On Penicillin‐Binding Protein 1b Affinity‐Labeling Reagents

Abstract

Dedicated to Professor Duilio Arigoni on the occasion of his 75th birthdayThe synthesis of some 3-aryl-3-(trifluoromethyl)3H-diazirine and benzophenone-based photoaffinity labels is reported. The photolabile group is bound to a scaffold that also accommodates functional groups to which an indicator unit (biotin) and the bioactive ligand can be attached orthogonally. To three of the labels, moenomycin was conjugated with the aim to provide tools for the identification of the moenomycin binding site within the transglycosylase domain of the enzyme PBP 1b. Some preliminary photoaffinity-labeling experiments were carried out.Introduction. ± Peptidoglycan is an essential cell-wall constituent of almost all eubacteria. It is a heteropolymer consisting of glycan strands cross-linked via short peptide chains. The final events of peptidoglycan biosynthesis at the outside of the cytoplasmic membrane are the formation of the macromolecular architecture by two major types of reaction, i) formation of the polysaccharide strands (transglycosylation) and ii) formation of peptide cross-linkages between the glycan chains (transpeptidation) (for review, see [1]). A representation of the transition state of the transglycosylation reaction is shown in Fig. 1, a [2]. Both the glycosyl donor (the growing peptidoglycan chain) and the glycosyl acceptor (the disaccharide-derived lipid II) (see [1]) are attached to the outer face of the cytoplasmic membrane via a C 55 anchor. The transglycosylation reaction is catalyzed by a number of multimodular bifunctional polymerases (that catalyze also the transpeptidation reaction) designated as class-A high-molecular-mass penicillin-binding proteins (PBPs). Of these, PBP 1b from Escherichia coli has been studied in great detail [3]. It comprises 844 amino acid residues and contains a short cytosolic N-terminus, a membrane span, the D198-G435 glycosyl-transferase module, and the Q447-N844 acyl-transferase module. Within the glycosyl-transferase module, Glu233 has been shown to be central to the transglycosylation, and, in the active site of the acyl transferase, Ser510 is essential, the acylation of which forms the basis of the antibiotic properties of the b-lactams. Recently, the soluble extracellular region of PBP 1b from Streptococcus pneumoniae has been expressed and characterized biochemically [4]. From the most-recent kinetic characterization of the E. coli PBP 1b, it was deduced that a doubly charged metal ion bound to the diphosphate group assists departure of the leaving group, whereas the essential Glu233 acts as a base, removing the proton from the 4-OH group of the glycosyl acceptor (see Fig. 1, a) [5].

Characterization of moenomycin antibiotic complex by multistage MALDI-IT/RTOF-MS and ESI-IT-MS

“…The moenomycins interfere with the transglycosylation reaction by inhibition of some membrane-bound, nonpenicillin-binding, monofunctional glycosyltransferases (Mgts) and some class A high-molecular-mass penicillin-binding proteins (HMM-PBPs) [2,3,[7][8][9][10][11][12]. Class A HMM-PBPs, such as the PBP1b from Escherichia coli, are two-domain proteins that catalyze both the transglycosylation reaction and the transpeptidation reaction.…”

“…Class A HMM-PBPs, such as the PBP1b from Escherichia coli, are two-domain proteins that catalyze both the transglycosylation reaction and the transpeptidation reaction. While ␤-lactam antibiotics bind covalently to an activesite serine in the transpeptidase domain of this bifunctional enzymes, moenomycins are assumed to act as competitive inhibitors at the transglycosylase domain [3,[7][8][9][10]. Although several antibiotics interfere with the transglycosylation reaction, moenomycins are the only compounds known to date that directly interact with the transglycosylases rather than bind to their substrates [3].…”

A Long Research Story Culminates in the First Total Synthesis of Moenomycin A

“…the transglycosylation domain of E. coli PBP 1b) are the moenomycin antibiotics. It was found that the binding of moenomycin A ( 1 ) to E. coli PBP 1b is reversible,3 and photoaffinity labeling has recently provided evidence that binding occurs indeed at the transglycosylation domain 7. Moenomycin A ( 1 ) and related antibiotics8 were discovered following extensive industrial screening in the 1960s and 1970s 9.…”

Synthesis and fungicidal activity of novel 1,3‐disubstituted 1H‐diazirine derivatives

“…From the 60s, various substituted diaziridines and diazirines were synthesized [29–43]. Halogen derivatives are ideal precursors for spectroscopic studies of carbens [44–46]; several derivatives were suggested as alkylating agents [47] and potential reagents for photolabeling of biological receptor sites [48–57], some of the substituted diaziridines and diazirines were investigated as anticancer drugs [58, 59], potential monoamine oxidase inhibitors [60, 61] and anesthetics [62, 63]. At the same time, to our astonishment, diazirine and diaziridine derivatives practically are not investigated as chemical means of plant protection [64].…”