WO1989001487A1

WO1989001487A1 - Removal of n-terminal acylated amino acid residues from polypeptides

Info

Publication number: WO1989001487A1
Application number: PCT/US1988/002761
Authority: WO
Inventors: Mark L. Stolowitz; Beth A. Paape
Original assignee: Bio Affinity Systems Inc
Current assignee: Bio Affinity Systems Inc
Priority date: 1987-08-10
Filing date: 1988-08-09
Publication date: 1989-02-23
Anticipated expiration: 1990-02-10
Also published as: AU2388688A

Abstract

The present invention discloses a two-step process by which an N-terminal-alpha-acylated amino acid residue may be removed from a polypeptide with the concomitant release of the residual polypeptide, wherein the N-terminal amino acid residue is unmodified and suitable for chemical degradation for the purpose of determining its primary structure. In the first step of the process a polypeptide comprised of n+1 amino acid residues, and containing an N-terminal-alpha-acylated amino acid residue, is reacted with a dithiophosphetane reagent, in tetrahydrofuran, dioxane or acetonitrile, at room temperature or at reduced temperature. Removal of the byproducts of the reaction yields a polypeptide of n+1 amino acid residues wherein the N-terminal-alpha-acylated amino acid residue has been converted into the corresponding N-terminal-alpha-thioacylated amino acid residue. In the second step the thioacylated polypeptide is reacted with an anhydrous organic acid, at room temperature, resulting in the removal of the N-terminal amino acid residue as the 2-methylthiazol-5(4H)-one or 2H-thiazol-5(4H)-one, and the concomitant release of the residual polypeptide comprised of n amino acid residues wherein the new N-terminal amino acid residue is unmodified.

Description

REMOVAL OF N-TERMINAL ACYLATED AMINO ACID RESIDUES

FROM POLYPEPTIDES FIELD OF THE INVENTION

The present invention relates to the field of peptide sequencing, and more particularly, to a process for selectively removing N-terminal-alpha-acetylated or formylated amino acid residues from a peptide, to permit the subsequent sequential degradation of the residual peptide.

BACKGROUND OF THE INVENTION

A variety of post-synthetic enzymatic modifications of polypeptides and proteins have been reported whereby the N-terminal-alpha-amino group and various amino acid side chains are modified by acetylation, formylation, methylation and other less frequently encountered reactions. Post synthetic modification of the N-terminal-alpha-amino group can prove troublesome for the biochemical researcher, in that N-terminal modified amino acid residues are not subject to the Edman degradation, the chemical process by which polypeptides are degraded, one amino acid at a time, from the N-terminal, to determine their primary structure (amino acid sequence). The three most commonly encountered N-lerminal modifications include acetylation, formylation and pyrrolidone carboxylic acid formation (cyciization of an N-terminal glutamic acid residue). Of these modifications, the N-terminal formyl group may be removed by mild acid hydrolysis (Doolittle, R. F. in Advanced Methods in Protein Sequence Determination, Needleman, S., Ed., pages 38-54, Springer-Verlag (1973)), and the pyrrolidone ring may be cleaved enzymatically (Podell, D. N. and Abraham, G. N., Biochem. Biophys. Res. Comm., 81, 176 (1978). Efforts to remove the N-terminal acetyl group have generally proven unsuccessful to date. The methods that have been used have not been consistently successful, and there is a great demand for a reliable, efficient and simple process. As one approach, the primary structures of short N-terminal acetylated polypeptides have been determined by mass spectroscopic techniques. The N-terminal acetylation problem has been most formidable, in part, because acylation is one of the most commonly encountered post-synthetic modifications.

