CS163191A3 - Process for preparing derivatives of a growth hormone releasing factor and peptides being useful as intermediates in said process - Google Patents

Description

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A method for producing Factor-releasing growth hormone derivatives and peptides useful as intermediates in this method

Technical field

The present invention relates to a process for producing GRF (1-29) NH2, an interesting biologically active growth hormone releasing factor and analogs thereof, as well as novel peptides that are useful as intermediates in this method.

Background Art

The use of biologically active peptides for pharmaceutical purposes and in agriculture has increased the importance of the ability to synthesize such compounds on a larger scale. Three methods are available: a) chemical synthesis, b) enzymatic synthesis, and c) fermentation by genetically manipulated microorganisms. While short peptides are preferred to methods a) and b) or a combination thereof, it has become much and more obvious that long peptides will be made in the future by taking advantage of methoxide recombinant DNA. However, these methods do not allow for a variety of modifications, such as incorporation of D-amino acids and C-terminal amide groups, which may be important for biological activity. Subsequent enzymatic modification is desirable and such reactions have been studied only to a limited extent.

An enzyme has been described which catalyzes the hydroxylation of C-terminal glycyl residues, which gradually decomposes leaving the penultimate residue amidated (US Patent No. 4,708,934, EP 308067A, DK Application No. 4489/88). This glycinoxydase enzyme, which is dependent on Ca 2+, O 2 and ascorbate as cofactors, is considered an enzyme responsible for in vivo peptide amide formation. It has been used for the amidation of small scale peptides, but it is still a question of whether it exhibits low activity activity for large scale work. The enzyme as 2 isolated from natural sources such as rat medial thyroid carcinoma is very expensive.

Amidation can also be achieved by protease catalyzed condensation reactions using an amino acid amide or peptide peptide as a 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 often does not occur with long peptides. In addition, the precursor peptide may exhibit low solubility in such media. However, serine or thiol protease catalyzed transpeptidation reactions can be carried out with great yields, but it is a prerequisite that the enzyme exhibits a specific peptide bond activity close to the C-terminus. Endopeptidases are not generally suitable as they will usually cleave in the other peptide chain positions. The serine carboxypeptidases, on the other hand, exhibit a strict C-terminal peptide binding specificity and are capable of catalyzing the exchange of the C-terminal amino acid with the amino acid amide added to the reaction medium to compete as a nucleophile with water.

This property of serine carboxypeptidases was carried out by a group of researchers at the Carlsberg Research Center, leading to a group of patents intended to be co-authored by DK Application No. 1443/79 represented by EP Pat. No. 17,485, US Patent No. 4,806,534 and its paternal US Patent No. 4 339 543 and W080 / 02151, among others DK Patent No. 155,613. These patents were based on the surprising finding that exopeptidases were useful as catalysts for enzymatic peptide synthesis, whereas prior art work exclusively with endopeptidases. Depending on the nature of the reactants (substrate and nucleophilic components) and reaction conditions, in particular pH, serine and thiol carboxypeptidases may catalyze peptide synthesis by elongation or transpeptidation. The preferred enzyme is carboxypeptidase Y (CPD Y) from yeast. 3 Basic and follow-up research has been described in a number of articles (refs. 14-10), all of which are incorporated herein by reference.

The general principle of enzymatic synthesis of peptides by transplantation in the presence of serine or thiol carboxypeptidases is disclosed in U.S. Patent No. 4,806,473 and in its parallel Danish Patent No. 155,613. generally discover and claim the production of peptide amides wherein A is an N-terminal amino acid protected or optionally an N-terminal protected peptide moiety and B is an L-amino acid residue, by reacting its substrate component, optionally N-terminal protected peptide AX-OU, where A is as defined above and X is an amino acid with a nucleophilic (amine) component of HB-NH2 in the presence of a 1-specific serine or thiol carboxypeptidase enzyme of yeast or animal, vegetable or microbial origin in an aqueous solution dispersion. pH from 5 to 10.5. As further explained in ref. 14, the preferred pH is about neutral when the peptide amide is desired.

As a representative example of U.S. Pat. No. 4,806,473, the reaction of Bz-Phe-Gly with Leu-N1 · 2 in the presence of CPD-Y at pH 7.6 and 26 ° C may be mentioned, leading to the formation of Bz-Phe-Leu-NN2 in 90% yield. . Further examples are included in DK Patent No. 155,613 inter alia by the reaction of Ac (Ala) - with Leu-NH 2 in the presence of CPD-Y leading to Ac (Ala) - Leu-NH 2 in 70% yield.

Further experiments are described in refs. 14-18, which support the pioneer character of these early abortions and the general applicability of serine carboxypeptidases as catalysts for C-terminal peptide modification.

In order to obtain a better understanding of the present invention, which is described in more detail below, it is considered appropriate to provide a general discussion of transpeptidation principles and competition reactions. The C-terminal amidation of peptides by the serine carboxypeptidase catalyzed transpeptidation reaction is dependent on: a) solubility of the peptide in an aqueous medium in which the enzyme is active and relatively stable, b) availability of C-terminus by enzymatic cleavage c) availability of serine carboxypeptidase of suitable substrate preference .

The type of reactions that may occur is shown in Scheme 1 with the substrate R-NH-ABC-OH and the nucleophile. Enzyme binds to the C-terminal peptide bond by formation of acylenzyme product, which can subsequently be deacylated by nucleophile to form an amidated transpeptidation product (TI), competing with water producing the hydrolysis product (H1). The TIk H1 ratio can be increased by increasing the concentration of the reactive, deprotected form of the nucleophile, that is, by increasing either the pH or adding the amount of nucleophile. The nature of the cleavage group, H-C-QH, had, in the case of CPD-Y, the same significant effect on this ratio (14, 15), however, with no clear trend. Since this amino acid residue did not form part of the desired amidated product (TI), it can in principle be chosen freely.

The serine carboxypeptidase may also catalyze the formation of an extended condensation product (Cl) in competition with Hla TI. However, at optimal pH values for transpeptidation, such reactions are vigorously disadvantageous in aqueous solution and consequently, in equilibrium, the amount of Cl that can be formed is limited to a few percent, even at nucleophilic concentrations exceeding 1H. Moreover, the rates of such reactions are normally very low compared to the compromising attack on the C-terminal peptide bond, and thus, condensation products are rarely a problem. However, this reaction may be reversed in cases where the C-terminal sequence of the peptidase does not fit the substrate preference of the enzyme, with the result that 5 form a small amount of condensation products in the initial phase of the reaction and then disappear with time when the substrate is consumed .

The hydrolysis product (H1) can be transformed into the road type by reaction to T2, H2 and TI products, followed by similar reactions with H2, etc. However, new products can also arise when the enzyme acts on the C-terminal amide bond of T1, i.e. ammonia, its cleaved group, producing another transpeptidation product (TT1) or hydrolysis product (HT1). Therefore, in order for these products to appear in quantification amounts, the amidase activity against TI is required to be significant compared to the peptidase activity towards the substrate, and this is normal only at pH> 9 where the peptidase activity is low and the amidase activity is high or in the where the peptide activity is due to a lack of preference for the C-terminal peptide bond.

