysis, which corresponded to the assumed product peak and/or by HPLC comparison with a chemically synthesized reference product. In all cases, HPLC systems were employed where the products could be clearly separated from other compounds. Sometimes HPLC at several pH-values were performed. Concentrated ammonia stock solutions were made to form the bulk of the reaction media. Normally at room temperature and at atmosphere pressure of around 1 bar, they were made basically in either of the two ways: either from a concentrated solution of ammonia in water which was mixed with a concentrated or aqueous acid solution or from an ammonium salt which following dissolution in water was mixed with a solid alkali base. Following cooling to room temperature and if necessary dilution by water pH was adjusted using the relevant acid or base. Thus 10 ml of 25% aqueous ammonia mixed with either 6.5 ml of glacial acetic acid (HOAc) or 3.7 ml of concentrated formic acid yields solution of 8 M and 10 M ammonia/ammonium concentrations when adjusted to pH 9.2 with concentrated acid, and likewise at the same pH a 4.5 M ammonia/ammonium solution may be obtained by mixing about 26.7 g NH.C1 and 8.1 g NaOH in 100 ml water. Ammonia concentrations are later corrected for decreases as a result of the addition of other solvents, reactants or additives. Most reactions were performed in closed vessels, which were normally closed at room temperature. pH recorded was thus the value measured at this temperature and the initial reaction pressure was usually above 1 bar by an unmeasured margin smaller than 1 bar, i.e. 1 - 2 bar. Unless otherwise mentioned, all experiments were carried out at a volume of 1 ml, or sometimes 0.5 ml, in Eppendorph plastic micro test tubes with safety lid lock, inserted into an Eppendorph 5427 thermomixer. Other tubes, i.e. glass tubes, have shown similar results. In a typical short model peptide reaction a 10 mM solution in an organic solvent, water or mixtures of these was made up and poured into a tenfold volume excess of 2 - 10 molar ammonia solution at the appropriate pH for reaction containing desired salt or additive like guanidium chloride. In case of CPD-Y the reaction was initiated by addition of a suitable amount of dry 20% w/w CPD-Y on citrate or a similar 333 uM CPD-Y solution of purified Carboxypeptidase Y produced by Carlbiotech Ltd. A/S, Copenhagen. Recombinant secreted CPD-Y was a kind gift from Dr. Jacob R. Winter of Carlsberg Research Center and was purified as described in example 18. An appropriate volume was added to initiate the reaction at correct molarity of enzyme, normally 5 - 30 μM. The reaction mixture was then shaken in the thermomixer for duration of the experiment, usually from 1 - 5 hours, while aliquots of 10 to 20 μl were extracted from HPLC analysis at regular intervals. It was checked that no precipitation, i.e. loss of UV absorptive groups in the analysis, occurred. In some instances aliquots were withdrawn and high amounts of organic solvent added to stop the reaction, or the reaction was quenched by addition of concentrated HC1. During the time course of reactions, these were monitored for substrate consumption, amide product obtained, primary hydrolysis byproduct and amide byproduct formed by secondary aminolytic cleavage of the proline residue in the primary hydrolysis byproduct or amide product. The relative molar percentages of these four types of compounds, substrate, amidated product, primary hydrolysis byproduct and amidated byproducts were calculated from the relative area counts obtained by integration of the HPLC UV absorptions obtained at 254 or 230 nm, respectively, using appropriate