Although N-terminal acetylated polypeptides have proven difficult to analyze, it has been shown that N-terminal thioacetylated polypeptides, prepared by thioacetylation of an unmodified polypeptide, are subject to rapid acid catalyzed cyciization and removal of the N-terminal amino acid residue. Thioacetylating reagents have been proposed as alternatives to isothiocyanates for the sequential degradation of polypeptides from their N-terminal, and the details of their cyciization are well documented (Barrett, G. C, Chem. Comm., 487 (1967); Previero, A. and Pechere, J. F., Biochem. Biophys. Res. Comm., 40, 549 (1970); Mross, G. A. and Doolittle, R. F., Fed. Proc. 30, 1241 (1971)). Consequently, if a process could be developed whereby an N-terminal acetylated polypeptide could be converted into the corresponding N-terminal thioacetylated polypeptide, the removal of the N-terminal amino acid residue would then be possible.

In recent years, peptides containing N-terminal-alpha-thioformylated and internal thioamidc linkages (Sauve, G., Rao, V. S., Lajoie, G. and Belleau, B., Can. J. Chem., 63, 3089 (1985)), have been prepared by reaction of appropriately blocked short peptides with Lawesson's reagents (dithiophosphetane reagents)(Cava, M. P. and Levinson, M. I., Tetrahedron. 41(22), 5061 (1985)). Additionally, the regioselective use of a Lawesson's reagent in the preparation or poiypcptides containing internal thioamidated glycine residues has also been reported (Lajoie, G., Lepinc, F., Maziak, L. and Belleau, B., Tetrahedron Lett., 24(36), 3815 (1983).

However, it has not heretofore been recognized that Lawesson's reagents may be successfully and simply utilized to convert the carbonyl moeity of an alpha-acylated

N-terminal amino acid residue into the corresponding thioacylated moiety, which may then be subsequently cyclized and cleaved to leave the residual peptide.

SUMMARY OF THE INVENTION

The present invention relates to a two-step process by which an N-terminal-alpha- acetylated or formylated amino acid residue (acylated amino acid residue) may be removed from a polypeptide with the concomitant release of the residual polypeptide, wherein the N-terminal amino acid residue of the residual polypeptide is unmodified and suitable for sequential degradation for the purpose of determining its primary structure.

In the first step of the process a polypeptide comprised of n+1 amino acid residues, and containing an N-terminal-alpha-acylated amino acid residue, is reacted with a dithiophosphetane reagent, in an aprotic polar organic solvent, such as, for example tetrahydrofuran, dioxane or acetonitrile, at room temperature or at reduced temperature. Removal of the byproducts of the reaction yields a polypeptide of n+1 amino acid residues wherein the N-terminal-alpha-acylated amino acid residue has been converted into the corresponding N-terminal-alpha-thioacyiated amino acid residue.

In the second step of the process the thioacylated polypeptide is reacted with an anhydrous organic acid, at room temperature, resulting in the removal of the N-terminal amino acid residue as the 2-methylthiazol-(4H )-one or 2H -thiazoI-(4H )-one, and the concomitant release of the residual polypeptide comprised of n amino acid residues wherein the new N-terminal amino acid residue is unmodified.

It is an object of this invention to provide a simple and efficient method of removing the N-terminal-alpha-acylatcd amino acid residue of a polypeptide to permit the subsequent sequential degradation of the residual polypeptide.

It is another object of this invention is to provide a new use for dithiophosphetane reagents.

DETAILED DISCUSSION

Polypeptides suitable for removal of N-terminal-alpha-acylated amino acid residues include compounds of general formula I, wherein X is either hydrogen or methyl and preferably methyl, n is equal to or greater than 1 and is preferably less than 20, and R is representative of the various amino acid side chains.

Formula I

Reaction of a polypeptide of general formula I with a stoichiomctric equivalent of a dithiophosphetane reagent, described in more detail below, in dioxane, acetonitrile, chloroform or toluene, and preferably in tetrahydrofuran, at room temperature or at reduced temperature, and preferably in the range 0°C to 25°C, for several hours, yields a polypeptide of general formula II, wherein the N-terminal-alpha-acylated amino acid residue has been converted into the corresponding N-terminal-alpha-thioacylated amino acid residue. The byproducts of this reaction must be separated from the thioacylated polypeptide chromatographically.