It will be appreciated that the action of the serine carboxypeptidase at the L-terminus of the peptide in the presence of the amino acid amide may result in numerous products of which only T1 is desired.

SCHEME MA I

OJ -NH-fl-B-C-D-NHg (Cl) R-NH-fl-D-D-OH (HT1)

transpcptidation reaction c: condensation rBa] <oe nucleus il: H-D-NHg h: hydrelyation reaction 7

While reactions with N-blocked dipeptides of the appropriate composition relative to the substrate substrate preference in some cases result in TI yields of up to 100 ¾ (14, 15, 17, 10), the situation is different with larger molecules.

Thus, in the patent group represented by U.S. Pat. U.S. Patent No. 4,645,740 and Danish Patent No. 140,714 to the present Attorney and incorporated herein by reference discloses a process for enzymatically replacing B-30 amino acids in inserts, especially converting porcine insulin to human insoluble by replacing B-30 alanine amino acid with threonine using serine carboxypeptidase, preferably yeast carboxypeptidase (CPD-Y) or carboxypeptidase P from Penicilliumjanthinellum (CPD-P) catalyst.

When porcine insulin was reacted with threonine amide in the presence of CPD-Y at pH 7.5, a complicated reaction product mixture (Example 4 patents) was obtained. The mixture was analyzed by ion exchange chromatography and three spikes were detected. The spikes were further analyzed by enzymatic digestion and amino acid analysis to produce the following (Ins-Pro-Lys-Ala-OH, which was used to name the porcine insulin starting material), and the symbols in Scheme I were used as illustrations.

Peak 1: 21% of which 65% Ins-Pro-Lys-Thr-NH2 (TT1) 35% Ins-Pro-Lys-Thr-NH2 (TI) Peak 2: 61% of which 52% Ins-Pro- Lys-Ala-OH (unreacted porkinsulin) (Ξ) 26% Ins-Pro-Lys-Thr-OH (human insulin) (HT1) 22% Ins-Pro-Thr-NH2 (T2) 8

Peak 3: 18%

Decomposition products.

Thus, in the known method, only 21% of the T1 and TT1 blends were obtained. The predominant fraction consisted of a mixture of unreacted S, HT1 and T2.

Also, significant amounts of decomposition products were obtained. In another example, at pH 9.5, 80% porcine insulin was reacted to form what appeared to be a human insulin amide (TI), which was still hydrolyzed in situ to human insulin (HT1). A large number (20%) responded to the creation of H2.

The above-mentioned insulin modification is further analyzed by references 23 and 16.

While the formation of human insulin amide by the general principles of the above-described transpeptidation (S -> T1) is certainly not possible, it is not readily monitored. Apparently, reaction conditions may be selected which, in contrast, appear to be suitable for T1 formation, but such features have not been reported.

Also, the separation of different reactants may be more or less difficult. Since T1 lacks a negative charge at the C-terminus, it can be readily separated from H1 in the preparative scale by ion exchange chromatography. However, provided that the side chain of the C-terminal amino acid residue is uncharged, T2, T3, etc. as well as C1 and TT1 exhibit the same charge and therefore cannot be separated from T1 by ion exchange chromatography. In such cases, preparative HPLC with a reverse phase column appears to be the only and much less attractive. It is therefore much more desirable to suppress the formation of such products by selecting the optimal pH, nucleophile concentration, and most importantly selecting the serine carboxypeptidase with the most suitable substrate preference (Table 1), which rapidly and in the highest possible yield converts the substrate to TI and produces, if at all. some as low as possible yield of undesirable by-products. Referring to the above observations, incorporation of C-terminal amide groups into peptides by trans-peptidation in the presence of a serine carboxypeptidase using the appropriate amino acid amide as a nucleophile, as widely described and claimed in US Patent No. 4,306,473 and other related patents, is highly by a suitable method effectively applicable to any peptide.

However, the process is not always sufficiently selective and requires purification procedures to remove the products of the various side reactions, especially when other peptides are used, in which case it is difficult to determine the optimal conditions for suppressing side reactions. In the early publications of the original inventions published shortly after the administration of the aforementioned patent applications, efforts have been made to analyze the effect of the C-terminal (cleaved) amino acid and the penultimate amino acid.

Thus, in reference 14 Breddam et al., Using Leu-NI.alpha. Nucleophile and Gly, Ala, Ser, Val. Leu and Phe of its cleavage groups suggested, based on the obtained yields, that only in cases where the cleavage group is one of the smallest amino acids, that is, Gly, Ala, or Ser is the reaction successful and at least for the simple substrates tested, there was no dependence on the penultimate residue (who is Ala,

Phe and Gly). In reference 15 Breddam et al., Using Gly-NI / 2 as nucleophile and Z-Ala-X as substrate, Gly, Ala, Ser, 10

Arg, Pro, Lys, Asn, His, Val, Met, Phe and Asp modified in an earlier statement to the effect that when using Gly-NH 2 as nucleophile, the yield is strongly dependent on the C-terminal (cleavage group) of the amino acid. The yields varied from 10 to 100% with the lowest yields obtained with substrates where the hydrophobic acid (Val, Met, Phe) serves as a leaving group. It should be noted that the yields with basic acids (Asn, Lys) are comparable to those with hydrophilic acids (Ala, Ser), Lys is even better than Ser.

Regarding the penultimate amino acid residue of the substrates, the effect of using a series of N-blocked dipeptides with different penultimate amino acids (Ala, Val, Leu, Ile, Phe and Val), its cleavage group, was studied. Using Gly-NH2 as nucleophile, it was evident that the binding yields that varied from 45% for Ile to 5% for Phe are dependent on the penultimate amino acid residue, but no clear trend could be found. In reference 23, some of the experiments establishing US Pat. No. 4,645,740 and their related patents were discarded. Here, porcine insulin Ins-Pro-Lys-Ala was reacted with, inter alia, Thr-NH 2 and it was found that Ins-Pro-Lys-0H was a better substrate than Ins-Pro-Lys-Ala-OH, since Ins-Pro-Thr- NH 2 was produced in higher yields than Ins-Pro-Lys-Thr-NH 2. In conclusion, Lys was better off than Ala in this reaction.

There was also significant oligomerization to form Pro-Lys-Thr-Thr-NH 2.

These results were further confirmed in reference 16 using Bz-Lys-Ala-OH as a model peptide in addition to porkinsulin. The final report was that for future use of the CPO-Y (serine carboxypeptidase used in the experiments) in the peptidase reactions, it is important to be aware of the possibility that by-products may be formed. 11

In addition to the above studies of applicability of serine carboxypeptidase in C-terminal modifications of the insulin, another experiment was reported with amidation of longer peptides using CPD-Y as a catalyst.