correction factors for difference in absorption. These are listed in the examples as substr., yield, hydr. and oth., respectively. In the case of longer substrates than tripeptides, the latter term has been calculated to include also possible secondary hydrolysis and aminolysis products, and for the human Calcitonin also byproducts formed by known chemical side reactions in the long peptide, i.e. α,0-shifted aspartic acid or oxidized methionine produced during the course of reaction, which could also be separated in the systems employed. It was not attempted to counter the oxidation reaction by addition of reductive additives. The time course of a typical reaction according to example 8 is illustrated in fig. 1. The relative amount of amidated product is in itself a valid parameter for the feasibility of the process. To help optimize reactions, a further variable was introduced and calculated. Thus, RATIO is calculated from the following formula: 100*YIELD RATIO = % (100 - SUBSTR. ) which indicates the catalytical efficiency in forming product from substrate and may also be indicative of the feasibility of introducing a recycling step in which unreacted substrate is recovered and put through a renewed reaction. This value is also listed in the tables as are the ammonia compounds SALT which were adjusted by the pH adjusting agent PHADJ to obtain the final ammonia concentration CNH 3 . Other additives are listed as ADD and their concentrations as CADD. The temperature for performing the experiment is listed as TEMP. Finally, the initial substrate is often listed in the examples as a core peptide named PEPTIDE with a separate leaving group listed as LEAVING and the initial substrate concentration as CPEPTIDE. Example 1 Scale amidation of Z-Thr-Pro-Met-OH In a 100 ml glass flask, 25 mg Z-Thr-Pro-Met-OH was dis¬ solved in 1.05 ml DMF, and 50 ml 8 M ammonia/ammonium solution was added, wherein pH had been adjusted to 9.2 using glacial acetic acid. The glass flask was placed in a stirred water bath thermostated at 37°C and following the addition of 0.78 ml of a 0.3 mM solution of Carboxy¬ peptidase Y to initiate the reaction, the flask was sealed with a plastic screw-cap lid and left in the wa¬ ter bath for 75 minutes, after which HPLC showed 76% of the amidated Z-Thr-Pro-NH 2 product, 20% of the hydro- lysis byproduct Z-Thr-Pro-OH and 4% remaining substrate. Example 2 Amidation of Z-Ala-Pro-Met-OH 0.23 mg Z-Ala-Pro-Met-OH was placed in an Eppendorph plastic tube and dissolved in 40 μl of DMSO and 945 μl of 8 M ammonia/ammonium solution, in which pH had been adjusted to 9.2 using glacial acetic acid, was added, followed by addition of 95 mg of guanidinium chloride. The reaction was initiated by addition of 15 μl of a 0.3 mM solution of Carboxypeptidase Y and was shaken in the Eppendorph mixer thermostated at 37°C for the duration of the experiment, following closing of the lid lock. After 1290 minutes, HPLC showed 85% of the amidated product Z-Ala-Pro-NH„, 2% of the hydrolysis byproduct Z- Ala-Pro-OH, 1% of the amide byproduct Z-Ala-NH„ and 12% unreacted substrate. Example 3 Amidation of Z-Thr-Pro-Met-OH 0.46 mg Z-Thr-Pro-Met-OH was placed in an Eppendorph plastic tube and dissolved in 20 μl of DMSO and 965 μl of 10 M ammonia/ammonium solution, in which pH had been adjusted to 9.2 using concentrated formic acid, was added. The reaction was initiated by addition of 15 μl of a 0.3 mM solution of Carboxypeptidase Y and was shaken in the Eppendorph mixer thermostated at 37 C C for the duration of the experiment, following closing of the lid lock. After 90 minutes, HPLC showed 88% of the amidated product Z-Thr-Pro-NH 2 , 12% of the