Formula II

Subsequent reaction of a polypeptide of general formula II with an anhydrous organic acid, such as, for example acetic acid, formic acid or heptafluorobutyric acid and preferably trifluoroacetic acid, in an aprotic organic solvent or neat, at room temperature for several minutes, results in the removal of the N-terminal-alpha-thioacylated amino acid residue as the 2-methylthiazol-5(4H )-one or 2H -thiazol-5(4H )-one, general formula Ilia, where X is CH₃ for the former compound, and H for the latter compound, and the concomitant release of a polypeptide comprised of n amino acid residues, general formula III b, wherein the new N-terminal amino acid residue is unmodified.

Formula IlIa Formula Illb

The dithiophosphetane reagent utilized in this process is of general formula IV, wherein A may be an aryl or alkyl substituted aryl moeity, such as, for example naphthyl, phenyl or toluyl; B may be either O or S; and C may be an alkyl, aryl or alkyl substituted aryl moeity, such as, for example methyl, ethyl, phenyl or toluyl.

Formula IV

When the quantity of the dithiophosphetane reagent is limited to the addition of a single stoichiometric equivalent, with respect to the polypeptide, the reagent exhibits a high degree of regioselectivity, confining its action to the least sterically hindered amide linkage. In polypeptides of general formula I, this is the N-terminal-alpha-acylated amino acid residue. When additional equivalents of dithiophosphetane reagent are utilized, other internal amide linkages may also be converted into the corresponding thioamide linkages.

Polypeptides of general formual I wherein the amino acid side chains (R) contain nucleophilic functionalities including amino, hydroxylic, thiol and carboxylic acid functionalities must be blocked prior to reaction with the dithiophosphetane reagent, as these functionalities are known to be reactive toward the dithiophosphetane reagent. A variety of suitable blocking reactions are known in the art. Preferred blocking reactions arc those reactions which result in the introduction of carbamate, carbonate or ester moieties, as these moieties are known to be less reactive toward the dithiophosphetane reagent then are amide linkages. Both reversible and irreversible blocking reactions may be utilized, and the blocking groups may be removed prior to reaction of the thioacylated peptide with anhydrous organic acid. If acid-labile blocking groups are utilized, reaction of a polypeptide of general formula II with anhydrous organic acid will result in both the removal of the N-terminal-alpha-thioacylated amino acid residue and the removal of the blocking groups.

Polypeptides of general formula I wherein the amino acid side chains (R) contain amide linkages, such as, for example the amino acid side chains of asparagine and glutaminc, may be reactive toward the phosphetane reagent, if the steric enviornment is suitable. Reaction yields the corresponding thioamide amino acid side chains. After reaction of the thioacylated polypeptide with anhydrous organic acid, these thioamide amino acid side chains may be converted back to the corresponding amide amino acid side chains by reaction of the polypeptide with silver nitrate or by treatment of the polypeptide with a mild alkaline solution in the presence of oxygen. For a general review of various blocking reactions known in the art, which are suitable for use in conjunction with peptide sequencing, sec Tarr, G. E., in Methods of Protein Microcharaclerization, Shively, J. E. (Ed.), pages 162- 165, Humana Press (1986).

The process of removal of the N-terminal-alpha-acylated amino acid residue may be facilitated by first immobilizing the polypeptide on a solid support. In this manner, the byproducts of the reaction of the dithiophosphetane reagent may be easily removed by washing the immobilized peptide on a scintered-glass filter. The removal of the thiazolinone, which results from the reaction of the polypeptide with anhydrous organic acid, is similarly facilitated. Additionally, poiypcptides exhibiting limited solubility in those solvents suitable for use with the dithiophosphetane reagent may be processed in this manner.