Thus, in EP-B2-197794 and in parallel US Patent No. 4,709,014 (Tamaoki), human calcitin-Leu peptide was reacted with ammonia as a nucleophile using CPD-Y as a catalyst under conditions similar to those used by Breddam and others in ref. 15. The above reference to reference 15 was limited to amino acid amides as nuclophils, while the main purpose of the article was to compare different nucleophiles, including ammonia.

Tamaoki obtained human calcitoninamide in a yield of 24.7%, with a residue of 57% of unreacted substrate and 17.2 ¾ of non-amidated by-products (including human calcitonin).

In their more general aspects, Tamaoki's patents disclose a method of producing a peptide having a C-terminal proline amide that consists in reacting in an aqueous solution of a peptide substrate having a C-terminal Pro-Leu, Pro-Ile,

Pro-Val or Pro-Phe with carboxypeptidase Y in the presence of ammonia.

Without any desire to approve the outcomes of doing Tamokim, it should be noted that the use of hydrophobic amino acids (Leu, &lt; Desc / Clms Page number 4 &gt;

Ile, Val, and Phe) provide better yields than Gly when Source is the penultimate amino acid. However, the yields of amidation products in Tamaoki's using Cbz-Ala-Pro-X-OH as the substrate where X is Leu, Leu, Val, Phe and Ile were only 35.1%, 12 43%, 15.4% , 13.5%, 22.6%. The remainder was, in the referenced range, unreacted starting material and non-amidated by additional Cbz-Ala-Pro-OH products.

As previously described, the present invention relates to a process for the production of releasing factor growth hormone derivatives and analogs thereof. Growth hormone releasing factor (GRF) or soma-tocrinin, stimulating growth hormone output in glandulapitnitaria, is the 44-residue amidated peptide 5 10 H 2 N-Tyr-Ala-Asp-Ala-Ile-P &amp; Th-Thr-Asn-Ser-Tyr -Arg-Lys- 15 20 25

Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp- 30 35

Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg- 40

Gly-Ala-Arg-Ala-Arg-Leu-NH 2 which can be used to stimulate vertebrate growth (31). Both amide groups as well as the 15 amino acid residue closest to the C-terminal can be removed without suppressed activity, but each leads to a gradual decrease in biological activity. . However, a large portion of the activity may be re-ameliorated to these truncated peptides. One such derivative of great importance is GRF (1-29) NH2, which can be considered as a bioactive core of GRF.

Since the determination of GRF 91-29 activity, NH 2 numerous structural linear or ylic analogues have been prepared by exchanging, omitting or modifying one or more of the 29 L-amino acids in the native GFR chain.

Particularly interesting analogues have been prepared by Heimer et al. (32), Roche Research Center, New Jersey, USA, see 13 / des NHZ Tyr1, D-Ala2, Ala15 / -GRF (1-29) NH2, which is 100 times more potent than the original compound in vivoa cyclo (Asp8-llyl) - (Al145 / GFR (1-29) NH2, which is less potent than the linear compound in vitro but retains considerable bioactivity and induces a significant reduction in growth hormone in pigs. Heimer and others are included in the references.

Heimer and others have also focused on a number of other modifications. Replacing the Gly with a helix forming the amino acids Val, Leu, Aib, and Ala resulted in analogs of increasing biological activity, while substitution of Sar, a helix by a cleavage residue, resulted in significant loss of biological activity.

GRF (1-29) NH 2 analogues with replacement of Tyr 1 desNH 2 Tyr 3 or incorporation of D-Ala 2 also resulted in increased biological activity. Other analogs are described in the patents and patent applications listed below, all of which are incorporated herein by reference. EP-A-352014 (Salk Inst. For Biol. Studies) shows a number of possible modifications of GRF and GRF (1-29) NH2 in 1, 2, 3, 8, 12, 13, 15, 18, 21, 22, 24, 25, 27 and 28 and where Cys, Abu, Asp, Glu, Orn, Lys, Dab or Dap (all L or D) may be substituted for the current Arg context. Apparently, the abundant peptides are more active with better resistance to enzymatic degradation than native GRF. EP-A-307860 (Roche) discloses a series of linear and cyclic GRF analogs relative to those previously discussed. EP-A-292334 (SalI) describes a number of possible modifications of GRF and GRF (1-29) NH2 in 1, 2, 3, 8, 10, 12, 13, 15, 18, 21, 22, 24, 25, 27 and 28 positions. •IN. 14 FR-A-2594832 (Sanofi) describes a modified GRF (I-29) NHO, 19 27 where Val can be exchanged with Ile, Met can be exchanged with 28 with Nle and / or Ser can be exchanged with Asn and in which at least 1 and up to 6 amino acids, preferably at positions 2, 5, 12, 17, 21 or 27, are replaced by alpha-aminoalkyl amino acid, preferably alpha-amino-4-pentynoic acid, alpha-amino-4- hexylnoic acid and 2,6-diamino-4-hexynoic acid, which may be substituted. EP-A-216517 (Salk) corresponding to US-A-4689318 discloses raduGRF (1-29) NH2 analogues wherein at least 4 to amino acids 5-13, 15, 18-21 and 26 are different from the native sequence and preferably comprise Alaz. US-A-4628043 (Salk) discloses another group of analogs wherein inter alia Tyr 1 can be replaced by Met, Leu, D-Tyr, Ο-His or Hisa Gly 4 can be replaced by D-Ala.

Other analogs are described in EP-A-188214 (Tulane Educatioal French) / D-Ala2, Arg12'21 / GRF (1-29) NH2, RP-A-177819 (Roche), US-A-4528190 (Salk ), US-A-4513586 (Salk), EP-A-121764 (Roche) with Met2? replaced by Leu or Nle, OP-A-63, / 287799 (Tulane Educational French) revealing, inter alia, Pr-Tyr1, Pr-Lys12'21 / GRF (1-29) NH2 as 106-fold more polymorphic than GRF (1 -29) NH2. Generally speaking, one skilled in the art of amidation of GRF (1-29) OH and its analogues would certainly expect the general method of US Patent No. 4,806,473 using GRF (1-28) -X, where X is an L-amino acid such as a substrate of Ang-NH2 as a nucleophile operating in the presence of serine carboxypeptidase, but had no clear choice of applicable amino acid X, and would expect a series of competing reactions leaving it uncertain about yields and the necessary purification procedures to obtain the desired amidated end product. 15

SUMMARY OF THE INVENTION

The present invention is based on the surprising finding that by selecting its cleaved group X in the GRF (1-2B) starting material, a number of amino acids that logically do not fit into any of the preferential patterns proposed by Bred-Dam and others and Tamaoki, discussed above, see acyclic alpha-aminocarboxylic acid having uncharged hydrophilic side chain at least methyl group size, GRF (1-29) NH 2 can be obtained in surprisingly high yields. Preferred amino acids are Thr, Ala, Ser, Clu and Asn, especially Thr and Ala. Very small amounts of unreacted substrate and byproducts resulting from complicating reactions are observed, especially considering the experience of the human insulin amide described above and Tamaoki's results with calcitonin. To some extent, these observations are based on model tripeptides containing the native GRF (27-28) sequence and various cleavage groups, as well as on the synthetic GRF (1-28) Ala-peptide, as described below.

