[28] Peptide chloromethyl ketones as labeling reagents

• Published in: Methods in Enzymology Vol. 222; pp. 503 - 513
• Main Authors: Williams, E.Brady, Mann, Kenneth G.
• Format: Book Chapter Journal Article
• Language: English
• Published: United States Elsevier Science & Technology 1993
• Subjects: Amino Acid Chloromethyl Ketones - chemical synthesis; Amino Acid Chloromethyl Ketones - metabolism; Amino Acid Chloromethyl Ketones - pharmacology; Biotin; Blood Coagulation Factors - metabolism; Endopeptidases - metabolism; Fibrinolysin - metabolism; Humans; Indicators and Reagents; Spectrometry, Fluorescence - methods; Structure-Activity Relationship; Thrombin - metabolism
• ISBN: 9780121821234; 0121821234
• ISSN: 0076-6879; 1557-7988
• DOI: 10.1016/0076-6879(93)22031-A

This chapter focuses on the peptide chloromethyl ketones as labeling reagents. The synthesis of the chloromethyl ketone group generally involves the reaction of a blocked amino acid with a chloroformate to produce a mixed anhydride. In these reactions, the principal difficulties are avoiding hydrolysis of the α-chloro ketone and the cyclization reactions of the peptide derivatives. Hydrolysis of the chloromethyl ketone to the hydroxymethyl derivative can occur rapidly in basic aqueous media; and nucleophilic reagents, such as amines and thiols, can react rapidly in nonaqueous solvents. For these reasons, a major factor in designing synthesis, purification, and storage strategies is the minimization of times when the derivatives are subject to basic conditions. The most commonly used method for attaching peptides to the chloromethyl ketone-derived amino acid is mixed anhydride coupling. The rapid reaction rates, nonaqueous solvents, and minimum amounts of base required are favorable. The chapter describes the applications of labeled peptide chloromethyl ketones, such as the determining activity of protease/zymogen molecules, the exhaustive removal of protease from zymogen preparation, and the fluorescence polarization techniques.