Preferred solid supports include those controlled pore glass and polystyrene supports utilized in the solid phase sequencing of immobilized polypeptides. A variety of immobilization chemistries arc known in the art, and have been recently reviewed (Laursen, R. A. and Machleidt, W., Methods of Biochemical Analysis. 26, 201 (1980) and Machlcidt, W., in Modern Methods in Protein Chemistry, Tschesche, H. (Ed.), pages 303-326, de Gruyter, Berlin ( 1983)). Solid supports which contain nucleophilic functionalities on their surface after immobilization of the polypeptide must be blocked prior to reaction with the dithiophosphetane reagent. The immobilization of short polypeptides (of general formula I wherein n is less then 5) may require the use of a linker moeity between the polypeptide and the solid support so as to insure the steric accessibility of the N-terminal-alpha-acylated amino acid residue.

Polypeptides which are immobilized prior to the removal of the N-terminal-alpha-acylated amino acid residue, are suitable for sequencing by solid phase techniques immediately following reaction with anhydrous organic acid. The reaction of the thioacylated polypeptide with anhydrous organic acid may be conducted in the reaction column of a solid phase peptide sequencer as the first step of a chemical degradation cycle. The thiazolinone released by the action of anhydrous organic acid may be collected in the effluent of the reaction column and identified by amino acid analysis. EXAMPLE l

Removal of the N-terminal amino acid residue from N-acetyl-Met-Leu-Phe

The dithiophosphetane reagent 2,4-bis-(4-phenoxyphenyl)-1,3,2,4-dithiaphosphetane 2,4- disulfide, was prepared form phenyl ether and P₄S₁₀ as previously described (Lecher, H. Z., Greenwood, R. A., Whitehouse, K. C. and Chao, T. H., J. Am. Chem. Soc., 78, 5018 (1956), and recrystallized from toluene prior to use.

The chemotactic peptide, N-acetyl-Met-Leu-Phe (20 mg) was esterified by reaction with anhydrous methanolic HCI (1 ml, 1 N HCl/CH₃OH) at 50°C for 10 minutes, then dried in vacuo at room temperature for 30 minutes. The peptide methyl ester (N-acetyl-Met-Leu-Phe-OCH₃) was dissolved in tetrahydrofuran (1 ml) and cooled on an ice-water bath to 0°C. The dithiophosphetane reagent (1.1 equivalents) was added to the peptide methyl ester and the reaction was allowed to proceed for one hour at 0°C. The reaction was then removed from the ice-water bath and allowed to proceed for an additional 2 hours at room temperature. The reaction product was placed on a silica gel column (1.0 X 5.0 cm) equilibrated with hexane and the decomposition products of the phosphetane reagent were eiuted with hexane. The column was then eluted with 7:3 hexane: ethylacetate and the thioacetylated peptide methyl ester collected. The solvent was removed in vacuo, and the thioacetylated peptide methyl ester was treated with trifluoroacetic acid (I ml) for 30 minutes at room temperature. Trifluoroacetic acid was removed by evaporation under a stream of nitrogen.

Analysis of the reaction product by reverse phase high performance liquid chroma tography with fluorescence detection, after reaction of the product with 9-fIuorenyImethyIchloroformate, confirmed the presence of an analyte with a retention time identical to that of the dipeptide Leu-Phe-OCH₃, as determined by comparison with an authentic sample of the dipeptide prepared independently. The efficiency of removal of the N-terminal-alpha-acctylated amino acid residue was estimated to be 87%, as determined by integration of the chromatographic peak as compared to a known quantity of the authentic sample. The identity of the 2-methylthiazol-(4H )-one was determined by amino acid analysis.