hydrolysis byproduct Z-Thr-Pro-OH and no remaining substrate. Example 4 Amidation of Z-Thr-Pro-Met-OH 0.46 mg Z-Thr-Pro-Met-OH was placed in an Eppendorph plastic tube and dissolved in 20 μl of DMF and 965 μl of 4.5 M ammonia/ammonium solution, in which pH had been adjusted to 9.2 using solid sodium hydroxide and ammonium chloride, was added. The reaction was initiated by addition of 15 μl of a 0.3 mM solution of Carboxypeptidase Y and was shaken in the Eppendorph mixer thermostated at 37°C for the duration of the experiment, following closing of the lid lock. After 143 minutes, HPLC showed 74% of the amidated product Z-Thr- Pro-NH 2 , 10% of the hydrolysis byproduct Z-Thr-Pro-OH and 16% remaining substrate. Example 5 Amidation of Z-Thr-Pro-Met(0)-0H 0.46 mg Z-Thr-Pro-Met(0)-0H, in which (0) designates the side chain sulfone, was placed in an Eppendorph plastic tube and dissolved in 905 μl of 8 M ammonia/ammonium solution, in which pH had been adjusted to 9.2 using glacial acetic acid, was added, followed by addition of 95 mg guanidinium chloride. The reaction was initiated by addition of 15 μl of a 0.3 mM solution of Carboxypeptidase Y and was shaken in the Eppendorph mixer thermostated at 37"C for the duration of the experiment, following closing of the lid lock. After 90 minutes, HPLC showed 33% of the amidated product Z-Thr- Pro-NH 2 , 8% of the hydrolysis byproduct Z-Thr-Pro-OH, 2% of the amide byproduct Z-Thr-Pro-NH 2 , and 57% remaining substrate. Example 6 Amidation of salmon calcitonin(16-32)-Thr-0H 1.0 mg SAL(16-32)-Thr-0H was placed in an Eppendorph plastic tube and dissolved in 20 μl of DMSO and 435 μl of 8 M ammonia/ammonium solution, in which pH had been adjusted to 9.2 using glacial acetic acid, was added. The reaction was initiated by addition of 45 μl of a 0.3 mM solution of Carboxypeptidase Y and was shaken in the Eppendorph mixer thermostated at 37°C for the duration of the experiment, following closing of the lid lock. After 300 minutes, HPLC showed 51% of the amidated product R-Thr-Pro-NH„, 24% of the hydrolysis byproduct R-Thr-Pro-OH, 16% of other byproducts and 9% remaining substrate. In the above, R designates the salmon calcitonin 16-30 sequence. Example 7 Amidation of salmon calcitonin(16-32)-Tyr-0H 1.0 mg SAL(16-32)-Tyr-0H was placed in an Eppendorph plastic tube and dissolved in 20 μl of DMSO and 435 μl of 8 M ammonia/ammonium solution, in which pH had been adjusted to 9.2 using glacial acetic acid, was added. The reaction was initiated by addition of 15 μl of a 0.3 mM solution of Carboxypeptidase Y and was shaken in the Eppendorph mixer thermostated at 37 β C for the duration of the experiment, following closing of the lid lock. After 1050 minutes, HPLC showed 44% of the amidated product R-Thr-Pro-NH 2 , 13% of the hydrolysis byproduct R-Thr-Pro-OH, 31% of other byproducts and 12% remaining substrate. In the above, R designates the salmon calcitonin 16-30 sequence. Example 8 Amidation of various salmon calcitonin ragment SAL(16- 32)-X peptides to yield SAL(16-32)-NH 2 . a• * 5 Leav- CPEP- CPD-Y Time Yield Substr. Hydr. Oth. Ratio ing TIDE (X) (mM) (μM) (min) (%) (%) (%) (%) (%) a) Reaction conditions: 7.7 M ammonia/ammonium, PHADJ: HOAc, pH 9.2, 37°C in Eppendorph mixer, 4% DMSO 0 b) No DMSO added, 1 M guanidinium chloride. c) Time course of reaction is illustrated in Figure 1. 