AU4688799A - Creación de Inventos y Patentes con Inteligencia Artificial

AU4688799A – Non-enzymatic chemical amidation process
– Google Patents

AU4688799A – Non-enzymatic chemical amidation process
– Google Patents
Non-enzymatic chemical amidation process

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Publication number
AU4688799A

AU4688799A
AU46887/99A
AU4688799A
AU4688799A
AU 4688799 A
AU4688799 A
AU 4688799A
AU 46887/99 A
AU46887/99 A
AU 46887/99A
AU 4688799 A
AU4688799 A
AU 4688799A
AU 4688799 A
AU4688799 A
AU 4688799A
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AU
Australia
Prior art keywords
peptide
terminal
amino
oxy
unsubstituted
Prior art date
1998-06-17
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)

Abandoned

Application number
AU46887/99A
Inventor
Binyamin Feibush
Stephen R. Jones
Lincoln A. Noecker
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Magainin Pharmaceuticals Inc

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Magainin Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
1998-06-17
Filing date
1999-06-17
Publication date
2000-01-05

1999-06-17
Application filed by Magainin Pharmaceuticals Inc
filed
Critical
Magainin Pharmaceuticals Inc

2000-01-05
Publication of AU4688799A
publication
Critical
patent/AU4688799A/en

Status
Abandoned
legal-status
Critical
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Classifications

