Bifunctional activity labels for selection of filamentous bacteriophages displaying enzymes

• Published in: Bioorganic & medicinal chemistry Vol. 3; no. 7; pp. 907 - 915
• Main Authors: Vanwetswinkel, Sophie, Touillaux, Roland, Fastrez, Jacques, Marchand-Brynaert, Jacqueline
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.07.1995
• Subjects: activity label; Bacterial Proteins; Bacteriophages - genetics; beta-Lactamase Inhibitors; beta-Lactamases - analysis; beta-Lactamases - genetics; Biotin - analogs & derivatives; Biotin - chemical synthesis; biotinyl spacer; Carrier Proteins - analysis; Carrier Proteins - antagonists & inhibitors; Carrier Proteins - genetics; Cross-Linking Reagents - chemical synthesis; d-carboxypeptidase; enzyme inhibition; Enzyme Inhibitors - chemical synthesis; Hexosyltransferases; Muramoylpentapeptide Carboxypeptidase - analysis; Muramoylpentapeptide Carboxypeptidase - antagonists & inhibitors; Muramoylpentapeptide Carboxypeptidase - genetics; Penicillanic Acid - analogs & derivatives; Penicillanic Acid - chemical synthesis; Penicillin-Binding Proteins; Peptidyl Transferases; Phage enzyme; Recombinant Proteins - analysis; β-lactamase; activity label; d; enzyme inhibition; β-lactamase; d-carboxypeptidase; biotinyl spacer; Phage enzyme
• ISSN: 0968-0896; 1464-3391
• DOI: 10.1016/0968-0896(95)00084-T

Two bifunctional activity labels of β-lactamases or penicillin binding proteins have been prepared. They feature a penicillin sulfone derivative, i.e. a suicide substrate of serine β-lactamases, or a penicillin derivative connected to a biotin moiety through a spacer containing a disulfide bridge. The biotinyl spacer 4 was prepared by coupling biotin to ε-amino-caproic acid, then to cystamine, and purified by transient protection with t-Boc. The penicillin sulfone inhibitor 13 was prepared by chemoselective sulfonylation of methoxymethyl 6-aminopenicillinate with pentafluorophenoxy- or benzyloxy-carbonylmethylsulfonyl chloride ( 9), followed by permanganate oxidation. Both direct coupling of the activated ester 13b and indirect coupling of the acid 13c obtained by benzyl ester deprotection, afforded the biotinylated sulfone inhibitor 16. The acid 6 resulting from reaction of the biotinyl spacer 4 with glutaric anhydride was activated as pentafluorophenyl-ester 7 and reacted with 6-aminopenicillanic acid to afford the penicillin binding protein label 18. Selection of the most active β-lactamase displayed on phage from a mixture containing less active enzymes could be accomplished in three rounds of labeling and affinity chromatography using suicide inhibitor 16. Two bifunctional activity labels of β-lactamase,s (n=2) or PBPs (n=0) have been prepared. They feature a β-lactam inhibitor connected to a biotin moiety through a spacer (25–35 Å length) containing a disulfide bridge.