Maxime Lampilas

The catalytic, glycosyl transferase and acyl transferase modules of the cell wall peptidoglycan‐polymerizing penicillin‐binding protein 1b of Escherichia coli

The penicillin‐binding protein (PBP) 1b of Escherichia coli catalyses the assembly of lipid‐transported N‐acetyl glucosaminyl‐β‐1,4‐N‐acetylmuramoyl‐l‐alanyl‐γ‐d‐glutamyl‐(l)‐meso‐diaminopimelyl‐(l)‐d‐alanyl‐d‐alanine disaccharide pentapeptide units into polymeric peptidoglycan. These units are phosphodiester linked, at C1 of muramic acid, to a C55 undecaprenyl carrier. PBP1b has been purified in the form of His tag (M46‐N844) PBP1bγ. This derivative provides the host cell in which it is produced with a functional wall peptidoglycan. His tag (M46‐N844) PBP1bγ possesses an amino‐terminal hydrophobic segment, which serves as transmembrane spanner of the native PBP. This segment is linked, via an ≅ 100‐amino‐acid insert, to a D198‐G435 glycosyl transferase module that possesses the five motifs characteristic of the PBPs of class A. In in vitro assays, the glycosyl transferase of the PBP catalyses the synthesis of linear glycan chains from the lipid carrier with an efficiency of ≅ 39 000 M−1 s−1. Glu‐233, of motif 1, is central to the catalysed reaction. It is proposed that the Glu‐233 γ‐COOH donates its proton to the oxygen atom of the scissile phosphoester bond of the lipid carrier, leading to the formation of an oxocarbonium cation, which then undergoes attack by the 4‐OH group of a nucleophile N‐acetylglucosamine. Asp‐234 of motif 1 or Glu‐290 of motif 3 could be involved in the stabilization of the oxocarbonium cation and the activation of the 4‐OH group of the N‐acetylglucosamine. In turn, Tyr‐310 of motif 4 is an important component of the amino acid sequence‐folding information. The glycosyl transferase module of PBP1b, the lysozymes and the lytic transglycosylase Slt70 have much the same catalytic machinery. They might be members of the same superfamily. The glycosyl transferase module is linked, via a short junction site, to the amino end of a Q447‐N844 acyl transferase module, which possesses the catalytic centre‐defining motifs of the penicilloyl serine transferases superfamily. In in vitro assays with the lipid precursor and in the presence of penicillin at concentrations sufficient to derivatize the active‐site serine 510 of the acyl transferase, the rate of glycan chain synthesis is unmodified, showing that the functioning of the glycosyl transferase is acyl transferase independent. In the absence of penicillin, the products of the Ser‐510‐assisted double‐proton shuttle are glycan strands substituted by cross‐linked tetrapeptide–pentapeptide and tetrapeptide–tetrapeptide dimers and uncross‐linked pentapeptide and tetrapeptide monomers. The acyl transferase of the PBP also catalyses aminolysis and hydrolysis of properly structured thiolesters, but it lacks activity on d‐alanyl‐d‐alanine‐terminated peptides. This substrate specificity suggests that carbonyl donor activity requires the attachment of the pentapeptides to the glycan chains made by the glycosyl transferase, and it implies that one and the same PBP molecule catalyses transglycosylation and peptide cross‐linking in a sequential manner. Attempts to produce truncated forms of the PBP lead to the conclusion that the multimodular polypeptide chain behaves as an integrated folding entity during PBP1b biogenesis.

Convergent stereospecific total synthesis of monochiral Monocillin I related macrolides

Abstract The first total synthesis of Monocillin I related macrolides has been achieved by a convergent and stereospecific route involving the palladium - catalyzed coupling of a functionnalized chiral vinylstannane with the appropriate dimethoxy bromomethyl isocoumarin. Cleavage conditions of the isocoumarin ring were then found for the preparation of a precursor hydroxy acid. The 14-membered macrolide was obtained in the Mitsunobu reaction conditions, and the required natural conjugated dienone epoxide system of Monocillin I was generated stereospecifically from that macrocyclic precursor.

Synthesis of a trisaccharide analogue of moenomycin A12 Implications of new moenomycin structure-activity relationships

Abstract A trisaccharide analogue of moenomycin A, 9a, has been synthesized and has found to be antibiotically inactive. This compound differs from an active compound, 9b, solely by the exchange NHAc→OH in unit C. A binding model for moenomycin-type transglycosylase inhibitors at the enzyme penicillin binding protein is proposed.

Convergent stereospecific total synthesis of Monocillin I and Monorden (or Radicicol)

Abstract The first total syntheses of the antifungal resorcylic macrolides Monocillin I and Monorden (or Radicicol) have been achieved by a convergent stereospecific route. TBDMS phenol ethers were found to be suitable for all the scheme and were removed in the ultimate step under mild conditions (aqueous borax/THF/methanol), hence allowing to get the natural macrolides in good yields, with no degradation. An efficient conversion of di-OTBMDS Monocillin I into Monorden is also reported.

Some selective reactions of moenomycin A.

A number of new moenomycin A derivatives have been prepared. Their antibiotic properties highlight the very specific recognition of moenomycin A at the transglycosylase binding site which is the basis of the transglycosylase inhibiting property of moenomycin A (4a).

Moenomycin analogues with modified lipid side chains from indium-mediated Barbier-type reactions

Abstract From moenomycin A both the chromophore part and the lipid side chain were degraded by ozonolysis to give an analogue with a glycolaldehyde unit in 2-position of the glyceric acid moiety. The aldehyde was converted to a number of homoallylic alcohols by indium-mediated Barbier-type reactions with allylic and benzylic halides. With exception of the phytyl bromide-derived reaction product all compounds were antibiotically inactive.