Based on these experiments and the general correlation of the mesimodel peptides and other peptides shown in the above articles, it is therefore appropriate to conclude that the method is not limited to the production of peptides containing native GRF (1-29) sequence, but at least where one or more amino acids in the positions 1 to 26 are replaced by other amino acids in both D- and L-form and cyclic amino acids which can be protected or derivatized, i.e. analogs of the types disclosed in the abovementioned references.

Correspondingly, the present invention relates to a process for the preparation of growth hoprone releasing factor derivatives, see GRF (1-29) NH2 and analogs thereof, characterized by bringing 16 into the substrate component highly.

GRF'-Met-Ser-X where GRF denotes the native GRF (1-26) sequence or analogs thereof including GRF (n-26) fragments, where n is 1-8, and X is an α-cyclic alpha-aminocarboxylic acid residue of non-naïve the thyrophilic side chain at the smallest size of the methyl group, with N-Arg-Nl ^ ^ as the nucleophilic component in the presence of an L-specific serine or thiolcarboxypeptidase enzyme from yeast or animals, plant or other microbial origin in aqueous solution or dispersion having pH from 6 to 9 and, if desired, by binding the desired N-terminal (1- (n1)) fragment chemically or enzymatically.

For enzymatic binding of the fragment, an endo-peptidase enzyme having its own specificity with respect to the C-terminal (1- (n-1)) fragment, i.e. papain or chymo-trypsin, may be used.

The chemical binding of the fragment can be accomplished by the introduction of suitable protecting groups in solution phase in the presence of suitable catalysts, for example DCC together with additives, for example KOBt. A preferred group of substrate components are GRF " -Met-Ser-X, wherein GRF " refers to a native GRF (1-26) sequence wherein 1 to 4 amino acid residues may be embedded or omitted or des-alpha-NH4 derivatives thereof aX is as defined above.

Useful carboxypeptidases in the method of the invention are L-specific serine or thiol carboxypeptidases. Such enzymes may be produced by yeast fungi, or may be of animal, plant or other microbial origin. 17

A particularly suitable enzyme is yeast fungal carboxypeptidase Y (CPD-Y). This enzyme is described in earlier patents by others with reference to john and others (ref. 28), which in particular has developed a suitable affinity chromatography purification method on affinity resin containing a polymeric resin matrix with bound benzylsuccinyl groups. CPD-Y, which is a serine enzyme, is available in large quantities and exhibits relatively high stability. Further details are provided in ref.

In addition to CPD-Y, which is currently the preferred enzyme, the method of the invention is feasible with other carboxypeptides such as those listed below:

Enzym Origin

Mushrooms kaeboxypeptidase S-carboxypeptidase S-2-carboxypeptidase (s) carboxypeptidase (s) from

Penicillium janthinellumPenicillium janthinellumAspergillus saitoi .Aspergillus oryzae

Plants

carboxypeptidase (s) C carboxypeptidase C ^

Phaseolain carboxypeptidase M-I and M-II carboxypeptidase W-II carboxypeptidase from orange peel orange leaves

Citrus natsudaidai Hayata (lemon) bean leaves germinated barley wheat bran germinated packed tomatoes yellow apples melons bromelin (pineapple) powder 18 - Tight, the relationship between a number of the above carboxypeptidases is discussed by Kubota and others (ref. 33).

As indicated above, the process of the invention is carried out at a pH of 6.0 to 9.0, preferably at a pH of 6.5 to 8.0, most preferably from 7.5 to 8.0. The preferred pH, which is often very narrow, depends on the enzyme used.

The selected pH should be maintained over time.

PH control should be accomplished by including the appropriate buffer for the selected pH range in the reaction medium.

The pH can also be maintained by the addition of an acid such as HCl, or a base such as NaOH, during the reaction. This can normally be achieved by using a pH-stat.

However, conditions may also be affected by changes in enzyme concentration, reaction time, etc.

The reaction is carried out in an aqueous reaction medium, if desired, may contain up to 80% by volume of the organic solvent. Preferred organic solvents are alkanools such as methanol and ethanol, glycols such as ethylene glycol or polyethylene glycols, glycerol, alkanoic acids, for example acetic acid, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, dioxane and dimethoxyethane. The choice of reaction medium composition depends in particular on the solubility, temperature and pH of the reaction components and the reaction products and the stability of the enzyme.

The reaction medium may also contain a component that provides insolubility to the enzyme, but retains a substantial portion of the enzymatic activity, such as ion exchange resin. Alternatively, the enzyme may be immobilized in a known manner, for example, by binding to a matrix such as bound dextran or aragosa, or to silica, polyamide or cellulose, or by enhancement in polyacrylamide, alginate or fibers. In addition, the enzyme may be modified chemical methods to improve its stability or enzymatic properties. Preferably, a chelating agent such as EDTA is included in the reaction medium; it may also include salts.

The concentration of the two reaction participants can vary widely, as explained below. A preferred starting concentration for the substrate component is 0.1-20 mM, preferably 1-10 mMa for a nucleophile component of 0.02-2.0M, preferably 0.2-1.5M.

The enzyme activity can also vary, but the concentration is preferably 10 to 10 M. Most preferred activity depends on the other on the substrate chain and the concentration, the nucleophile concentration, reaction time, reaction temperature, pH and the presence of organic solvents and / or salts . The amount of enzyme should in each case be adjusted to provide the necessary in-conversion to produce an optimal absolute amount of product.

According to the invention, the reaction temperature is 10 to 50 ° C, preferably 20 to 40 ° C. The most preferred reaction temperature for danousynthes can be determined by experiments, but depends mainly on the concentration of the nucleophile component and the concentration of the enzyme. A suitable temperature will usually be about 20 to 30 ° C, preferably about 25 ° Cberouc to calculate the consideration for enzyme activity and stability.