EXAMPLE Il Removal of the N-terminal amino acid residue from immobilized N-acetyl-Met-Leu-Phe

The chemotactic peptide, N-acetyl-Met-Leu-Phe (10 mg, 45 mmol) was dissolved in N.N-dimethylformamide (1.0 ml) and activated by reaction with diisopropylcarbodiimide (6.3 mg, 50 mmol). Boc-1,4-phenylenediamine (x.x mg, xxx mmol) was added to the solution. After 30 minutes, the solvent was removed in vacuo and the elongated peptide was treated with trifluoroacetic acid (1.0 ml) for 30 minutes to remove the Boc protecting group, after removal of the trifluoroacetic acid by evaporation under a stream of nitrogen, the elongated peptide was dissolved in N,N-dimethylformamide (0.7 ml) and triethylamine (0.05 ml) and added to diisothiocyanato glass, (100 mg, 200-400 mesh, 75

Angstrom). The reaction mixture was incubated at 40°C for 2 hours with occasional agitation. The immobilized peptide was washed on a scintered-glass filter with

N,N-dimethyIformamide (20 ml) and resuspended in N,N-dimethylformamide (1.0 ml).

Propylamine (0.02 ml) was added to the mixture to block reactive isothiocyanate groups on the surface of the disiothiocyanato glass. After an additional 30 minutes of incubation at

400C, the immobilized peptide was washed on a scintered-glass filter with

N,N-dimethylformamide (20 ml), acetone (20 ml) and tetrahydrofuran (20 ml). An immobilized peptide standard was retained for subsequent analysis.

The dithiophosphetane reagent 2,4-bis-(4-phenoxyphenyl)-1,3,2,4-dithiaphosphetane 2,4-disulfide (1.2 equivalents) in tetrahydrofuran (1.5 ml) was added to the immobilized peptide, and the reaction mixture was allowed to stand for one hour, with occasional agitation, at 0°C in an ice-water bath. The reaction mixture was then removed from the ice-water bath and the reaction was allowed to proceed for an additional 2 hours at room temperature. The immobilized peptide on washed on a scintered-glass filter with tetrahydrofuran (20 ml), then treated with trifluoroacetic acid (1 ml) for 30 minutes at room temperature. The trifluoroacetic acid, containing the 2-methylthiazol-5(4H )-one, was washed from the immobilized peptide with tetrahydrofuran (20 ml).

Samples of the immobilized peptide standard and the product of the reaction with the dithiophosphetane reagent were hydrolyzed in constant boiling hydrochloric acid (6 N H Cl) for 12 hours at 120°C, then analyzed by amino acid analysis. The methionine content of the sample reacted with the dithiophosphetane reagent followed by trifluoroacetic acid was 89% less then that of the immobilized peptide standard.

EXAMPLE III

Removal of the N-terminal amino acid residue from immobilized alph-aMSH

Alpha-MSH (N-acetyl-Ser-Tyr-Scr-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂) was acylated by reaction with acetyl chloride in trifluoroacetic acid (5% v/v, 0.5 ml), for 15 minutes at room temperature, then dried in vacuo. To the residue was added methanolic hydrochloric acid (1 ml, 1 N HCl/CH₃OH), and the solution was incubated for 10 minutes at 50°C, to esterify carboxylic acid side chains, then again dried in vacuo.

The residue, after blocking of amino acid side chains, was dissolved in NN-dimethylformamide ( 1.0 ml) and triethylamine (0.05 ml) and added to diisothiocyanato glass ( 100 mg, 200-400 mesh, 75 Angstrom). The mixture was incubated for 3 hours at 40°C with occasional agitation. The immobilized peptide was washed on a scintered-glass filter with N,N-dimethylformamide (20 ml) and resuspended in N,N-dimethylformamide (1.0 ml). Propylamine (0.02 ml) was added to the mixture to block reactive isothiocyanate groups on the surface of the disiothiocyanato glass. After an additional 30 minutes of incubation at 40°C, the immobilized peptide was washed on a scintered-glass filter with N,N-dimethylformamide (20 ml), acetone (20 ml) and tetrahydrofuran (20 ml).