5 0 5 a) Reaction conditions: 1 mM peptide, 7.7 M CNH 3 (ammonia/ammonium), PHADJ: HOAc, pH 9.2, 37°C in Eppendorph mixer, 1 M guanidinium chloride. b) No guanidinium chloride added. c) pH 7.8. d) Comparison example. Example 10 Amidation of Z-Thr-Pro-Met in various solvents and ammonia/ammonium mixturesa) Sol- CNH 3 Salt+ Time Yield Substr.Hydr.Oth. Ratio vent PHADJ (M) (min) (%) (%) (%) (%) (%) Dioxane d) 7.7 NH 3 + 180 70 14 16 0 82 HOAc Dioxane b**d) 4.3 NH 4.C1+ 1020 21 79 - - 100 NaOH Dioxane c)d) 4.0 NH 4,C1+ 60 64 14 20 2 75 NaOH DMSO 4.3 NH 4 C1+ 150 70 21 9 0 88 NaOH a) Reaction conditions: ImM peptide, 4% solvent, 5 μM CPD-Y, pH 9.2, 37 β C in Eppendorph mixer. b) 3.3 μM CPD-Y added. c) 33 μM CPD-Y added. d) Dioxane was peroxide free grade. Example 11 Amidation of Z-Thr-Pro-Met at various concentrations of DMSO a ^ DMSO CNH 3 CPD-Y Time Yield Substr. Hydr. Oth. Ratio Cone. (%) (M) (μM) (min) (%) (%) (%) (%) (%) a) Reaction conditions: SALT/PHADJ: NH 3 /H0Ac, pH 9.2, 1 mM peptide, 37°C in Eppendorph mixer. b) 0.5 mM peptide. Example 12 Amidation of Z-Thr-Pro-Met in various ammonia/ammonium mixtures containing DMFa) CNH SALT/PHADJ Time Yield Substr. Hydr. Oth. Ratio (M) (min) (%) (%) (%) (%) (%) a) Reaction conditions: 1 mM peptide at pH 9.2, 5 μM CPD-Y, 2% DMF, 37°C in Eppendorph mixer. b) 20 μM CPD-Y added. Example 13 Synthesis of human calcitonin (1-32)-Met-0H by enzymatic peptide synthesis ______ ^ __ About 2 mg of human calcitonin α carboxylic methyl ester containing some impurities was dissolved together with 89 mg of L-Methionine and 95 mg of guanidinium chloride in 850 μl H 2 0 and 40 μl DMSO and pH was adjusted to 8.8 using 2 M sodium hydroxide in a pH-stat vessel thermo¬ stated at 37°C. The reaction was performed in this ves¬ sel. The reaction was initiated by addition of 15 μl of a 0.3 mM CPD-Y solution and was complete within 30 min. The mixture was adjusted to pH 2.5 and applied to a re- verse phase C. R HPLC column, from which the product was eluted using a 0.1% TFA/aqueous acetonitrile gradient to yield 1.2 mg (60%) upon drying under nitrogen. Amino acid analysis (ratio) Asp + Ala (5.1), Glu (1.9), Ser (0.9), Gly (4.0), His (0.6), Thr (4.9), Pro (2.1), Tyr (1.1), Val (0.9), Met (1.9), Cys (1.2), He (1.0), Leu (2.3), Phe (3.2). The chemical breakdown of cysteine and the confirmed presence of two methionine residues is noted. a) Reaction conditions: 1 mM peptide, 7.7 M CNH„ (ammonia/ammonium), PHADJ: HOAc, pH 9.2, 1 M guanidinium chloride, 37°C in Eppendorph mixer. b) No guanidinium chloride added. c) No guanidinium chloride added and 4.3 M CNH„, PHADJ/SALT: NaOH/NH Cl. d) 25°C. e) Comparison example. Example 15 Amidation of Z-Ala-Pro-Met at various pH values and concentrations 3 pH CPEP- CNH 3 Time Yield Substr. Hydr.Oth. Ratio TIDE (mM) (M) (min) (%) (%) (%) (%) (%) a) Reaction conditions: SALT/PHADJ: NH 3 /H0Ac, 1 M guanidinium chloride, 5 μM CPD-Y + additional 10 μM after 1100 minutes, 37 C C in Eppendorph mixer. b) 16 μM CPD-Y initially. Example 16 Amidation of Z-Ala-Pro-Met-OH in different solvents at various tem eraturesa) a) Reaction conditions: 4.2 M CNHg, SALT/PHADJ: NH 4 Cl/NaOH, pH 9.2, 1 mM peptide. b) 0-7 mM peptide, peroxide free dioxane. Example 17 Amidation of Z-Ala-^ro-Met and Z-Thr-Pro-Met in various ammonia/ammonium mixturesa) Pep- CNH 3 Salt+ pH Time Yield Substr.Hydr.Oth. Ratio tide PHADJ -MetOH (M) (min) (%) (%) (%) (%) (%) ZThrPro 7.9 H 3 + 8.2 85 48 0 52 0 48 HOAc ZAlaPro 4.3 NH 3 + 9.2 67 55 4 39 2 57 HOAc ZAlaPro 4.3 NH 4„C1+ 9.2 225 41 45 14 0 74 NaOH ZAlaPro 4.5 H 3 + 9.2 120 17 83 0 0 100 b) NH 4 N0 3 ZAlaPro 7.5 NH 3 + 9.2 92 43 38 19 0 70 b) NH„N0 4 3 ZAlaPro 9.0 H 3 + 9.2 40 8 92 0 0 100 b)c) NH 4 N0 3 a) Reaction conditions: 1 M peptide, 5 μM CPD-Y, 37°C in Eppendorph thermomixer. b) 17 μM CPD-Y, 4% DMSO c) 30°C Example 18 a " t Amidation ' for ZThrProMetOH and ZAlaProMetOH catalyzed by different forms ' of carboxypeptidases from yeast in different ammonia/ammonium mixtures adjusted to pH 9.2 with acetic acid. Peptide CPD Enzyme Time Yield Substr.Hydr.Oth- Ratio -Met (μM) form (min) (%) (%) (%) (%) (%) ZThrPro 2 NEb) ZThrProd) 2 SRb) ZAlaProe) NEb) ZAlaPro e) 2 SR b) a) 1 mM peptide, 37°C, salt: NH 3 , PHADJ: HOAc b) NE = Native Extracted CPD-Y prepared and purified according to Johansen, J.T., Breddam, K. and Ottesen, M. (1985) Carlsberg Res- Commun. 