Similar variations occur for the reaction time, which is more dependent on other reaction parameters, especially the enzyme concentration. The standard reaction time of the invention is 1-3 hours. 20 -

The amino acid abbreviations for the amino acid derivatives of the peptide are given in the IUPAC-IUB Guidelines. Commission onBiochemical Nomenclature and the amino acids are in the L-form unless otherwise indicated. The binding site for the C-terminal amino acid residue of the substrate is designated S1 and those proamino acid residues away from the cleavage amino acid. the bonds are labeled with S 1, S 2, ... S 2. The substrates of the substrate are all denoted by P1, P1, P1 ... P1 in accordance with the reference sites (reference 1). The following additional abbreviations are used: AcOH, Acetic acid, B2, N-benzyl, OCCI, N, N'-dicyclohexylcarboimide,

Dh bz, 3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl, DICI, diisopropylcarbodiimide, DMAP, 4-N, N-dimethylaminopyridine, DMF, N, N-dimethylformamide, EDTA, ethylenediaminetetraacetic acid , Fmoc, flurenyl-9-γ-methylcarbonyl, Ft, fractionation, RGF, growth factor releasing factor, HPLC, high performance liquid chromatography, Mtr, 2,3,6-trimethyl-4-methoxyphenylsulfonyl, Pfp, pentafluorophenyl, TFA , trifluoroacetic acid, THF, tetrahydrofuran. Before the process of the invention is illustrated by examples, starting materials, measurement methods, etc., it will be explained in general terms. The starting materials of DMF were purified by fractional vacuum distillation. THF was passed through an active alumina prior to use. Fmoc-amino acids were purchased from Milligen and converted to Dhbt esters by reaction with carbodiimides and Dhbt-OH (Fluha) in THF (20). Resin synthesizing peptides (Macrosorb SPR) were purchased from Sterling Organics. Sequence analysis was performed on gas phase ABI sequencer and d Durrum D-500 equipment was used to analyze amino acids. HPLC equipment was from Waters Associates. Samples 21 were hydrolyzed for 24 hours in 6N hydrochloric acid and evaporated prior to amino acid analysis. H-Arg-NH2-2HCl, Bz-Met-OH, the other amino acids AMDEROAT AMINO ACID AND GRF (1-29) -NH2 were obtained from Bach, Switzerland. CPD-Y is commercially available from applicants. Bz-Met-Ser-Ala-OH, Bz-Met-Ser-Leu-OH, Bz-Met-Ser-Thr-OH, Bz-Met-Ser-Arg-OH, Bz-Met-Ser-Gly-OHa 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).

General Procedures for Preparing Tripeptide Substrates of BzMetSerX, X = Ala, Gly, Ser, Thr, Leu, Arg, Asn,

Glu, Met 10.7 g of benzylmethionine methyl ester was dissolved in 240 ml of DME and 36.6 g of serine isopropyl ester was added followed by 400 ml of H 2 O. Then the pH was adjusted to 8.5 using aqueous sodium hydroxide solution and 0.8 ml of 0.1 M EDTA was added. Thereafter, the reaction was started by adding 10 ml of a 0.2 µl by weight solution of crude papain pre-treated with 0.1 M tetramerkaptoethanol at 35 ° C. The reaction mixture was stirred for 3 hours and 25 ml of enzyme solution was added after 30 minutes and more was added after one hour. 25 ml of enzyme solution. Thereafter, the pH was reduced to 3 by addition of aqueous HCl and the mixture was filtered and reversed in reverse phase HPLC on a Waters Preppak 50060 / µm C ^g column using an alcohol gradient of 3 in 50 µM acetic acid. The combined fractions containing the pure product benzoylmethionylserine isopropyl ester were combined and brought to dryness under reduced pressure to give 9.0 g of BzMetSerOiPr (54%). Example of BzMetSerThr Synthesis: 2.0 g of BzMetSerOiPr were dissolved in 30 ml of DMP and added to a solution of 24.0 g of threonine in 34 ml of 1 0 and 2 ml of 0.1 N EDTA at which the pH was adjusted to 9.0 s using a sodium hydroxide solution. The reaction was then induced by addition of 2.8 ml, 0.35 mH CPD-Y solution and the mixture was stirred at pH 4.5 for 4.5 hours at room temperature. The reaction reagent was stopped by adding 10 M HCl solution to pH 3.0 au: after filtration, the mixture was purified by reverse phase HPLC on Waters Deltapak 300 µl / µm columns using an alcohol gradient in 50 mM acetic acid pH 3 .

The combined fractions corresponding to pure products were brought to dryness under reduced pressure to give 0.63 g of BzMetSerThrOH (34%) with an analytical purity of more than 95% by HPLC at 220 µm.

BzMetSerAla, BzMetSerSer, BzMetSerGly, BzMetSerLeu, BzMeSerArg, BzMetSerAsn, BzMetSerGlu, and BzMetSerMet were prepared in an analogous manner using the free amino acids Ala, Ser, Gly, Leu, Arg, Asn, Gin, and Met as nucleotides instead of Thr and the similarly pure products.

Assembly of human GRF (1-28) -Ala-0H

The column was filled with diatomaceous earth supported polydi-methylacrylamide methyl ester resin, Macrosorb SPR-100 (500 µg, 0.05 mmol functional groups) derivatized with ethylenediamine for acylated 4-hydroxymethylphenoxyacetic acid. Fmoc-Ala-O-Pfp (137 mg, 275 µmol) and DMAP (6 mg, 6 µmol) were dissolved in DMF and recirculated through the resin (500 mg) overnight. The reaction mixture was transferred with DMF and acetic anhydride (50 µl) and DMAP ‘(3 mg) was added. After minutes of recirculation, the reaction mixture was again transferred and the circuit was carefully washed with DMF.

The standard cycle of synthesis consisted of a 10 minute deprotection with piperidine in DMF (20%), DMF washing, introduction and recirculation of the acylating agent, and washing was introduced into the deprotection point. During the wash periods, all flaps and loops were operated. All bindings were accomplished by a single addition of Fboc amino acid Dbbt esters (3 eq,) as described by Ttherton and others (20) with t-butyl based side chain protection and Mtr protection of arginine. Binding times were determined by the fading of the insignificant yellow color induced on the resin formation of the ion pair between amino groups and 0hb-OH (22). The two-hour binding time was sufficient for most reactions. The slower binding was observed for Arg (20) (6 hours) and the prosequence of Ala (4) to Leu (14) (5-10 hours).

Deprotection and cleavage from the resin was carried out with TFA containing phenol (5%) for 16 hours. Evaporation and trituration with diethyl ether, 3 x 25 mL, resulted in the isolation of the crude peptide (210 mg). This was analyzed in reverse phase HPLC (5yU Vydac) using a gradient of 30% to 70% B (A = 0.1% TFA, B = 10% water and 0.1% TFA in acetonitrile). , probably due to the presence of methionine sulfoxide in one of them. 200 mg of peptide was treated with TFA containing thioanisole (10%) for two hours. Then only one major component was found by analytical HPLC as described above. After evaporation with diethyl ether, the peptide was dissolved in 10 mL of DMF / 1% AcOH (2/3, 10 mL) and chromatographed on G15 column with 1 L of AcOH. The fractions containing the main component were collected and lyophilized. This was separated by preparative HRLC to 25 mm x 300 mm 15 / U in reverse phase column (Waters delta-preparation. The sample was divided into two portions, each of which was dissolved in 20 mL of 15 µL DMF in water. per column and eluted at 10 ml / min for 10 min with 30% B and then with a gradient (20% -70% B) over 30 min The main tip eluted after 32 min and was lyophilized to give a total of 44 mg of peptide. showed: Asp 2.93, Thr 0.94, Ser 2, B2, Glu 2.23, Gly 1.08, Ala 3.91, Val 1.05, Met 1.0, Ile 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.