C—CHEMISTRY; METALLURGY

C07—ORGANIC CHEMISTRY

C07K—PEPTIDES

C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates

C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used

C07K14/4723—Cationic antimicrobial peptides, e.g. defensins

C—CHEMISTRY; METALLURGY

C07—ORGANIC CHEMISTRY

C07K—PEPTIDES

C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length

C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general

C07K1/124—Hydrazinolysis

Description

WO 99/65931 PCT/US99/13626 NON-ENZYMATIC CHEMICAL AMIDATION PROCESS CROSS REFERENCE TO RELATED APPLICATIONS This application claims benefit of priority under 35 U.S.C. § 119(e) to U.S. 5 Provisional Application Ser. No.: 60/089,635 filed June 17, 1998, which is herein incorporated by reference in its entirety. BACKGROUND OF THE INVENTION FIELD OF THE INVENTION 10 This invention relates to non-enzymatic methods of preparing peptides amidated at the C-terminal position. Such peptide C-terminal amides are useful in various pharmaceutical compositions such as, for example, antimicrobicides. DESCRIPTION OF THE RELATED ART 15 Recombinant DNA techniques have been used to produce many naturally occurring peptides. In addition, recombinant DNA techniques have made possible the selection, amplification and modification of such peptides. For example, alteration of the DNA coding sequence results in changes in the amino acid sequence of recombinantly produced peptides, thereby altering their function. However, some 20 modifications to a recombinantly produced peptide cannot be accomplished by altering the DNA sequence. The C-terminal carboxyl group in many naturally occurring antimicrobial peptides, for example, often exists as an amide (R-CONH 2 ). In this form, C-terminal amidated peptides exhibit improved antimicrobial activity. Cuervo, J.H. et al., Peptide 25 Res., 1, pp. 81-86, 1988; Lee, J.Y. et al., Proc. Natl. Acad. Sci. 86, pp. 9159-9162, 1989. However, it is not possible to produce a C-terminal amidated peptide using current recombinant expression techniques. Rather, peptides are converted to the amidated form using biological means subsequent to recombinant expression of the peptide in a peptidylglycine form. 30 One such biological means is the in vivo amidation of the C-terminal carboxyl WO 99/65931 PCT/US99/13626 2 group of a precursor peptide using a C-terminal a-amidating enzyme. In such a reaction, the precursor peptide is a peptidylglycine substrate of the general formula X R-Gly, where Gly represents a glycine residue, R represents an amino acid, and X represents the remaining part of the peptide. Treatment of the peptidylglycine substrate 5 with a C-terminal a-amidating enzyme yields a peptide C-terminal amide of general formula X-R-CONH 2 . Peptidylglycine a-amidating enzymes have been used to effect such an amidation. Bradbury, A.F. et al., Nature, 298 (5875), pp. 686-688, 1982. However, such an enzymatic method is limited to those peptides that possess a C terminal glycine residue. Other a-amidating enzymes have been characterized and 10 been used to amidate recombinantly produced peptides in vitro. Eipper, B.A. et al., Peptides, 4(6), pp. 921-8, 1983; Murthy, A.S., J. Biol. Chem. 261, pp. 1815-1822, 1986; Engels, J.W. et al., Protein Eng. 1, pp. 195-199, 1987. Disadvantageously, enzymatic amidation methods are time consuming, costly, unpredictable, and, oftentimes, substrate specific. In addition, such methods require an additional step of 15 purification of the final amidated peptide. Reductive cleavage of nitrogen-nitrogen bonds with Raney nickel and hydrazine to form amides has been achieved with non-peptide substrates such as N, N’-diacylated hydrazines. Robinson et al., Canadian Journal of Chemistry, Vol. 39, pp. 1171-1173, 1961. Few non-enzymatic, chemical amidation procedures for production of peptide C 20 terminal amides are known in the art. U.S. Patent 5,589,364 describes the non enzymatic chemical amidation of peptide methyl esters in a single step using ammonia. However, the use of ammonia can be quite harsh and thus prohibits the application of such a non-enzymatic chemical amidation method with relatively non-reactive esters (e.g. simple alkyl esters and non-activated aryl esters) and/or with base-sensitive 25 substrates under mild conditions without the formation of undesired side products. Furthermore, nonoptimal reaction conditions such as elevated pressures must be used and slower reaction rates lead to the formation of undesired lactams and polymeric by products. Accordingly, there still exists a need in the art for a simpler, gentler, more 30 versatile, non-enzymatic method for the preparation of peptide C-terminal amides that WO 99/65931 PCT/US99/13626 3 can be applied to a variety of peptide substrates. SUMMARY OF THE INVENTION The invention answers this need by providing a non-enzymatic method of 5 preparing a peptide C-terminal amide comprising the steps of: reacting a peptide C terminal carboxylic acid ester with an N-amino or N-oxy amine derivative to form the corresponding peptide C-terminal N-amino or N-oxy amide derivative; and converting the corresponding peptide C-terminal N-amino or N-oxy amide derivative under conditions sufficient to the corresponding peptide C-terminal amide. 10 The invention also provides a non-enzymatic method of preparing a peptide C terminal amide comprising the steps of: reacting a fusion peptide C-terminal carboxylic acid ester with an N-amino or N-oxy amine derivative to form a peptide C-terminal N amino or N-oxy amide derivative; and converting the peptide C-terminal N-amino or N-oxy amide derivative under conditions sufficient to the corresponding peptide C 15 terminal amide. BRIEF DESCRIPTION OF THE FIGURES Figure 1: Reaction scheme for conversion of : (a) MSI-344 (GIGKFLKKAKKFGKAFVKILKK corresponding to SEQ ID NO:1) 20 to (b) MSI-78: (GIGKFLKKAKKFGKAFVKILKK corresponding to SEQ ID NO:2 which contains a C-terminal amide group). Figure 2: HPLC analysis following conversion of MSI-344-methyl ester to MSI-1918 (GIGKFLKKAKKFGKAFVKILKK corresponding to SEQ ID NO: 5 which contains a C-terminal hydroxamic acid group) with hydroxylamine. 