41, p. 1-14 c) SR = Secreted Recombinant CPD-Y expressed and prepared according to Nielsen, T.C., Holmberg, S. and Peterson, J.G. (1990) Appl. Microbiol. Biotechnol- 33, p. 307-312 and purified as under b) . d) 2% DMF CNH 3 : 4.3 M e) 4% DMSO C H 3 : 7.5 M f) Traces- Example 19 CPD-Y catalyzed amidation of ZAlaProThr-OH in ammonia/ammonium mixtures pH-adjusted with acetic acida) and further containing various anionic and nonionic detergents at 0.5% (w/v). Detergent Time Yield Substr.Hydr.Oth. Ratio (min) (%) (%) (%) (%) (%) 0.5% N-Lauroylsarcosine 412 31 26 43 0 42 0.5% Octanesulfonic Acid 300 0.5% Brij 35 300 0.5% Tween 20 300 a) Reaction conditions: 8.0 M CNHg, PHADJ: HOAc, pH 9.2, ImM ZAlaProThr, 4% DMSO, 17 μM CPD-Y, 37 β C in Eppendorph thermomixer. Example 20 CPD-Y catalyzed amidation of ZAlaProMet-OH in ammonia/ammonium mixtures pH-adjusted with acetic acida) and further containing various zwitterionic or anionic detergents at 5% (w/v). Detergent Time Yield Substr.Hydr.Oth. Ratio (min) (%) (%) (%) (%) (%) 5% CHAPSO 35 60 40 100 5% Pentanesulfonic Acid 1200 89 10 89 a) Reaction conditions: 8.0 M CNH 3 , PHADJ: HOAc, pH 9.2, ImM ZAlaProMet, 4% DMSO, 17 μM CPD-Y, 37 β C in Eppendorph thermomixer. Example 21 CPD-Y catalyzed amidation of ZAlaProTyr-OH in ammonia/ammonium mixtures pH-adjusted with acetic acida) and further containing various additives. a) Reaction conditions: 8.0 M CNH, PHADJ: HOAc, pH 9.2, ImM ZAlaProTyr, 4% DMSO, 5 μM CPD-Y, 37 β C in Eppendorph thermomixer. Example 22 CPD-Y catalyzed amidation of various peptides of structure ZAlaPro-X-OH in various acetic acid/ammonia/ammonium mixturesa) in the pH range 8.5 to 9-0 a) Reaction conditions: 1 mM ZAlaPro-X, 1 M guanidine hydrochloride, 4% DMSO, 17 μM CPD-Y, 37°C in Eppendorph thermomixer. Example 23 Amidation of human calcitonin 1-32-Met-OH to form human calcitonin amide 1.1 mg of cyclic human calcitonin 1-32 elongated by methionine acid was directly dissolved in a glass test tube in 20 μl of DMSO, 0.47 ml of a 25% (w/w) ammonia solution pH adjusted to 9.2 by glacial acetic acid was added to give an unclear solution. 47 mg guanidinium chloride was then dissolved herein. The mixture was thermostated to 37°C in an Eppendorph thermomixer, after which the reaction was initiated by addition of 0.3 mM CPD-Y to yield a CPD-Y concentration of about 5 μM. Following shaking of the closed reaction vessel for about half an hour at 37°C, the solution was shown to contain 42% human calcitonin amide by HPLC as well as 21% of the hydrolysis byproduct calcitonin free acid, the remainder being unreacted substrate and some breakdown products. Example 24 Amidation of human calcitonin 1-32-Thr-OH to form human calcitonin amide 1.9 mg of cyclic human calcitonin 1-32 elongated by threonine acid was directly dissolved in a test tube in 40 μl of DMSO and 0-92 ml of a 25% (w/w) ammonia solution pH adjusted to 9.2 by glacial acetic acid and 96 mg guanidinium chloride was added to the solution. The mixture was thermostated to 37 β C in an Eppendorph thermomixer, after which the reaction was initiated by addition of 0-3 mM CPD-Y to yield a CPD-Y concentration of about 8 μM. Following shaking of the closed reaction vessel for forty minutes at 37°C, about 1/3 of the substrate had been converted and the solution was shown to