Transpeptidation reactions

The transpeptidation reactions reported in Table II below were carried out as follows: the nucleophile (H-Arg -,, 2 HCl) was dissolved in 6 mM EDTA and the pH adjusted with NaOH solution to the selected pH-reaction. 1 ml of a nucleophile solution from a 40-80 mH solution in either DMF (Bz-peptides) or 5% acetic acid (GRF (1-2S) -Ala-OH) followed by the enzyme from the aqueous solution. at the pH of the state (1 ml) so far, reactions with GRF (1-28) -Ala-0H were performed in a small degree of 90.1 ml) with a nucleophile acting as a buffer. When a 5µl acetic acid substrate was added, a decrease in pH was observed.

To compensate, the pH of the nucleophile solution was increased prior to the addition of the substrate. These reaction conditions were slightly modified in the experiments reported in Tables III and IV as explained. HPLC analyzes During the reaction, the µl / µl portion was removed from the reaction mixture and the reagent composition was determined by HPLC. The following elution system was used: 50 mM triethylammonium phosphate, pH 3.0 (A-buffer) and 50 mM triethylammonium phosphate, pH 3.0 in 80% CH 2 CN (B-buffer) using different linear and concave gradients. -blocked tripeptides Nova-Pak 5 / U C-18 reverse phase 25 column from Waters was used, while Vydac C-18 reverse phase column was used for GRF (1-28) -Ala-0H. A gradient was found that could separate the N-benzylated tripeptide substrates as well as possible reaction products including referenced by-products. This allowed for a detailed analysis of the reaction mixture. A similar system was not developed by longer peptides. However, maximum resolution was obtained and each product was collected and subjected to acid hydrolysis and amino acid analysis. Separations were performed at room temperature and monitored by UV absorbance at 254 nm (Bz-peptides) or 280 nm (GRF).

The percentage composition of the reaction mixture was determined by the direct integration of the integrated spike regions since all components were Bz, or, in the case of GRF, tyrosine, the dominant chromophore at the appropriate wavelengths. The amino acid fraction (Ft) was expressed as the ratio between T1 and all other products formed, i.e., non-consumed substrates were neglected in the calculation.

The invention is further illustrated by the accompanying drawings

FIG. 1. Time course of CPD-Y catalyzed transposition to GRF (1-2B) -Ala-OH using H-Arg-MH2 as nucleotide. The reaction conditions are shown in Table IIO, GRF (1-28) -Ala-OH (S),, GRF (1-29) -NH2CT1, Q, GRF (1-28) -0H (χχ),, GRF (1-28) -Arg-Arg-NH 2 (TT 1).

FIG. 2: Separation of products from GRF (1-28) -Ala-0H after treatment with CPO-Y for 147 minutes in the presence of H-Arg-NH2. The Vydac, C-18, 5 / U columns were used, using the TEAP buffer system described above and 30-45% B gradients over 30 minutes. L: GRF (1-28) -Arg-Arg-NH 2, 2: GRF (1-29) - NH 2, 3: GRF (1-28) -0H, 4: GRF (1-28) -Ala-0H . 26 In the initial screening experiments, the serine carboxypeptidase catalyzed transfection reaction leading to gRF (1-29) -NH 2 was studied in model reactions with the following N-benzoylated tripeptides: Bz-Met-5er-Arg-OH, Bz-Met- Ser-Leu-OH and Bz-Met-Ser-Ala-OH using H-Arg-NH 2 as nucleophile. The transpeptidation product is in all three cases Bz-Met-Ser-Arg-NH2 corresponding to Bz-GRF (27-29) -NH2. with their expected utility in transpeptidation reactions. Thus, -Arg-OH was considered suitable for CPD-W-II (18,21,26) and probably CPD-S1, for which good results were also obtained using such Leu OH as the cleavage group with ArgNH2 as nucleophile ( while the SerOH formed poorly. Ala-OH and probably Leu-OH were considered suitable for CPD-Y (14,15) in terms of activity (14,15). As for the transpeptidation suitability of these groups, the prior art has left no ambiguous or inconsistent guidance for CPD-Y, as previously described. Thus, while in some references (14, 15) the preferred cleavage moieties are small hydrophilic amino acid residues, other references (U.S. Patent No. 4,709,014) to a particular group of large hydrophobic amino acid residues, and Ala, which in terms of size and hydrophilicity having an intermediate position among natural amino acids, it has been found to be poor in only one long peptide report, i.e. pork insulin (US Patent No. 4,645,740), the total yield being less than 10% of the correct product. In this regard, it should be noted that hydrophilic amino acids are defined as amino acids having side chains much more hydrophilic than cysteine, indicated by hydrophilicity values reported by Hopp and Woods (Proc. Nat. Acad. Sci. USA, 78 (6). 3824-3828-181 (ref. 34) Good initial results with Ala versus Leu in this 27 model furthered studies using a variety of hydrophilic amino acid residues (i.e., Gly, Ser, Thr, Asn, and Gln) to establish size requirements for a possible successful product formation, as well as establishing the degree of hydrophilicity needed to test Met its cleavage group having a hydrophilicity value between Leu and Ala.

Table I

Substrate Preferences of Serine Carboxypeptides for Hydrolysis of PXY-OH Peptide Substrates X, Y CPD-Y CPD-W-II CPD-MI CPD-S-1 Group (Amino Acids) P | 1, Arg, Lys 2 Glu, Asp 3 Ser, Thr, Gln, Asn, His, Gly, Cys 4 Tyr, Phe, Trp 5 Ala, Met, Leu 6 Val, Ile 7 Pro

D D D D

A A B C A

B A C C C

CAD C DD D E E E

C EB DA CA EB E

AND

D

D

AND

B

B

E

The preferences are given in order of order: A> B> C> D> E, this is rapid hydrolysis and E extremely slow "/ Km" in the corresponding mild corresponding hydrolysis. The data is from references 17, 18, 21, 26, 30.

Optimal conditions for serine carboxypeptidase catalyzed transpeptidation reactions can be determined in each case by experiments. The yield of transpeptidation is generally influenced by the rise in pH due to the depiotonation of the amino group of the amino acid amide of the nucleophile, but simultaneously 28 peptidase activity decreases, leading to lower conversion rates and amidase activity increasing, leading to degradation of the product. The rise in pH can be compensated by the rise in nucleophile concentration, but it also reduces the rate of conversion as the nucleophile acts as an inhibitor (25). With respect to pH, the compromise is between 7.5 and 8.0 except for CPD-S-1 which is unstable above pH 7.0.

The transpeptidation reaction with CPD-W-II and Bz-Met-Ser-Arg-QH was initially carried out at pH 7.6 and 10 mM H-Arg-NH 2, the low concentration of nucleophile was monitored by efficient binding of nucleophiles with positively charged side chains to this enzyme. After 47 minutes, 91% of the substrate was converted to the following products (see Table II): 64% Bz-Met-Ser-Arg-NH2 / T1), 20% Bz-Met-Ser-OH (H1) and 6% Bz-Met -H (H2). Bz-Met-Arg-NH2 (T2) as well as other possible products (see Scheme 1) were absent. The transpeptidation fraction was 0.71 and this value was also obtained at pH 8.0. With 2.5 [mu] m, 25 mM H-Arg-NH2, the transpeptidation fractions were 0.55, 0.64, and 0.72 respectively, suggesting that values of about 0.7 could not be exceeded.