25 Figure 3: HPLC analysis following reduction of MSI-1918 [SEQ ID NO: 5] to MSI-78 [SEQ ID NO:2] with hydrazine and Raney nickel (Ni). Figure 4: HPLC analysis following conversion and cleavage of MSI-1850 methyl ester to MSI-1918 [SEQ ID NO: 5]. Figure 5: Scheme depicting downstream processing methods for MSI-1922 30 (MKAIFVLLEHHHHHLKDAQTNSSSNNNNNNNNNNLGIEGRISEFNGIGKFLKK WO 99/65931 PCT/US99/13626 4 AKKFGKAFVKILKK corresponding to SEQ ID NO:4). Figure 6: HPLC analysis following conversion and cleavage of MSI-1922 methyl ester to MSI-1918 [SEQ ID NO: 5]. Figure 7: HPLC analysis following reduction of MSI-1918 [SEQ ID NO: 5] to 5 MSI-78 [SEQ ID NO:2] with hydrazine and Raney nickel as described in Example 3. DETAILED DESCRIPTION OF THE INVENTION The invention provides a non-enzymatic method of preparing a peptide C terminal amide comprising the steps of: reacting a peptide C-terminal carboxylic acid 10 ester with an N-amino or N-oxy amine derivative to form the corresponding peptide C terminal N-amino or N-oxy amide derivative; and converting the corresponding peptide C-terminal N-amino or N-oxy amide derivative under conditions sufficient to the corresponding peptide C-terminal amide. According to the invention, the peptide C-terminal carboxylic acid ester may be 15 any synthetic, recombinant, or naturally occurring peptide C-terminal carboxylic acid ester, i.e. any synthetic, recombinant, or naturally occurring peptide having a carboxylic acid moiety at the C-terminal position. In a preferred embodiment of the invention, the peptide C-terminal carboxylic acid ester is of general formula (I): 20
RI-C(O)OR
2 (I). In formula (I), R, is an amino acid sequence and R 2 is a linear or branched, substituted or unsubstituted Ci-Cio alkyl group, or a substituted or unsubstituted C 3
C
7 cycloalkyl,
C
7
-C
1 0 aralkyl, C 6
-C
12 aryl or heteroaryl group. R 1 may be any amino acid sequence. In 25 a preferred embodiment of the invention, to avoid competing amidation reactions, the amino acid sequence of R, does not contain any amino acids having carboxylic acid group containing side chains (e.g. glutamic acid, aspartic acid). In addition, as would be understood by one of skill in the art, the amino acid sequence may be chosen to avoid problems such as, for example, catalyst poisoning by amino acid groups 30 containing, for example, disulfide linkages. Preferably, R, is a about 2-150 amino acid WO 99/65931 PCTIUS99/13626 5 sequence, more preferably, a about 2-110 amino acid sequence, and most preferably, a about 2-50 amino acid sequence. Preferably, R 2 is a linear or branched, substituted or unsubstituted Ci-Cio alkyl group, more preferably, a methyl group. Possible heteroatoms for the heteroaryl group include N, 0, and S. Possible substituents include 5 those substituents known in the art (e.g. alkyl, hydroxyl, nitro, chloro, bromo, iodo, cyano, amino) as long as they do not interfere with the overall C-terminal amidation process. In a more preferred embodiment of the invention, the peptide C-terminal carboxylic acid ester is a recombinant peptide C-terminal carboxylic acid ester. In another more preferred embodiment of the invention, the peptide C-terminal carboxylic 10 acid ester is a recombinant peptide C-terminal carboxylic acid ester of formula (I) where R 1 has the following amino acid sequence:
H
2 N-GIGKFLKKAKKFGKAFVKILKK [SEQ ID NO:1]. 15 The peptide C-terminal carboxylic acid ester for use in a method of the invention may be prepared by any means known in the art. For example, the peptide C-terminal carboxylic acid ester may be prepared from the corresponding peptide C-terminal carboxylic acid precursor using esterification methods known in the art. Bodanszky et al., Peptide Synthesis, 2nd Edition, John Wiley & Sons, 1976. The peptide C-terminal 20 carboxylic acid precursor may be any peptide C-terminal carboxylic acid. As with the peptide C-terminal carboxylic acid ester, the peptide C-terminal carboxylic acid may be a synthetic peptide, a recombinant peptide or a naturally occurring peptide, as understood by one of skill in the art. Preferably, the peptide C-terminal carboxylic acid is a recombinant peptide C-terminal carboxylic acid. 25 The N-amino or N-oxy amine derivative may be any N-amino or N-oxy amine derivative having at least one free amine hydrogen known in the art, i.e. any amine having at least one free amine hydrogen and is N-substituted with an amino or oxy group. In a preferred embodiment of the invention, the N-amino or N-oxy amine derivative is of formula (II): 30 WO 99/65931 PCT/US99/13626 6
NH(R
3
)(YR
5 ) (II). In formula (II), Y is NR 4 or 0 and R 3 , R 4 , and R 5 are, independently, hydrogen, a linear or branched, substituted or unsubstituted Ci-Cio alkyl group, or a substituted or 5 unsubstituted C 3
-C
7 cycloalkyl, C 7 -Co aralkyl, C 6 -Cl aryl or heteroaryl group. Possible heteroatoms of the heteroaryl group and possible substituents are each as described above. In a preferred embodiment of the invention, R 3 is hydrogen, Y is 0 and R 5 is hydrogen, methyl or a benzyl group. In another preferred embodiment of the invention, R 3 is hydrogen, Y is NR 4 and R 4 and R 5 are each a hydrogen. In a more 10 preferred embodiment of the invention, R 3 is hydrogen, Y is 0 and R 5 is hydrogen (i.e. hydroxylamine). According to the invention, the high nucleophilicity of the N-amino or N-oxy amine derivative offers the advantage that it may be used to rapidly convert relatively non-reactive peptide C-terminal carboxylic acid esters at about neutral pH to a peptide 15 C-terminal N-amino or N-oxy amide derivative, as described below, without having to protect the side chain amino or, if present, N-terminal amino groups or arginine guanidino side chain groups on other amino acid residues. In addition, the relatively low pKa (about 6.0-9.0, preferably, about 6.0-8.0) of the N-amino or N-oxy amine derivative allows for reaction under about neutral conditions, permits reaction with base 20 sensitive substrates, and prevents competitive amidation reactions. A peptide C-terminal N-amino or N-oxy amide derivative may be any synthetic, recombinant or naturally occurring peptide C-terminal N-amino or N-oxy amide derivative, i.e. any synthetic, recombinant or naturally occurring peptide having an N amino or N-oxy amide moiety at the C-terminal position. Most preferably, a peptide C 25 terminal N-amino or N-oxy amide derivative is any recombinant peptide C-terminal N amino or N-oxy amide derivative. In a preferred embodiment of the invention, the peptide C-terminal N-amino or N-oxy amide derivative is of formula (III):