The dithiophosphetane reagent 2,4-bis-(4-phenoxyphenyI)-1,3,2,4-dithiaphosphetane 2,4-disulfide (1.5 equivalents) in tetrahydrofuran (1.25 ml) was added to the immobilized peptide, and the reaction mixture was allowed to stand for one hour, with occasional agitation, at 0ºC in an ice-water bath. The reaction mixture was then removed from the ice-water bath and the reaction was allowed to proceed for an additional 5 hours at room temperature. The immobilized peptide was washed on a scintered-glass filter with tetrahydrofuran (20 ml), then treated with trifluoroacetic acid (1 ml) for 30 minutes at room temperature. The trifluoroacetic acid, containing the 2-methylthiazol-5(4H )-one, was washed from the immobilized peptide with tetrahydrofuran (20 ml).

The immobilized alpha-MSH was submitted for sequential degradation in a solid phase peptide sequencer and the amino acid sequence determined through the lysine residue at position 1 1. The initial yield on sequencing, as determined by comparison with an amino acid analysis of the immobilized peptide, was 67%. The average repetitive yield for 10 cycles of sequential degradation was 93%.

Claims

1. A process for removing an N-terminal-alpha-acylated amino acid from a peptide to enable the sequential degradation of the residual peptide, comprising the steps of:

a) providing a peptide of Formula I wherein X is selected from CH₂ and H, n is 1 to 20 and R is selected from all amino acid side chains;

Formula I

b) reacting the peptide of Formula I with a stoichiometrically equivalent amount of a dithiophosphetane reagent of Formula IV, wherein A is selected from aryl, alkyl and alkyl substituted aryl moieties, B is selected from O and S, and C is selected from aryl, alkyl and alkyl substituted aryl moieties, to form an N-terminal-alpha thioacylated peptide;

Formula IV c) isolating the N-terminal-alpha-thioacylated peptide of step b;

d) treating the isolated peptide product of step c with anhydrous organic acid to remove the N-terminal amino acid residue therefrom.

2. The process of Claim 1 wherein said dithiophosphetane reagent comprises formula IV wherein A is phenyl, B is oxygen, and C is phenyl (2,4-bis-(4-phenoxyphenyl)-1,3,2,4- dithiaphosphetane 2,4-disulfide).

3. The process of Claim 1 wherein the reaction of step b is performed in a polar organic solvent at a temperature in the range of 0° to 25° Centigrade.

4. The process of Claim 3 wherein the polar organic solvent is selected from dioxane, acetonitrile, chloroform, toluene and tetrahydrofuran.

5. The process of Claim 4 wherein the polar organic solvent is tetrahydrofuran.

6. The process of Claim 3 wherein said reaction of step b is run for 1 to 12 hours.

7. The process of Claim 1 wherein R groups reactive toward said dithiophosphetane reagent are blocked prior to step b.

8. The process of Claim 1 wherein said N-terminal-alpha-thioacylated peptide product of step b is separated from the other reaction byproducts by chromotagraphy.

9. The process of Claim 1 wherein said anhydrous organic solvent is selected from acetic acid, formic acid, heptafluorabutyric acid and trifluoroacetic acid.

10. The process of Claim 9 wherein said anhydrous organic solvent is trifluoroacetic acid.

11. The process of Claim 1 wherein said reaction of step d is performed in an aprotic organic solvent.

12. The process of Claim I wherein said reaction of step d occurs at room temperature.

13. The process of Claim 1 wherein said process occurs in solution.

14. The process of Claim 1 wherein said acylated peptide is immobilized on a solid support.

15. The process of Claim 14 wherein said acylated peptide is coupled to a linker moiety and said linker moiety is immobilized on said solid support.

16. The process of Claim 15 wherein said peptide comprises less than five amino acid residues.

17. The process of Claim I further comprising the step of sequencing the residual peptide.

18. The process of Claim 1 further comprising the steps of isolating the N-terminal-alpha-acylated amino acid after removal thereof and identifying said isolated N-terminal-alpha-acylatcd amino acid.