Bio-affinity Systems, Inc.

Varian Associates, Inc.

Analytichem International, Inc.

Mark L Stolowitz

Vyas M Dixit

Amy G Taketomo

Beth A Paape

Anne E DeVaux

Edward A Kesicki

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.

Method of thioacylation peptide sequencing with alcoholysis of

A protein micro sequencing method for use in conjunction with the thiocylation 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 derivatized polypeptide is subjected to cleavage by acid which affords a 2-substituted-5(4H)-thiazolinone. After removal of excess acid, the thiazolinone is reacted with a small excess of a fluorescent or enhanced ultraviolet absorbance reagent having a reactive carboxylic acid chloride, sulfonic acid chloride, chloroformate, isocyanate or anhydride functionality, in the presence of a tertiary amine catalyst, to yield the corresponding 5-O-acyl-2-(substituted)thiazole derivative, detectible by enhanced ultravoilet absorobance or fluorescence emission at extremely low concentration thereby providing a method of sequencing very small amounts of protein.

Method of thioacylation peptide sequencing with acylation of

The invention relates to the functionalization of particulate bonded phase chromatographic supports prepared by silanization of silica gel or controlled pore glass and containing pendant primary alkyl amine groups. Functionalization results from the activation of the amines by reaction with N,N'-carbonyldiimidazole (CDI), or a related azolide, in anhydrous organic solvent, followed by derivatization of the support. Derivatization results from reaction of the activated support with a functionalizing reagent consisting of a primary or secondary, alkyl or aryl amine in organic solvent, or from an aqueous solution of the amine or its salt. A urea linkage results through which the functionalizing reagent is covalently attached to the support. Derivatization can result from the addition of an excess of a single reagent, or as a consequence of the sequential addition of two or more functionalizing reagents. Chromatographic supports preparation in this manner yield materials suitable for affinity, covalent, ion exchange and hydrophobic interaction chromatography of biomolecules as well as for the preparation of immobilized reagents.

Bonded phase chromatographic supports

A solid phase N-hydroxysuccinimide reagent having the general formula: ##STR1## where X is particulate silica gel or controlled pore glass; n=3-7; Y is either (CH.sub.2).sub.n' or (CH.sub.2).sub.n' --NH--CO--CH.sub.2 wherein n'=2-6; Z may be in the form of OCOR, OSO.sub.2 R and OCO.sub.2 R, and Z' may be either Z or OH, wherein R is a chromophore, fluorophore or electrophore capable of detection in chromatographic separation technology is disclosed. Also disclosed is the method for making compounds of this type and the method for using these compounds to identify and quantitate analytes having primary or secondary amine or thiol functionalities.

Solid phase N-hydroxysuccinimide reagents

A solid phase oxime compound having the general formula: ##STR1## where X is particulate silica gel or controlled pore glass; n is 3 or 4, n' is 1-6; R' is an electron withdrawing group; and Y and Y' are selected from OH, OCOR', OSO.sub.2 R' and OCO.sub.2 R', where R' is a chromophore, fluorophore or electrophore, is disclosed. Also disclosed is the method for making compounds of this type and the method for using these compounds to detect and quantify analytes having primary or secondary amines or thiol functionalities.

Solid phase oxime reagent

A method of sequencing polypeptides is disclosed utilizing liquid-solid affinity chromotography. The method utilizes a reagent reactive at one position with the amino moiety of a terminal amino acid, and reactive at a second position with boronic acid. The reagent is coupled to the terminal amino acid. A scavenger molecule having a sulfhydryl group and an amine group reacts with the excess reagent and the excess scavenger and scavenger-reagent complex are removed on an immobilized organomercurial column. The terminal amino acid is cleaved from the polypeptide, and coupled to an immobilized boronic acid column. The amino acid is removed from the column and identified and the remainder polypeptide is recycled.

Growing community of inventors

Long Beach, CA, United States of America

Mark L Stolowitz