Transpeptidation reactions with CPD-Y on Bz-Met-Ser-Ala-OH and Bz-Met-Ser-Leu-OH were initially performed at pH 7.5 and 0.5 MH-Arg-NH 2, rather high concentration of nucleophile due to expected weak binding to CPD-Y, which has pronucleophile preference with hydrophobic side chains. The trans-peptidation fraction was 0.91 with Bz-Met-Ser-Ala-OH and 0.41 with Bz-Met-Ser-Leu-OH. The general trend of dependence on the nature of the amino acid of the cleavage group is in accordance with the foregoing observations, where the fraction of transpeptidation with CPD-Y in most cases was significantly and sometimes drastically higher where the cleavage group is small and / or hydrophilic. Ala-OH, compared to hydrophobic, i.e. -Leu-OH (14,15). However, the yields for Leu, unlike the results reported in ref. 14, p. 243, where Leu was used as a leaving group resulting in 0% transpeptidation yield. 29

Thus, initially only the reaction with Bz-Met-Ser-Ala-OH was carried out under other reaction conditions and as a base for further studies of transamidation of longer peptides. At 0.025 ΙΊ and 0.1 MH-Arg-NH 2, the transpeptidation fractions were lower, ie 0.77 and 0.87, respectively, and a 0.5 M concentration was therefore conserved. However, a small amount of transpeptidation was produced at this concentration. (2 undesired Bz-Met-Arg-NH 2 (T 2). The pH rise to 7.8 prevented the formation of this product and at the same time the transpeptidation fraction was increased by 0.98. CPD-S-1 was also tested in transpeptidation reactions with Bz-Met -Ser-Arg-OH and Bz-Met-Ser-Leu-OH, with 0.2 MH-Arg-N1 and pH 6.5 fractions of transpeptidation were 0.38 and 0.48, respectively. effect.

Initial results of transpeptidation reactions in N-benzylated tripeptide sorting assays seemed to indicate that the most effective amidation resulting in a good yield of suppressing unwanted peptides such as T2 could be achieved with CPD-Y as -Ala-OH cleaved. group. Consequently, GRF (1-28) -Ala-0H was selected as a test intermediate and was prepared by continuous flow of peptide synthesis. Grouping of GRF (1-28) -Ala-OH (2) was performed by solid-phase methodology described above and cleaved from the resin with TFA and phenol for 20 hours.

The reaction of GRF (1-28) -Ala-OH with CPD-Y in the presence of H-Arg-Nl ^ was carried out under the conditions given in Table II. The reaction was followed by HPLC and the time course is shown in Figure 1.

In approximately 2.5 hours, substantially all of the substrates were converted to GRF (1-28) -Arg-NH2 (T1), GRF (1-28) -OH (H1) and GRF (1-28) -Arg-Arg products -NH 2 (TTl). The HPLC chromatogram of the final reaction mixture is shown in Figure 2. The identity of GRF (1-29) -NFl 2 as the dominant product was further examined by co-chromatography with authentic GFR (1-29) -NH 2: only one tip was observed. Separate experiments without the addition of H-Arg-NH2 confirmed the retention time H1. Smaller components elute around the position of GRF (1-28) -Ala-OH, each forming less than 2% of the reaction mixture, were collected, but amino acid analysis did not identify any of them as C-terminally modified derivatives of GRF (1- 29).

At 147 minutes, the reaction mixture contained 84% GRF (1-28) -Arg-NH 2, i.e. GRF (1-29) -NH 2. Thus, with 1 g of CPD-Y, about 2.7 kg of GRF (1-29) -NH 2 can be produced using the reaction times and conditions set forth in Table II. However, CPD-Y foods are stable at pH 8.0 and 22 ° C, and therefore a much longer reaction time and correspondingly reduced enzyme concentration can be used. Since CPD-Y is readily isolated from baker's yeasts after autolysis (ref. 28) or from genetically manipulated yeast cells (ref. 29), the cost of enzymes is relatively low and the procedure described herein therefore appears to be a valuable alternative to using a much more rare glycine oxidase. - 31 - .mu.m. > iTi O ΓΊ i — I 03 <ø> -4 r- ~ r-- ~ coco n- ca 03

CM Ή.

C ta>! MO »—fit

Table II. Amidation of GRF (1-29) and its model peptides. tn Cto 't =

Q CL,

O

N 2 - •: ω r4 * <c <n n o. o oooooo o o oooo o o o o o o 1 - 1 03 03 tn co * c o o o cj what OJ OJ i - 1 OJ OJ *> 3 "CO 1—1 r — í <OOOC M3 ua <3 <3 ΓΟ OJ 1—1 CO ooo what C oO m τ — 1 mm co oo “m η mc OJ <ro * i —! I — 1 mmoo <Γ0 rl o tn O ca cm r - <ca ca OJ OJ O o to r — 1 OJ or —OfJOOOOOOOOOOOOOOoOnOnOnOnOnOrOOOOO1— i ~ ” ~ sz — SS “- Z — with LO zS Z- — from ——————————————————————————————————————————————————————————————————————————————————————————————————————— . ». OO OO OO OO OO OO OO OO OO OO "OO OO OO OO OO OO OO OO i i i i i i i i i i i i i i i 2: 3 co> * on> a> ·>> - OON l3 OO O oa oot “J t — i o o o o o o o o o o o o o o o oo o o o o o o o oo o

CM CM

CM CM CM CM

CM O · i 1. o> 1 1 1 X z—- that <0 O 1 r · * -53 uo r-1 c C <· '' i <1 ttl — J 1 1 1 <zl z zl 1 O ω ω ✓- ' I · CO 1 1 1 1 1 1 C C C C C C C C U U U U U U U U U U U.. · E E · O O 1 1 1----Q Q Q Q Q