R
1
-C(O)N(R
3
)(YR
5 ) (III). 30 WO 99/65931 PCT/US99/13626 7 In formula (III), R 1 , R 3 , Y, and R, are each as described above. According to the invention, a peptide C-terminal N-amino or N-oxy amide derivative may be prepared by reaction of an N-amino or N-oxy amine derivative with a peptide C-terminal carboxylic acid ester in a molar ratio of about 1:1, each as described 5 above. More preferably, an excess of the N-amino or N-oxy amine derivative is used to react with a peptide C-terminal carboxylic acid ester. The concentration of the reactant N-amino or N-oxy amine derivative ranges between about 2M – neat, preferably, between about 2-16 M, more preferably, between about 4-10 M. The reaction is conducted at a pH range of about 7-11, preferably, about 8-9.5, at about ambient 10 temperature until formation of the peptide C-terminal N-amino or N-oxy amide derivative, as described herein, is complete. Completion of formation of the peptide C terminal N-amino or N-oxy amide derivative may be determined by analytical techniques and methods known in the art (e.g. high performance liquid chromatography (HPLC), gas chromatography, capillary electrophoresis). As recognized by one of skill 15 in the art, the addition of solvents including, for example, denaturants (e.g. urea, guanidine) to solubilize the reactant peptide as necessary is envisioned as well. The newly formed peptide C-terminal N-amino or N-oxy amide derivative may also be further purified by means known in the art such as, for example, chromatography techniques (e.g. reverse phase HPLC, ion exchange chromatography). 20 The peptide C-terminal amide may be a synthetic, recombinant or naturally occurring peptide C-terminal aide, i.e. any synthetic, recombinant or naturally occurring peptide having an amide moiety at the C-terminal position. Most preferably, the peptide C-terminal amide is a recombinant peptide C-terminal amide. In a preferred embodiment of the invention, the peptide C-terminal amide is of formula (IV): 25
R
1
-C(O)NHR
3 (IV). In formula (IV), R, and R 3 are each as described above. According to a method of the invention, conversion of a peptide C-terminal N 30 amino or N-oxy amide derivative, as described above, is effected «under conditions WO 99/65931 PCT/US99/13626 8 sufficient» to the desired corresponding peptide C-terminal amide, as described above. «Under conditions sufficient» may include any reaction conditions that effect conversion of a N-amino or N-oxy amide moiety to an amide moiety. In a preferred embodiment of the invention, the peptide C-terminal amide results from the cleavage of 5 the N-N or N-O bond of the C-terminal N-amino or N-oxy amide moiety of the peptide C-terminal N-amino or N-oxy amide derivative, as described above. In a more preferred embodiment of the invention, such «under conditions sufficient» include reductive reaction conditions. Examples of suitable reductive reaction conditions include, but are not limited to, catalytic (e.g. Pd, Pt, Ni, Rh, Ru) hydrogenation (Babu 10 et al., Indian J. Technol., Vol. 5, pp. 327-328, 1967; exchange or transfer hydrogenation (e.g. hydrazine with catalysts containing Ni (e.g. Raney Ni), Fe, Cu, Sn, Mg, or Zn) (Robinson et al., Can. J. Chem. 39, pp. 1171-1173, 1961; Zajac, W.W. et al., J. Org. Chem. 36, pp. 3539-3541, 1971); cleavage by sodium or lithium in ammonia (Denmark, S.E. et al., J. Org. Chem. 55, pp. 6219-6223, 1990); metal-acid reduction 15 (e.g. reductants containing Zn, Sn, or Fe in acid) (Kirk and Othmer, Encyclopedia of Chemical Technology, Vol 2., Interscience, New York, 1963); and electrocatalytic reductions using electrodes containing Fe, Cu, Sn, or Ni (Cyr, A. et al., Electrochim. Acta, 35, pp. 147-152, 1990). More preferably, the peptide C-terminal amide is formed by means of catalytic hydrogenation, exchange or transfer hydrogenation, or metal-acid 20 reduction. Most preferably, conversion to the peptide C-terminal amide is achieved by exchange or transfer hydrogenation by addition of hydrazine and Raney Ni. In the event that the N-amino or N-oxy amine derivative is hydrazine (i.e. in formula (II), R 3 is hydrogen, Y is NR 4 and R 4 and R, are each a hydrogen), then conversion of the peptide C-terminal carboxylic acid ester to the peptide C-terminal amide may be 25 achieved as a one-pot synthesis by the addition of a catalyst such as, for example, Raney Ni and, if necessary, additional hydrazine, once formation of the peptide C terminal hydrazide (i.e. the reaction product of a peptide C-terminal carboxylic acid and hydrazine) is determined to be complete, as described above. As recognized by one of skill in the art, reaction conditions (e.g. reactant amounts, temperature, time, and pH) 30 may vary depending upon the type of substrate and reaction methods used. In general, WO 99/65931 PCT/US99/13626 9 however, the amount of reductant used ranges between about a molar equivalent to an excess based on the peptide C-terminal N-amino or N-oxy amide derivative, as described above. The reaction temperature may range between about 0-60’C, preferably, between about ambient temperature to about 45 C. The pH of the reaction 5 mixture may range between about 6-10, preferably, between about 7-9. The reaction pH may be adjusted and maintained by the addition of a buffer such as, for example, ammonium chloride. As recognized by one of skill in the art, reaction time may vary depending on the type and amount of catalyst used but, generally, formation of the desired peptide C-terminal amide is complete within about 24 hours. 10 The invention also provides a non-enzymatic method of preparing a peptide C terminal amide comprising the steps of: reacting a fusion peptide C-terminal carboxylic acid ester with an N-amino or N-oxy amine derivative to form a peptide C-terminal N amino or N-oxy amide derivative; and converting the peptide C-terminal N-amino or N-oxy amide derivative under conditions sufficient to the corresponding peptide C 15 terminal amide. According to the invention, the fusion peptide C-terminal carboxylic acid ester may be any synthetic or recombinant fusion peptide C-terminal carboxylic acid ester comprising a fusion partner segment and a peptide C-terminal carboxylic acid ester segment. In a preferred embodiment of the invention, the fusion peptide C-terminal 20 carboxylic acid ester is of formula (V):
