US4863870A
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Method of thioacylation peptide sequencing with alcoholysis of thiazolinones
Assignee: BIO-AFFINITY SYSTEMS Inc A CORP OF CA
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Filing: 1988-03-10
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Granted: 1989-09-05
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Expires: 2008-03-10
Abstract
Abstract A sequencing method which exploits the thioacylation degradation of polypeptides and proteins is disclosed. The process involves reaction of the N-terminal amino acid of a polypeptide with an excess of a thioacylating reagent. After sufficient time to insure quantitative coupling and removal of excess reagent, the N-thioacyl polypeptide is subjected to cleavage by acid which affords a 2-substituted-5(4H)-thiazolinone of the N-terminal amino acid. Subsequent addition of a large excess of an aliphatic primary or secondary alcohol, either directly to the cleavage acid or after its removal, yields the corresponding N-thioacyl amino acid ester, a stable compound suitable for chromatographic separation and subsequent detection by contemporary methods of high pressure liquid chromatography.
Inventors
- 👤 Mark L. Stolowitz
- 👤 Beth A. Paape
- 👤 Anne E. DeVaux
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 08:41
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/68; G01N33/6803; G01N33/6842; C; C07; C07K; C07K1/00; C07K1/12; C07K1/122; C07K1/128; G01N33/6818; G01N33/6824; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/141111
Claims Analysis
Total Claims: 14
Claim 1
Independent Claim
163 words
A method for determining the identity of an N-terminal amino acid of a polypeptide comprising the steps of: (a) providing a starting polypeptide having an N-terminal amino acid; (b) reacting said polypeptide with a thioacylating reagent of the form X-C(S)W wherein X is aryl or aryl-alkyl and W is a leaving group, to form a compound having the formula IV, wherein R represents an amino acid side chain and -(peptide) represents the starting peptide, not including the N-terminal amino acid; ##STR7## (c) cleaving the compound of formula IV with a volatile anhydrous acid to form a residual polypeptide, which comprises the starting polypeptide with the N-terminal amino acid removed, and a 2-substituted-5(4H)-thiazolinone compound of general formula V; ##STR8## (d) reacting the compound of general formula V with an aliphatic primary or secondary alcohol to yield a corresponding N-thioacyl amino acid ester of general formula VI wherein R' is an alkyl substituent; ##STR9## (e) identifying the N-thioacyl amino acid ester of general formula VI.
Claim 2
Independent Claim
163 words
A method for determining the identity of an N-terminal amino acid of a polypeptide comprising the steps of: (a) providing a starting polypeptide having an N-terminal amino acid; (b) reacting said polypeptide with a thioacylating reagent of the form X-C(S)W wherein X is aryl or aryl-alkyl and W is a leaving group, to form a compound having the formula IV, wherein R represents an amino acid side chain and -(peptide) represents the starting peptide, not including the N-terminal amino acid; ##STR7## (c) cleaving the compound of formula IV with a volatile anhydrous acid to form a residual polypeptide, which comprises the starting polypeptide with the N-terminal amino acid removed, and a 2-substituted-5(4H)-thiazolinone compound of general formula V; ##STR8## (d) reacting the compound of general formula V with an aliphatic primary or secondary alcohol to yield a corresponding N-thioacyl amino acid ester of general formula VI wherein R' is an alkyl substituent; ##STR9## (e) identifying the N-thioacyl amino acid ester of general formula VI.
Claim 3
Dependent Claim
18 words
The method of claim 1 wherein said polypeptide is immobilized on a solid support prior to step b.
Claim 4
Dependent Claim
32 words
The method of claim 1, wherein said thioacylating reagent is selected from the group consisting of X-C(S)SR', X-C(S)OR' and X-C(S)NR'R" wherein R' and R" are alkyl, aryl, acyl or substituted derivatives thereof.
Claim 5
Dependent Claim
25 words
The method of claim 3 wherein said thioacylating reagent is selected from the group consisting of carboxymethyl dithiobenzoic acid, cyanomethyl dithiobenzoate and carboxymethyl dithiophenylacetic acid.
Claim 6
Dependent Claim
16 words
The method of claim 1 wherein step b occurs in the presence of excess thioacylating reagent.
Claim 7
Dependent Claim
15 words
The method of claim 5 further comprising removing excess thioacylating reagent prior to step c.
Claim 8
Dependent Claim
25 words
The method of claim 1 wherein step b occurs in the presence of an acid scavenger in an alkaline aqueous solvent or an anhydrous solvent.
Claim 9
Dependent Claim
13 words
The method of claim 1 wherein said volatile anhydrous acid is trifluoroacetic acid.
Claim 10
Dependent Claim
27 words
The method of claim 1 further comprising separating the compound of formula V from said residual polypeptide and separating said acid from said compound of formula V.
Claim 11
Dependent Claim
24 words
The method of claim 1 wherein said aliphatic primary or secondary alcohol is selected from the group consisting of methanol, ethanol, 2-propanol and n-propanol.
Claim 12
Dependent Claim
14 words
The method of claim 10 wherein said aliphatic primary or secondary alcohol is methanol.
Claim 13
Dependent Claim
13 words
The method of claim 1 wherein X is phenyl, m-nitrophenyl, benzyl or p-nitrobenzyl.
Claim 14
Dependent Claim
9 words
The method of claim 12 wherein X is phenyl.
References Cited (Backward Citations)
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