contain 16% human calcitonin amide by HPLC at several pH values as well as only traces of the hydrolysis byproduct calcitonin free acid, the remainder being 67% unreacted substrate and some breakdown products. Example 25 Amidation of human calcitonin 1-32-Tyr-OH to form human calcitonin amide 1.8 mg of cyclic human calcitonin 1-32 elongated by tyrosine acid and further containing a little calcitonin acid was directly dissolved in a test tube in 40 μl of DMSO and 0.94 ml of a 25% (w/w) ammonia solution pH adjusted to 9.2 by glacial acetic acid and containing 96 mg guanidinium chloride was added to the solution. The mixture was thermostated to 37°C in an Eppendorph thermomixer, after which the reaction was initiated by addition of 0.3 mM CPD-Y to yield a CPD-Y concentration of about 20 μM. Following shaking of the closed reaction vessel for about half an hour at 37 β C, 37% of the substrate had been converted and it was shown by HPLC that 26% of this had been converted to human calcitonin amide, i.e. a total yield of about 10%, as well as only a little more of the hydrolysis byproduct calcitonin free acid, the remainder being 63% unreacted substrate and some breakdown products. Example 26 CPD-Y catalyzed amidation of ZThrProMet-OH with various substituted amines of structure H^N-R to give products of structure ZThrProNH-R a ' Amine -R Cone. ime Yield Subst.Hydr.Oth.Rat. (M) Ethyl amine -CH 2 CH 3 2.0 120 16 31 52 0 24 2-Ethanol amine -CH 2 CH 2 OH 3.0 90 59 15 26 0 69 Hydrazine -NH 2 2.0 75 80 5 15 0 84 Semi- carbazide b -NHC0NH 2 2.0 75 69 24 8 0 90 Benzyl amine c) -CH 2 CgH 5 0.1 34 54 35 8 3 83 a) Reaction conditions: 1 mM ZThrProMetOH, 5 μM CPD-Y, 4% DMSO, pH 9.2, pH adjusted with acetic acid, 35°C in Eppendorph Thermomixer. b) Used as hydrochloride, pH adjusted with NaOH. c) 100 μM CPD-Y, pH adjusted with HCl, reaction in pH- stat cup. REFERENCES: 1. Breddam, K. , Widmer, F. & Johansen, J.T. (1980) Carlsberg Res. Commun. 4_5, 237-247 2. Widmer, F., Breddam, K. , and Johansen, J.T., (1981), Carlsberg Res. Commun., 4_6, 97-106. 3. Breddam, K. , Widmer, F. & Johansen, J.T. (1981) Carlsberg Res. Commun. 46, 121-128 4. Breddam, K., Widmer, F. & Johansen, J.T. (1981) Carlsberg Res. Commun. 4_6, 361-372 5. Breddam, K. , Johansen, J.T. & Ottesen, M. (1984) Carlsberg Res. Commun. 9, 457-462 6. Breddam, K. (1985) Carlsberg Res. Commun. 50, 309-323 7. Breddam, K. , (1986), Carlsberg Res. Commun. 5_^, 83-128 8. Breddam, K. (1988) Carlsberg Res. Commun. 53, 309-320 9. Breddam, K. & Ottesen, M. (1984) Carlsberg Res. Commun. 49, 473-481 10. Johansen, J.T., Breddam, K. & Ottesen, M. (1976) Carlsberg Res. Commun. 41_, 1-14 11. Nielsen, T.L., Holmberg, S. & Petersen, J.G.L. Appl. Microbiol. Biotechnol (1990), 33, 307-312 12. Kubota et al. Carboxypeptidase C N (1973), J. Biochem. 74, no. 4, 757-770 13. Hopp & Woods, Proc. Natl. Acad. Sci. USA, 713,p. 3824- 3828 (1981) 14. D ochouska, A., et al., (1987), Cell, 50, 573-584 15. Cooper, A & Bussey H., (1989), Molecular and Cellular Biology, 9_, 2706-2714 16. Bech, L. M. , & Breddam, K. , (1988), Carlsberg Res. Commun., 53, 381-393

Claims: C A I M S 1. Process for the preparation of C-terminally modified peptides of the general formula Peptide - Pro - NH-R wherein R is selected from hydrogen, hydroxy, C._ 6 alkyl, hydroxy C, , alkyl and C 6 _ g aralkyl or R is NHR 1 wherein R-, is hydrogen, C, 6 alkyl, 5 _ g aralkyl or a group C0-R 2 , wherein R~ is selected from NH 2 , C, β alkyl and ,- g aralkyl c h a r a c t e r i z e d by reacting a substrate component of the general formula Peptide - Pro - X wherein X is an amino acid having an uncharged or positively charged side chain comprising at least two carbon atoms and further comprising at least one hetero atom selected from N, 0 and S, with a nucleophile component NH 2 -R, wherein R has the above meaning 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 7.5 to 10, and if desired converting a reaction product wherein R is different from hydrogen into a peptide amide. 