THE DEVELOPMENT OF A NEW, SPECIFIC PEPTIDE CLEAVAGE AT ASPARAGINE AND GLUTAMINE (DIAMINOBUTONIC ACID, DIAMIN OPROPANOIC ACID, IODOBENZENE DIACETATE)

JAMES KARL BLODGETT, Purdue University

Abstract

Obtaining the amino acid sequence of a protein is an important first step in the elucidation of its structure. The ability to cleave a polypeptide chain at specific locations for the purpose of generating a small number of large fragments is of critical importance in sequence analysis. That is, both the sequencing operation and eventual overlap studies necessary for sequence reconstruction are aided tremendously by such cleavages. The possibility of using asparagine and glutamine residues as sites for the specific cleavage of polypeptides has been examined in a variety of synthetic asparagine- and glutamine-containing model peptides. The specific cleavage process begins with an obligatory prior rearrangement of asparagine and glutamine residues to 2,3-diaminopropanoic acid (DAPA) and 2,4-diaminobutanoic acid (DABA) residues, respectively. This reaction, which is an acidic version of the Hofmann rearrangement of primary amides, is effected in near quantitative yield with I,I-bis(trifluoroacetoxy)iodobenzene. Alternatively, the reaction can be carried out with iodobenzene diacetate, which is the precursor to the trifluoroacetoxy-reagent. Following rearrangement, intramolecular transfer of the amino acid acyl residue from the (alpha)-amino group to the (beta)- or (gamma)-amino group of DAPA or DABA, respectively, is induced by exposing DAPA- and DABA-containing peptides to basic (pH 9-10) conditions for 24-30 hours at 60(DEGREES)C. This "transpeptidation" process, which is an equilibrium reaction with K(,eq) (TURN) 4, requires buffers known to be bifunctional catalysts (e.g. bicarbonate and phosphate). Subsequent application of the Edman degradation chemistry produces 60-80% cleavage yields in DAPA- and DABA-containing peptides. Cleavage of peptides at non-transpeptidized DAPA residues is possible when the cyclization step of the Edman degradation is run at 60(DEGREES)C in heptafluorobutyric acid. After 10 hours of reaction at 60(DEGREES)C, 50-70% cleavage occurs in DAPA-containing peptides and no cleavage occurs in DABA-containing peptides.

Degree

Ph.D.

Subject Area

Biochemistry

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