Bifunctional activity labels for selection of filamentous bacteriophages displaying enzymes.
Two bifunctional activity labels of beta-lactamases or penicillin binding proteins have been prepared. They feature a penicillin sulfone derivative, i.e. a suicide substrate of serine beta-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 epsilon-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 beta-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.
Vanwetswinkel
S
S
Laboratoire de Biochimie Physique et des Biopolymères, Université Catholique de Louvain, Belgium.
Touillaux
R
R
Fastrez
J
J
Marchand-Brynaert
J
J
D001426:Bacterial Proteins; D001435:Bacteriophages / Q000235:genetics*; D001710:Biotin / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D002352:Carrier Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics; D003432:Cross-Linking Reagents / Q000138:chemical synthesis*; D004791:Enzyme Inhibitors / Q000138:chemical synthesis*; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics; D010397:Penicillanic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D011994:Recombinant Proteins / Q000032:analysis; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000032:analysis* / Q000235:genetics
C096830:6-((biotinyl-N-epsilon-aminocaproyl-N-cystamine-N'-carbonyl)methylsulfonyl)aminopenicillanic acid S,S-dioxide; C096832:6-(biotinyl-N-epsilon-aminocaproyl-N-cystamine-N'-glutaryl)aminopenicillanic acid; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; D065093:beta-Lactamase Inhibitors; D001710:Biotin; D010397:Penicillanic Acid; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D001618:beta-Lactamases