LcJ

E

IA

-H tn o c

E ta + j

> H> f-l Q. C. ta 2 ta> o n Q. 3 ^ 2 ta ta v * to ta p>. C

CJ

Ui <

AND

O tq

C

I from ~ \ t

CM

I cst ca Ό32SH> oa 2o let ta 2 ta fa 2 <a

> N at ro i i tta t a

O c • Ή Γ3

N '>>

rH C. Π3 32

As previously shown, the initial results with N-benzoyl tripeptides Bz-Met-Ser-Ala-DH, Bz-Met-Ser-Arg-OH and Bz-Met-Ser-Leu-QH seemed to indicate that between these three the peptides of the most effective amidation would be achieved by sAla-OH as a leaving group, and this approach was further tested with the complete peptide sequence. In order to obtain a more thorough analysis of the appropriate cleavage groups for amidation, a further series of model peptides containing hydrophilic amino groups of different size of its cleavage group was synthesized. Thus, small Gly, medium Ser and greater Thr were selected, and trans-peptide assays were performed using Bz-Met- Ser-Ala-OH as a comparison. The results are shown in Table III below: T abu 1 ka III substrate CPD-Y composition time mg / ml min S TI H1 other Ft BzMetSerAlaOH 0.06 64 10.4 76.9 9.1 3.610.86 BzMetSerSerOH 0.62 51 17.0 61.0 3.1 18.0 0.74 BzMetSerGlyOH 1.23 89 13.6 48.3 1.5 36.6 0.56 BzMetSerThrOH 0.10 94 10.8 72.9 8.0 8.4 0.82

substrate concentration: 2 mM

concentration of Arg1-J1: 1000 mM pH: 7.90

It can be seen that, with respect to the product yield of the trans-peptidation fraction, surprisingly, Thr, which was not previously referred to as a cleavage group, is comparable to Ala over its larger size. while Oly leaves a larger amount of unreacted substrate and significant amounts of by-products. 33

In some reactions by the method of the present invention, the absolute amount of product formed depends on the degree of conversion and passes through the optimum that would justify stopping the reaction and recycling the remaining substrate, while others are fully responsible for the reaction to completion. In the Bz-Met-5er-X peptide model of the experiments carried out, Ala and Thr as the X-cleavage group appear to be in the last category, while Asn and Glu as cleavage groups would appear in the previous one. In Table IV below, some incomplete conversion results are reported using a large but hydrophobic Met as a reference example: T a b u 1 k and IV substrate CPD-Y composition time mg / ml min. S i TI H1 other F t BzMetSerAsnOK 1.60 52 47.0 40.0 3.0 10.0 0.76 BzMetSerGlnQH 1.30 38 29.0 52.6 4.4 15.0 0.73 BzMetSerMetOH 0, 03 25 30.0 32.2 33.0 4.3 0.46

Substrate Concentration: 2 mM, ArgNl · ^: 1000 mM, pH: 7.9.

It can be seen that between 50% and 70% of conversion, large amounts of product are produced relative to the amount of substrate consumed using large, hydrophilic amides Asn and Ginj as cleavage groups, while Met produces as weakly as Leu and Gly in Tables II and III and gives forming a plurality of hydrolysis by-products. it should be noted that this is despite the fact that L is shown in Table I where Met is grouped together with Leu and Ala, Met is an active substrate as a cleavage group, reducing enzyme requirements as apparent in Table IV. It may be noted that Table I also groups 34

Ser, Thr, Glu, and Asn.

Along with Gly as a group, but in a completely different category than Ala, Leu, Met. Thus, the results in Tables II, III and IV illustrate the lack of possibility to predict synthetic yields based solely on hydrolytic preferences listed in Table I.

The logical standard for evaluating the applicability of various possible cleavage groups would be the native GRF sequence in which the C-terminal sequence is -Met-Ser-Arg-OH.

Based on the above results, it can be concluded that the transamidation can be carried out with sufficient selectivity when using Ala, Thr, Ser, Asn and Glu as cleavage groups, while Leu, Gly and Met give rise to significant amounts of minor products. Thus, the hydrophilic side chain at the smallest methyl group size appears to be necessary for the desired selectivity and improved yield of the supernatural GRF sequence. This result could not be foreseen in the articles discussed above, and it means that it is indeed possible to select substrates having greater possibilities as protrane-amidation intermediates than native GRFG-29).

Claims (13)

- 37 - PATENT REQUIREMENTS 3 C r A process for the production of releasing factor growth hormone derivatives, see GRF (1-29) .NH2 and analogs thereof, characterized in that a substrate component of the formula GRF'-Met-Ser-X is reacted where GRF "denotes native GRF ( 1-26) a sequence or analogs thereof including GRF (n-26) fragments wherein n is 1-8, and X is an acyclic alpha-aminocarboxylic acid residue having an uncharged hydrophobic side chain at the smallest methyl group, s H-Arg-Nl ^ ^ as a nucleophilic component in the presence of an L-specific serine or thiolcarboxypeptidase enzyme of yeast or animal, plant or other microbial origin in an aqueous solution or dispersion having a pH of from 6 to 9 and is for example, by binding the desired N-terminal (1- (n1)) fragment chemically or enzymatically. The method of claim 1, wherein the substrate component is GRF "-Met-Ser-X, wherein GRF" refers to a native GRF (1-26) sequence wherein from 0 to 6 amino acid residues may be or are omitted or omitted from des-alpha-N1, and X is as defined above. 3. The method of claims 1 or 2 wherein X is selected from Ala, Ser, Thr, Asn, and Glu. 4. The method of claims 1 to 3 wherein the carboxypeptidase enzyme used is a yeast yeast carboxy-peptidase Y. 5. A process according to claim 4 wherein the carboxypeptidase is used which has been purified by affinity chromatography on an affinity resin consisting of a 1-30-polymeric resin matrix with a plurality of bound benzyl succinyl groups. 6. A method according to any one of claims 1 to 3 wherein the carboxypeptioase enzyme used is selected from the group consisting of Carboxypeptidase S1 and S-2 from Penicillium janthinellum, carboxypeptidase from Aspergillussaitoi or Aspergillus oryzae, carboxypeptidase C from orange leaves or orange peels, carboxypeptidase Citrus natsudaidai Hayata, phaseolain from bean leaves, carboxypeptidase MI and germ from germinating barley, carboxypeptidazaza I / II from wheat bran, carboxypeptidase from germinating germinates of cotton, tomatoes, melons and bromellin ( ananaan powder). 7. A method according to any one of the preceding claims wherein the immobilized carboxypeptidase enzyme is used. A process according to any one of the preceding claims, characterized in that an aqueous reaction solution containing from 0 to 30% organic solvent is used. 9. The process of claim 8 wherein the organic solvent used is selected from the group consisting of alkanols, alkanoic acids, dimethylsulfoxide, dimethylformamide, dioxane, tetrahydrofuran, dimethoxyethane, glycerol, ethylene glycol, and polyethylene glycol. 10. The method of claim 1 wherein GRF'-Met-Ser-X is used which has been produced enzymatically, by recombinant DNA methods, by chemical synthesis, or a combination thereof. 39 11. A GRF-related peptide of the formula GRF "Met-Ser-X, wherein GRF" refers to a native GRF (1-26) sequence wherein from 1 to 4 amino acid residues may be replaced or omitted, or as des-alpha-NHg derivatives and X is an acyclic residue of an alpha-aminocarbonic acid having an uncharged hydrophobic side chain of at least a dimethyl group. 12. The GRF related peptide of claim 11 wherein GRF " refers to a native GRF (1-26) sequence and X is as defined above. The GRF related peptide of claim 11 or 12, wherein X is Ala, Thr, Ser, Asn, or Gln. 14. The GRF related peptide of claim 13 wherein X is Ala or Thr. algae dulls: