Selection of beta-lactamases and penicillin binding mutants from a library of phage displayed TEM-1 beta-lactamase randomly mutated in the active site omega-loop.

A combinatorial library of mutants of the phage displayed TEM-1 lactamase was generated in the region encompassing residues 163 to 171 of the active site Omega-loop. Two in vitro selection protocols were designed to extract from the library phage-enzymes characterised by a fast acylation by benzyl-penicillin (PenG) to yield either stable or very unstable acyl-enzymes. The critical step of the selections was the kinetically controlled labelling of the phages by reaction with either a biotinylated penicillin derivative or a biotinylated penicillin sulfone, i.e. a beta-lactamase suicide substrate; the biotinylated phages were recovered by panning on immobilised streptavidin. As labelling with biotinylated suicide substrates tends to select enzymes that do not turnover, a counter-selection against penicillin binding mutants was introduced to extract the beta-lactamases. The selected phage-enzymes were characterised by sequencing to identify conserved residues and by kinetic analysis of the reaction with benzyl-penicillin. Several penicillin binding mutants, in which the essential Glu166 is replaced by Asn, were shown to be acylated very fast by PenG, the acylation being characterised by biphasic kinetics. These data are interpreted by a kinetic scheme in which the enzymes exist in two interconvertible conformations. The rate constant of the conformational change suggests that it involves an isomerisation of the peptide bond between residues 166 and 167 and controls a conformation of the Omega-loop compatible with fast acylation of the active site serine residue

In vitro selection for catalytic turnover from a library of beta-lactamase mutants and penicillin-binding proteins.

A library of mutants of the RTEM beta-lactamase displayed on phage was created; it contained penicillin binding proteins (PBPs) as well as a small fraction of active beta-lactamases. The library was submitted to a selection process to extract the beta-lactamases i.e. the enzymes that turnover efficiently. This was achieved by a two steps procedure. In the first step, the beta-lactamases were labelled by reaction with a biotinylated suicide inhibitor while the PBPs were blocked by incubation in the presence of benzylpenicillin. In the second step, the labelled active phage-enzymes were separated by affinity chromatography on streptavidin coated beads. (C) 1997, Elsevier Science Ltd

Synthesis of new sulfonylamido-penicillanic acid sulfones inhibitors of beta-lactamases.

Three new sulfonylamido-penicillanic acid sulfones have been prepared by reaction of 6-aminopenicillanic esters with the monoester or monoamide derivatives obtained in nucleophilic substitution reactions by alcohol or aniline on the carboxyl chloride function of sulfoacetic dichloride followed by oxidation. These penicillin sulfones are converted to beta-lactamases suicide inhibitors by removal of the C3 ester protecting group. This synthetic strategy can give access to sulfonamidopenam sulfones bearing a variety of 6-amino side chain. These inhibitors inactivate the RTEM beta-lactamase rapidly. The kinetics of inactivation are consistent with the partitioning of an acylenzyme intermediate between two main pathways: regeneration of free enzyme and irreversible inactivation, little transient inactivation is observed. A slow inhibition by the product of enzymatic hydrolysis of the sulfones is also observed

Selection of the most active enzymes from a mixture of phage-displayed beta-lactamase mutants.

A mixture of phages displaying the wild type and four mutant Rtem beta-lactamases was submitted to three rounds of selection by incubation with a biotinylated suicide inhibitor followed by chromatography on streptavidin coated beads. The final mixture of selected phages was shown to be made up of two third of phages displaying the most active enzyme, present initially as a minor component and 25 percent of a mutant with a five fold fewer activity. This technique allows to select for catalytic efficiency. Copyright (C) 1996 Elsevier Science Lt

Solvolysis of the methoxymethyl protecting group in penicillin derivatives.

The methoxymethyl (MOM) moiety, used as protecting group for the carboxyl function of penicillin derivatives, their sulfoxides and sulfones, could be easily cleaved in aqueous methanol at room temperature. The rate of deprotection by solvolysis is not sensitive to the nature of the substituent in position 6, but depends on the state of oxidation of the thiazolidine sulfur. Copyright (C) 1996 Published by Elsevier Science Ltd

8-(n-methyl-n-tosylamino)bicyclo[4.2.0]octane-7-spiro-2'-oxacyclopropane

C17H23NO3S, M(r) = 321.4, monoclinic, P2(1)/c, a = 8.724 (3), b = 12.009 (3), c = 15.780 (4) angstrom, beta = 91.81 (3)-degrees, V = 1652.4 (9) angstrom3, Z = 4, D(x) = 1.29 g CM-3, lambda(Mo Kalpha) = 0.71069 angstrom, mu = 2.08 cm-1, F(000) = 688, T = 291 K, R = 0.071 for 1671 observed reflections. In the four-membered ring the nitrogen substituent is exo; the ring is far from planar, with a dihedral angle about one diagonal of 32 (1)-degrees

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

Synthesis and rearrangement of potential zinc beta-lactamase inhibitors.

On alkylation of the p-toluene sulfonic salt of benzyl 6-aminopenicillanate (6-APA) with benzyl 2-bromoacetate, the benzyl esters of N-(benzyloxycarbonylmethyl)-6-APA (1) and of N,N-di(benzyloxycarbonylmethyl)-6-APA (3) were obtained. Hydrogenolysis of 1 on Pd/C afforded the N (carboxymethyl)-6-APA (2) while hydrogenolysis of 3 gave a rearranged product (4). Both 2 and 4 are inhibitors of the zinc beta-lactamase II of B. cereus