R
7
-R
6
-C(O)OR
2 (V). In formula (V), R 2 is as described above, R 6 may be any synthetic, recombinant, or 25 naturally occurring amino acid sequence «beginning» (reading from left to right) (i.e. the N-terminal position of the amino acid sequence) with an unhindered amino acid, as described below, and R 7 is a fusion partner. Preferably, R 6 is a recombinant amino acid sequence. Preferably, the amino acid sequence of R 6 is a about 5-50 amino acid sequence, more preferably, a about 8-40 amino acid sequence, and most preferably, a 30 about 10-30 amino acid sequence. The fusion partner segment may be any fusion WO 99/65931 PCT/US99/13626 10 partner known in the art and would be understood by one of skill in the art to be a peptide. Examples of suitable unhindered amino acids include, but are not limited to, glycine, alanine or serine. Preferably, the unhindered amino acid is glycine. According to the invention, the junction (i.e. point of connection) of the fusion partner segment 5 and the peptide segment may be any consecutive amino acid sequence that may be cleaved with an N-amino or N-oxy amine derivative, as described herein. Preferably, the fusion partner segment «ends» (reading from left to right) (i.e. the C-terminal position of the fusion partner segment) with an asparagine or aspartic acid amino acid and the peptide segment «begins» (reading from left to right) with a unhindered amino 10 acid, as described above. In a preferred embodiment of the invention, the junction of the fusion partner segment and the peptide segment is a consecutive asparagine and glycine amino acid sequence. The peptide C-terminal carboxylic acid ester segment may be any peptide C-terminal carboxylic acid ester as described above. A fusion peptide C-terminal carboxylic acid ester may be prepared by means 15 known in the art including, for example, esterification of the corresponding fusion peptide C-terminal carboxylic acid precursor. Bodanszky et al., Peptide Synthesis, 2nd Edition, John Wiley & Sons, 1976. A fusion peptide C-terminal carboxylic acid precursor may be any fusion peptide C-terminal carboxylic acid precursor comprising a fusion partner segment, as described above, and a peptide C-terminal carboxylic acid 20 segment (i.e. a peptide C-terminal carboxylic acid ester segment, as described above, where the carboxylic acid ester moiety is a carboxylic acid moiety). A fusion peptide C-terminal carboxylic acid precursor may be prepared by any means known in the art. For example, a fusion peptide C-terminal carboxylic acid precursor may be prepared by recombinant means known in the art. In an alternative example, a fusion peptide C 25 terminal carboxylic acid precursor may be prepared by from a fusion partner and a synthetic, recombinant or naturally occurring peptide C-terminal carboxylic acid «beginning» with an unhindered amino acid, each as described above. Reaction of a fusion peptide C-terminal carboxylic acid ester with an N-amino or N-oxy amine derivative results in the formation of a peptide C-terminal N-amino or 30 N-oxy amide derivative, each as described above. Advantageously, according to a WO 99/65931 PCT/US99/13626 11 method of the invention, both cleavage of the fusion protein at the junction of the fusion partner segment and peptide segment, each as described above, and conversion of the C-terminal ester moiety of the peptide to a C-terminal N-amino or N-oxy amide moiety may be achieved in a single step. 5 According to the invention, a peptide C-terminal N-amino or N-oxy amide derivative may be prepared by reaction of a fusion peptide C-terminal carboxylic acid ester with an N-amino or N-oxy amine derivative, each as described above, in a molar ratio of about 1:1. More preferably an excess of the N-amino or N-oxy amine derivative is used to react with the fusion peptide C-terminal carboxylic acid ester. The 10 concentration of the reactant N-amino or N-oxy amine derivative ranges between about 2M – neat, preferably, about 2-16 M, more preferably, about 4-10 M. The reaction is conducted at a pH range of about 7-11, preferably, about 8-9.5 at about ambient temperature until cleavage and formation of the peptide C-terminal N-amino or N-oxy amide derivative, as described above, is complete. Completion of cleavage and 15 formation of the peptide C-terminal N-amino or N-oxy amide derivative may be determined by analytical techniques and methods known in the art (e.g. high performance liquid chromatography (HPLC), gas chromatography, capillary electrophoresis). As recognized by those of skill in the art, the addition of solvents including, for example, denaturants (e.g. urea, guanidine) to solubilize the reactant 20 peptide as necessary is envisioned as well. The newly formed peptide C-terminal N amino or N-oxy amide derivative may be further purified by means known in the art such as, for example, chromatography techniques (e.g. reverse phase HPLC, ion exchange chromatography). According to the invention, the peptide C-terminal N-amino or N-oxy amide 25 derivative may be converted «under conditions sufficient» to a peptide C-terminal amide, each as described above. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed. It is intended that the specifications and examples be considered exemplary only with the 30 true scope of the invention being indicated by the claims. Having provided this detailed WO 99/65931 PCT/US99/13626 12 information, applicants now describe preferred aspects of the invention. EXAMPLES: EXAMPLE 1. 5 Preparation of MSI-78 [SEQ ID NO:21 from MSI-344 [SEQ ID NO:11 via a two step chemical conversion. MSI-344 [SEQ ID NO:1] is a recombinantly produced peptide with an unmodified C-terminal carboxyl group, several lysine residues and an unprotected N terminal amino group. MSI-78 [SEQ ID NO:2] is the C-terminal amidated form of 10 MSI-344 [SEQ ID NO:1] and displays significantly greater antimicrobial activity than MSI-344 [SEQ ID NO:1]. The conversion of MSI-344 [SEQ ID NO:1] to MSI-78 [SEQ ID NO:2] is outlined in Figure 1. MSI-344 was first converted to MSI-344 methyl ester by deprotection of chemically synthesized MSI-344-t-butyl-oxy-carbonyl protected methyl ester in a mixture of hydrochloric acid, dioxane and methanol. The 15 reaction material contained approximately 90% MSI-344-methyl ester as determined by HPLC analysis. Conversion of MSI-344-methyl ester to MSI-1918 [SEQ ID NO: 51: MSI-344-methyl ester hydrochloride salt was added to 8 M hydroxylamine 20 diluted in deionized water and adjusted to pH 8.2 with acetic acid. The reaction was agitated for two hours at 20 C. The reaction was determined to be complete following HPLC analysis of sample aliquots from the reaction. The reaction was then diluted into 5% acetic acid and applied to an amberchrom column. The column was washed with 2% acetic acid to remove excess hydroxylamine. The peptide was eluted from the 25 column with a 0-70% gradient of acetonitrile in deionized water with a 2% constant concentration of acetic acid. All fractions determined to be ninhydrin positive were pooled and lyophilized to yield the crude acetate salt of MSI-1918 [SEQ ID NO: 5]. This crude product contained approximately 83% MSI-1918 [SEQ ID NO: 5] and 3% MSI-344 [SEQ ID NO:1] along with a number of unidentified impurities initially 30 present in the t-butyl-oxy-carbonyl protected methyl ester starting material as WO 99/65931 PCT/US99/13626 13 demonstrated by the HPLC analysis in Figure 2. The presence of MSI-344 [SEQ ID NO:1] in the reaction product resulted from hydrolysis of the methyl ester group to form the carboxylic acid. More importantly, the reaction products contained no peptide lactam side products or polymeric material which would result from reaction of the 5 unprotected amine groups present in the peptide. Protection of the E-amino group of lysine and the arginine guanidino side chain groups was not necessary to accomplish conversion of MSI-344-methyl ester to MSI-1918 [SEQ ID NO: 5]. For the HPLC trace displayed in Figure 2, chromatographic bands were identified through co-elution with the corresponding synthetic compound. Control 10 peak column retention times were as follows (dashed line): MSI-344 [SEQ ID NO: 1] was 9.160 minutes, MSI-344-methyl ester was 9.673 minutes, MSI-78 [SEQ ID NO:2] was 9.924 minutes and MSI-344-lactam side product was 10.242. Peak column retention times for the experimental reaction products were as follows (solid line): MSI-344 [SEQ ID NO:l] was 9.244 minutes, MSI-1918 [SEQ ID NO: 5] was 9.591 15 minutes while the unidentified impurities ranged from 7.247 to 7.772 minutes. Reduction of MSI-1918 ISEQ ID NO: 5] to MSI-78 [SEQ ID NO:21: The crude MSI-1918 [SEQ ID NO: 5] acetate salt product was converted to the hydrochloride salt. The crude MSI-1918 [SEQ ID NO: 5] hydrochloride salt was 20 dissolved in deionized water and methanol. Ammonium chloride was dissolved in this solution followed by the addition of Raney nickel. The reaction was vigorously agitated and warmed to 40’C. A 35% solution of hydrazine was then added to the flask, followed by additions at five and eight hours. Progress of the reaction was determined by HPLC analysis of sample aliquots following the initial addition of 25 hydrazine. At seven hours, the reaction was determined to be approximately 50% complete. The reaction was allowed to proceed overnight and at twenty-four hours the conversion appeared to be almost complete. The resultant material contained approximately 6.9% MSI-344 [SEQ ID NO:1] and 93% MSI-78 [SEQ ID NO:2] as demonstrated by the HPLC analysis in Figure 3. Again, the reaction products contained 30 no peptide lactam side product which would result from reaction of unprotected amine WO 99/65931 PCT/US99/13626 14 groups of lysine residues indicating that protection of e-amino or arginine guanidino side chain groups is not necessary to accomplish the reduction. For the HPLC trace displayed in Figure 3, peak column retention times for the experimental reaction products were as follows (solid line): MSI-344 [SEQ ID NO:1] 5 was 9.222 minutes, while MSI-78 [SEQ ID NO:2] was 9.979 minutes. Mass Spectrum (+ES) for MSI-78 [SEQ ID NO:2]: Caled. For C 1 22
H
21
N
32 0 22 : 2477. Found: 2477 (M+1). EXAMPLE 2 10 Cleavage of MSI-1850 [SEO ID NO:31 and conversion to MSI-1918 [SEO ID NO: 5] in a single step. MSI-1850 (QPELAPEDPEDEFNGIGKFLKKAKKFGKAFVKILKK corresponding to SEQ ID NO:3) is a recombinantly produced MSI-344-HSV fusion peptide with an unmodified C-terminal carboxyl group, several lysine residues and an 15 unprotected N-terminal amino group. The fusion peptide consists of fourteen amino acids derived from Herpes Simplex glycoprotein D on the N-terminal, with the remainder of the peptide comprising MSI-344 [SEQ ID NO:1]. The HSV region of the fusion protein contained several aspartic and glutamic acid residues while the MSI-344 [SEQ ID NO:1] region contained no amino acid residues with carboxylic acid side 20 chains. Cleavage of MSI-344 [SEQ ID NO:1] from its fusion partner can be accomplished with hydroxylamine because of the asparagine-glycine cleavage site which connects the two peptides. Conversion to the hydroxamic acid intermediate and cleavage from MSI-344 [SEQ ID NO:1] fusion partner can therefore be accomplished in a single step. 25 Preparation of the MSI-1850-ester is again necessary prior to cleavage and conversion to the hydroxamic acid intermediate. For esterfication of MSI-1850 [SEQ ID NO:3], the fusion peptide was dissolved in 2% hydrochloric acid in methanol and agitated under nitrogen for fourteen hours. The solvent was removed under vacuum and the residue was subsequently placed under high vacuum for two hours. The 30 reaction was not forced to completion but instead was processed as the crude product.
