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WO1989001487A1 - Elimination d'acides amines acyles residuels a terminal-n dans des polypeptides - Google Patents

Elimination d'acides amines acyles residuels a terminal-n dans des polypeptides Download PDF

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Publication number
WO1989001487A1
WO1989001487A1 PCT/US1988/002761 US8802761W WO8901487A1 WO 1989001487 A1 WO1989001487 A1 WO 1989001487A1 US 8802761 W US8802761 W US 8802761W WO 8901487 A1 WO8901487 A1 WO 8901487A1
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Prior art keywords
amino acid
terminal
peptide
alpha
reaction
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PCT/US1988/002761
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English (en)
Inventor
Mark L. Stolowitz
Beth A. Paape
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Bio Affinity Systems Inc
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Bio Affinity Systems Inc
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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
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C07K14/68—Melanocyte-stimulating hormone [MSH]
    • C07K14/685—Alpha-melanotropin
    • 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
    • C07K1/122—Hydrolysis with acids different from HF
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • 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).
  • N-lerminal modifications include acetylation, formylation and pyrrolidone carboxylic acid formation (cyciization of an N-terminal glutamic acid residue).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • aprotic polar organic solvent such as, for example tetrahydrofuran, dioxane or acetonitrile
  • 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 another object of this invention is to provide a new use for dithiophosphetane reagents.
  • 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.
  • the byproducts of this reaction must be separated from the thioacylated polypeptide chromatographically.
  • 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.
  • the reagent 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.
  • amide linkages such as, for example the amino acid side chains of asparagine and glutaminc
  • 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.
  • 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.
  • 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.
  • 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 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.
  • the dithiophosphetane reagent 2,4-bis-(4-phenoxyphenyl)-1,3,2,4-dithiaphosphetane 2,4- disulfide was prepared form phenyl ether and P 4 S 10 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/CH 3 OH) 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-OCH 3 ) 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.
  • 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.
  • 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
  • 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
  • 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.
  • Alpha-MSH (N-acetyl-Ser-Tyr-Scr-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH 2 ) 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/CH 3 OH), and the solution was incubated for 10 minutes at 50°C, to esterify carboxylic acid side chains, then again dried in vacuo.
  • 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 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.

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  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
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  • Toxicology (AREA)
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Abstract

Selon un procédé à deux étapes d'élimination d'un acide aminé alpha-acylé résiduel à terminal-N dans un polypeptide et de libération concomitante du polypeptide résiduel, on obtient un acide aminé résiduel à terminal-N non-modifié et propre à subir une dégradation chimique à des fins de détermination de sa structure primaire. Pendant la première étape du procédé, on fait réagir un polypeptide composé de n + 1 acides aminés résisuels et contenant un acide aminé alpha-acylé résiduel à terminal-N avec un réactif dithiophosphétane, dans du tétrahydrofurane, du dioxane ou de l'acétonitrile, à la température ambiante ou à une température réduite. Une fois les sous-produits de la réaction éliminés, on obtient un polypeptide ayant n + 1 acides aminés résiduels, dans lequel l'acide aminé alpha-acylé résiduel à terminal-N s'est converti dans l'acide aminé alpha-thioacylé résiduel correspondant à terminal-N. Pendant la deuxième étape du procédé, on fait réagir le polypeptide thioacylé avec un acide anhydre organique, à la température ambiante, ce qui entraîne l'élimination de l'acide aminé résiduel à terminal-N sous la forme de 2-méthylthiazol-5(4H)-one, ainsi que la libération concomitante du polypeptide résiduel composé de n acides aminés résiduels, où le nouvel acide aminé résiduel à terminal-N n'est pas modifié.
PCT/US1988/002761 1987-08-10 1988-08-09 Elimination d'acides amines acyles residuels a terminal-n dans des polypeptides Ceased WO1989001487A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US083,860 1979-10-11
US8386087A 1987-08-10 1987-08-10

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WO1989001487A1 true WO1989001487A1 (fr) 1989-02-23

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AU (1) AU2388688A (fr)
WO (1) WO1989001487A1 (fr)

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DOOLITTLE, Analytical Biochemistry, 78, 491-505, Published 1977. *
JENSEN, Tetrahedron, 42 (23), 6555-6564, Published 1986. *

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