2. Process according to claim 1, c h a r a c t e r i z e d by reacting hydrazine NH„-NH as the nucleophile component, with a substrate having its N-terminal and possible side chain amino groups protected, so as to form a protected peptide hydrazide, converting the protected hydrazide into an azide, reacting the protected azide with ammonia to form a protected peptide amide and removing the protective groups. 3. Process according to claim 2 for the preparation of a calcitonin, c h a r a c t e r i z e d by using a calcitonin-X, wherein X has the above meaning, as the substrate component. 4. Process according to any of claims 1 to 3, wherein X is selected from Met, Thr, Tyr, Met(o), His, Gin, Asn, Arg, Lys and Trp. 5. Process according to claims 1 to 4, wherein the nucleophile is selected from ammonia, ethyl amine, hydrazine and semicarbazide. 6. The process according to any of claims 1 to 5, wherein the carboxypeptidase enzyme used is a carboxypeptidase from yeast. 7. Process according to claim 6, wherein the enzyme used is carboxypeptidase Y. 8. The process according to claim 7, wherein a carboxy- peptidase Y is used which has been purified by affinity chromatography on an affinity resin comprising a polymeric resin matrix with a plurality of coupled benzylsuccinyl groups. 9. The process according to any of the preceding claims, wherein an immobilized carboxypeptidase enzyme is used. 10. The process according to any of the preceding claims, wherein an aqueous reaction solution containing from 0 to 25% of organic solvent is used. - so ¬ il. The process according to claim 10, wherein the organic solvent used is selected from the group consisting of dimethyl sulfoxide, dimethyl formamide, alkanols, alkanoic acids, dioxane, tetrahydrofurane, dimethoxy ethane, glycerol, ethylene glycol and polyethylene glycols. 12. The process according to claims 1 or 4, wherein ammonia is added to the reaction medium as a concentrated solution or in liquid form. 13. The process according to claims 1 or 4, wherein the ammonia concentration in the reaction medium is from 4.0 to 12.0 M, preferably 5 - 8 M. 14. The process according to any of the preceding claims, wherein a pH adjustment agent is used selected from low molecular organic acids, preferably acetic acid or formic acid. 15. The process according to any of the preceding claims, wherein the reaction medium comprises a gelation inhibiting agent, preferably guanidium hydrochloride. 16. The process according to any of the preceding claims wherein the reaction is carried out at a pressure of 1 - 3 bar, preferably 1 - 2 bar in a closed vessel. 17. The process according to claim 1, wherein a peptide- Pro-X is used, which has been produced enzymatically, by recombinant DNA-methods, by chemical synthesis or a combination of these. 18. A calcitonin-related peptide of the formula Peptide' -Pro-X' wherein Peptide' denominates the native 1-31 amino acid sequence of human, salmon or eel calcitonin and X' is Met, Lys, Arg, Trp, Tyr or Thr. 19. Human calcitonin-( 1-32)-X"-0H, wherein X" is Met, Tyr or Thr.

Inventors: AASMUL-OLSEN STIG, CHRISTENSEN KURT ASBAEK, WIDMER FRED, AASMUL-OLSEN, STIG, CHRISTENSEN, KURT ASBAEK, WIDMER, FRED
Applicants: CARLBIOTECH LTD AS, CARLBIOTECH LTD. A/S
Application year: 1992
Main classification: C07K7
IPC codes: A61K38, C07K1, C07K14, C12N9, C12P21