WO 99/65931 PCT/US99/13626 15 MSI-344 [SEQ ID NO:1] does not contain any carboxylic acid side chains, therefore only the C-terminal is converted to the methyl ester. MSI-1 850-methyl ester along with a reference material consisting of phenylacetic acid was dissolved in deionized water. One volume of the solution was 5 added to one volume of 8 M hydroxylamine and adjusted to pH 8.3 with hydrochloric acid and the reaction was allowed to proceed for twenty-four hours at ambient temperature. The HPLC analysis in Figure 4 demonstrates that the reaction product contained MSI-1918 [SEQ ID NO: 5], indicating that both cleavage from the fusion partner occurred along with conversion to the hydroxamic acid. The reaction product 10 also contained MSI-344 [SEQ ID NO:1] which resulted from cleavage of MSI-1850 [SEQ ID NO:3] which had not been fully converted to the methyl ester in the previous esterfication reaction because it was not allowed to proceed to completion. MSI-1918 [SEQ ID NO: 5] is then converted to MSI-78 [SEQ ID NO:2] according to the reduction procedure described in Example 1. 15 EXAMPLE 3 Three step conversion of MSI-1922 [SEQ ID NO:4] to MSI-78 [SEO ID NO:2] Esterification of MSI-1922 [SEQ ID NO:41: The peptide MSI-1922 [SEQ ID NO:4], Figure 5, (1.01 g, 43% active moiety by 20 HPLC) was added to a 100 mL round bottom flask. Methanol (50 mL) and p toluenesulfonic acid (2.0 g) were added to the flask with vigorous magnetic stirring. The flask was equipped with a Soxhlet extractor (125 mL capacity). The thimble (25 x 80 mm) was filled with 3A molecular sieves and 25 mL methanol was added to the extractor. This was done to prevent the volume holdup of the extractor from 25 concentrating the reaction too much. The reaction was brought to a vigorous reflux under N 2 . The reflux was sufficient to cycle solvent through the extractor at ~ 4-5 min intervals. The reaction was allowed to proceed for 24 h. The reaction was worked-up by chilling the reaction to 0-4 C, adding 1 volume of t-butyl methyl ether (50 mL), and allowing it to stand at this temperature for 10 min. The suspension was then centrifuged 30 at 1600 x g for 10 min. The pellet was partially dried in vacuo for 30 min @ 30 inches WO 99/65931 PCT/US99/13626 16 Hg. Note: The reaction can easily be run at concentrations > 50 mg/mL. Dilution in this case was necessary due to the glassware used. The Soxhlet extractor procedure pushes the esterification to completion as evidenced by the ratio of MSI-1918 [SEQ ID 5 NO:5] to MSI-344 [SEQ ID NO:1], in the reaction sample from the cleavage reaction described below (see attached chromatogram, Figure 6). Incomplete esterification will be detected as the carboxylic acid, MSI-344 [SEQ ID NO:1], after cleavage. Hydroxylamine Conversion to Hydroxamic Acid and Cleavage: 10 MSI-1922 methyl ester, 2, Figure 5, was suspended in 15 mL 10 M NH 2 OH / 8M urea and the suspension was homogenized with vigorous stirring. The suspension was stirred for 2h before dilution with 15 mL of 8 M urea. This solution was shaken at rt for 48 h and the cleavage reaction was worked-up by acidification to pH ~ 6.0 with acetic acid. The solution was then diluted and centrifuged for loading onto an ion 15 exchange column (SP-Sepharose, elution with a gradient of sodium chloride in 50 mM phosphate buffer). The final purified yield of MSI-1918 [SEQ ID NO:5] as the TFA salt was 128.8 mg (62% yield, assuming the product is 66% active moiety, based on elemental analysis). Mass Spectrum (+ES) for MSI-1918 [SEQ ID NO:5]: Calcd. For C 122
H
2 10
N
32 0 23 : 2493. 20 Found: 2493 (M+1). Reduction of MSI-1918 [SEQ ID NO:51 to Amide (MSI-78 [SEQ ID NO:2]): MSI-1918 [SEQ ID NO:5] (550 mg as the TFA salt) was dissolved in 11 mL of deionized water in a 50 mL polypropylene centrifuge tube. Raney Ni (Activated Metals 25 A-5000, 100 mg) was added to the reaction vessel. The reaction was magnetically stirred and warmed to 40’C. The hydrazine solution (17.5% w/v as a 50/50 molar ratio of hydrazine hydrochloride/hydrazine pH 8.1) was loaded into a 10 mL syringe and placed on a syringe pump. The pump was set to dispense 6.29 mL of the hydrazine buffer over 2h. The evolution of gas was vigorous after several minutes of hydrazine 30 addition and foaming of the peptide solution became problematic. At several time WO 99/65931 PCT/US99/13626 17 points during the course of the reaction, n-butanol was added to reduce the foaming. After a total of 700 tL of n-butanol was added, the foaming was controlled. After 2h of addition, the reaction was sampled for HPLC analysis and the addition of hydrazine was stopped. The reaction was allowed to stir for an additional 1.5 h until the HPLC 5 result confirmed that the reaction was already completed at the 2 h time point (see attached chromatogram, Figure 7). The reaction was worked-up by removal of the magnetic stir bar and removal of most of the Raney Ni by the magnet. The solution was then desalted and converted to the TFA salt by absorbing it on a C 18 column ( 50 mL, 30 g, Bakerbond 40 gm, prep LC packing) and washing with 0.2% TFA in deionized 10 water (500 mL). The peptide was then eluted with 60/40 acetonitrile/water containing 0.1% TFA. The ninhydrin positive eluant was lyophilized to yield 482.9 mg of MSI-78 [SEQ ID NO:2] as the TFA salt (88 % yield). EXAMPLE 4 15 Hydrazinolysis of a peptide C-terminal methyl ester A peptide C-terminal methyl ester (e.g. MSI-344-OMe) is dissolved in an aqueous solution containing a hydrazine/hydrazine hydrochloride buffer (pH 8-10). The hydrazine solution is prepared by adding the desired acid to a solution of 35% hydrazine (or a more concentrated solution can be prepared from hydrazine 20 monohydrate or neat hydrazine if desired). The solution is magnetically stirred at room temperature removing an aliquot at regular intervals for HPLC analysis. Complete conversion of the peptide C-terminal methyl ester to the peptide C-terminal hydrazide is monitored by HPLC. Once the reaction is complete, the reaction mixture is chilled in an ice water bath and neutralized by addition of acid. The crude peptide C-terminal 25 hydrazide is then desalted and purified by ion exchange or reverse phase chromatography. The purified peptide C-terminal hydrazide is reduced to the corresponding peptide C-terminal amide by addition of hydrazine (pH 8) in water with Raney Nickel catalysis. Alternatively, in a one-pot procedure, the crude peptide C terminal hydrazide is reduced directly to the amide by the careful addition of an aliquot 30 of Raney Ni, or by addition of the reaction mixture to Raney Ni. For saftey reasons, WO 99/65931 PCT/US99/13626 18 this is only practical if the concentration of hydrazine used in the conversion is relatively low or the excess hydrazine free base is removed in vacuo. It should be understood that the foregoing discussion and examples merely 5 present a detailed description of certain preferred embodiments. It will be apparent to those of ordinary skill in the art that various modifications and equivalents can be made without departing from the spirit and scope of the invention. All the patents, journal articles and other documents discussed or cited above are herein incorporated by reference.

Claims (20)

1. A non-enzymatic method of preparing a peptide C-terminal amide comprising the steps of: reacting a peptide C-terminal carboxylic acid ester with an N-amino or N-oxy 5 amine derivative to form the corresponding peptide C-terminal N-amino or N-oxy amide derivative; and converting under conditions sufficient said peptide C-terminal N-amino or N oxy amide derivative to the corresponding peptide C-terminal amide. 10

2. A method of claim 1, wherein said peptide C-terminal carboxylic acid ester is of general formula (I): R 1 -C(O)OR 2 (I) 15 wherein R, is an amino acid sequence; and R 2 is a linear or branched, substituted or unsubstituted Ci-C10 alkyl, a substituted or unsubstituted C

3 -C 7 cycloalkyl, C 7 -Ci 0 aralkyl, C 6 -C 1 2 aryl or heteroaryl group. 20 3. A method of claim 2, wherein said amino acid sequence comprises about 2-150 amino acids and R 2 is a methyl group.

4. A method of claim 3, wherein R, is: 25 H 2 N-GIGKFLKKAKKFGKAFVKILKK [SEQ ID NO:1].

5. A method of claim 2, wherein said N-amino or N-oxy amine derivative is of formula (II): 30 NH(R 3 )(YR 5 ) (II) WO 99/65931 PCT/US99/13626 20 wherein Y is NR 4 or 0; R 3 , R 4 , and R, are, independently, hydrogen, a linear or branched, substituted or unsubstituted Ci-C1o alkyl, or a substituted or unsubstituted C 3 -C 7 cycloalkyl, C 7 -CIO 5 aralkyl, C 6 -C 1 2 aryl or heteroaryl group.

6. A method of claim 5, wherein R 3 is hydrogen, Y is 0 and R 5 is hydrogen.

7. A method of claim 5, wherein said peptide C-terminal N-amino or N-oxy amine 10 derivative is of formula (III): R 1 -C(O)N(R 3 )(YR 5 ) (III) wherein 15 R 1 is an amino acid sequence; Y is NR 4 or 0; R 3 , R 4 , and R 5 are, independently, hydrogen, a linear or branched, substituted or unsubstituted Ci-Cio alkyl, or a substituted or unsubstituted C 3 -C 7 cycloalkyl, C 7 -CI 1 aralkyl, C 6 -C 12 aryl or heteroaryl group. 20

8. A method of claim 7, wherein said peptide C-terminal amide is of formula (IV): R 1 -C(O)NHR 3 (IV) 25 wherein R 1 is an amino acid sequence; and R 3 is hydrogen, a linear or branched, substituted or unsubstituted Ci-Ci alkyl, or a substituted or unsubstituted C 3 -C, cycloalkyl, C7-CiO aralkyl, C 6 -C 1 2 aryl or heteroaryl group. 30 WO 99/65931 PCTIUS99/13626 21

9. A method of claim 1, wherein said converting step comprises the addition of hydrazine and Raney nickel.

10. A method of claim 9, wherein said converting step further comprises the 5 addition of a buffer.

11. A method of claim 1, wherein said peptide C-terminal carboxylic acid ester is a recombinant peptide C-terminal carboxylic acid ester; said peptide C-terminal N-amino or N-oxy amide derivative is a recombinant peptide C-terminal N-amino or N-oxy 10 amide derivative; and said peptide C-terminal amide is a recombinant peptide C terminal amide.

12. A non-enzymatic method of preparing a peptide C-terminal amide comprising the steps of: 15 reacting a fusion peptide C-terminal carboxylic acid ester with an N-amino or N-oxy amine derivative to form a peptide C-terminal N-amino or N-oxy amide derivative; and converting under conditions sufficient said peptide C-terminal N-amino or N oxy amide derivative to the corresponding peptide C-terminal amide. 20

13. A method of claim 12, wherein said fusion peptide C-terminal carboxylic acid ester is of formula (V): R 7 -R 6 -C(O)OR 2 (V) 25 wherein R 2 is a linear or branched, substituted or unsubstituted Ci-Cio alkyl, a substituted or unsubstituted C 3 -C 7 cycloalkyl, C 7 -CIe aralkyl, C 6 -C 2 aryl or heteroaryl group; and R 6 is an amino acid sequence wherein the N-terminal position of said amino 30 acid sequence is an unhindered amino acid selected from the group consisting of WO 99/65931 PCT/US99/13626 22 glycine, alanine, and serine; and R 7 is a fusion partner wherein the C-terminal position of said fusion partner is an amino acid selected from the group consisting of asparagine and aspartic acid. 5

14. A method of claim 13, wherein the junction of R 6 and R 7 comprises a consecutive asparagine and glycine amino acid sequence.

15. A method of claim 13, wherein said N-amino or N-oxy amine derivative is of formula (II): 10 NH(R 3 )(YR 5 ) (II) wherein Y is NR 4 or 0; 15 R 3 , R 4 , and R 5 are, independently, hydrogen, a linear or branched, substituted or unsubstituted Ci-Cio alkyl group, or a substituted or unsubstituted C 3 -C 7 cycloalkyl, C7 CIO aralkyl, C 6 -C 12 aryl or heteroaryl group.

16. A method of claim 15, wherein R 3 is hydrogen, Y is 0 and R 5 is hydrogen. 20

17. A method of claim 15, wherein said peptide C-terminal N-amino or N-oxy amide derivative is of formula (III): R 1 -C(O)N(R 3 )(YR 5 ) (III) 25 wherein R 1 is an amino acid sequence; Y is NR 4 or 0; and R 3 , R 4 , and R 5 are, independently, hydrogen, a linear or branched, substituted or 30 unsubstituted Ci-Cio alkyl group, or a substituted or unsubstituted C 3 -C 7 cycloalkyl, C7- WO 99/65931 PCT/US99/13626 23 CIO aralkyl, C 6 -C 2 aryl or heteroaryl group.

18. A method of claim 17, wherein said C-terminal amidated peptide is of formula (IV): 5 Ri-C(O)NHR 3 (IV). wherein R 1 is an amino acid sequence; and R 3 is hydrogen, a linear or branched, substituted or unsubstituted Ci-Cio alkyl 10 group, or a substituted or unsubstituted C 3 -C 7 cycloalkyl, C 7 -C 10 aralkyl, C 6 -C 2 aryl or heteroaryl group.

19. A method of claim 16, wherein said converting step comprises the addition of hydrazine and Raney nickel. 15

20. A method of claim 12, wherein said fusion peptide C-terminal carboxylic acid ester is a fusion recombinant peptide C-terminal carboxylic acid ester; said peptide C terminal N-amino or N-oxy amide derivative is a recombinant peptide C-terminal N amino or N-oxy amide derivative; and peptide C-terminal amide is a recombinant 20 peptide C-terminal amide.

AU46887/99A
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Non-enzymatic chemical amidation process

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Method for producing peptide hydrazide, peptide amide, and peptide thioester

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