Labeling lactam antibiotics

Proteins identified by their ability to bind labelled (3-lactam antibiotics in vivo and in vitro. The intrinsic activities of PBPs include transglycosylase/transpepti-dase, carboxypeptidase and endopeptidase activities required for the formation of the bacterial murein sacculus forming the bacterial cell wall. The enzymes are located in the cytoplasmic membrane. 

Nafcillin, oxacillin, cloxacillin, and dicloxacillin are more resistant to bacterial (3-lactamases than is penicillin G. Consequently, these antibiotics are effective against streptococci and most community-acquired penicillinase-producing staphylococci. Methicillin, which is no longer marketed in the United States, is another penicillinase-resistant antibiotic similar to nafcillin and oxacillin. For historical reasons, staphylococci resistant to oxacillin or nafcillin are labeled methicillin resistant. Many hospitals are reservoirs for MRSA and methi-cillin-resistant Staphylococcus epidermidis (MRSE). These nosocomial pathogens are resistant in vitro to all (3-lactam antibiotics. 

Fig. 7 Dose response curve (n = 9) for penicillin G (black circle) and for cephapirin red. circle) in acetonitrile-water (99 1) for imprinted polymer (MIP, 2.5 mg L 1)/labeled P-lactam antibiotic (PAAP, 250 nmol L ), adapted from [36]...

The amount of label bound to the MIP in the absence of the analyte is known as B0 and B is the amount of label bound to the MIP in the presence of each concentration of analyte. The plot of the ratio B/B0 as a function of the log[analyte], or the log [interferent], is a sigmoid curve such as the one shown in Fig. 7 for the penicillin G assay described above. As the concentration of penicillin G increases in the sample, the amount of bound PAAP decreases as does the B/B0 ratio. Another p-lactam antibiotic not derived from penicillin, such as cephapirin, did not show any cross-reactivity (Fig. 7). 

It must be underlined that, for the development of a successful FILA based on the use of non-related tracers, the latter should also show sufficient affinity for the specific binding sites of the imprinted polymer otherwise the assay will not be selective. For instance, in order to facilitate the competition between the labeled derivative and the analyte, Moreno-Bondi et al. have developed a FILA for the analysis of penicillins [34, 36] using novel fluorescently labeled [5-lactam antibiotics with a close resemblance to the analyte (Fig. 12) [95]. 

The biosynthesis of the p-lactam antibiotic penicillin (Fig. 65), and also of cephalosporin, involves incorporation of L-valine and the question arises as to which of the two diastereotopic terminal methyl groups of the valine occupies which position in the penicillin. (In the case of cephalosporin, the question is as to which methyl group is incorporated into the six-membered ring and which becomes the methylene group of the carbinyl acetate.) The problem has been solved by two groups 65d,141) by synthesis of specifically 13C methyl labeled valine (cf. Fig. 42, and p. 35) which was then biosynthetically incorporated in the antibiotics. The position of the 13C in the resulting antibiotic molecules was determined by 13C NMR spectroscopy. 

Penem[2- C] FCE 22101, 219, a j -lactam antibiotic, has been synthesized in eight steps in overall radiochemical yield of 21%, 98% radiochemical purity and specific activity of 641 MBqmmoP (17.3 mCi mmol ) using sodium salt of [1- C]glycolic acid as the starting labelled material (equation 91). The acetoxymethyl ester FCE 22891 220 has been prepared in 41% yield by condensing 219 with bromomethyl acetate. 

Since cephalexin is transported by the dipeptide transport system [75,133], the question arose whether or not the reduction of cephalexin transport activity following CPH treatment could be caused by either reduction in the number of transport sites or an impairment of the transport system for -lactam antibiotics and dipeptides 77]. Using photoaffinity labeling, two membrane polypeptides of brush border membrane of molecular weight of 130,000 and 95,000 were identified as constituents of the dipeptide transport system 77]. The results of this study demonstrated that CPH-treatment of rats greatly reduced the photoaffinity la-... 

Synthesis of samples of L-cysteine 134 stereospecifically labeled with tritium was first achieved by ourselves in connection with studies on the biosynthesis of the -lactam antibiotics penicillin and cephalosporin (133, 134). The key steps in this synthesis were conversion of the imines 130 and 130, = H, to... 

The technique of carbon-13 labelling in the study of antibiotic biosynthesis utilising NMR detection methods has been reviewed with specific reference to the -lactam antibiotics [204). The biosynthesis of penicillins and cephalosporins has recently been reviewed and stable isotopes have played a significant role in these studies [208]. 

Whitney, J. G., D. R. Brannon, J. A. Mabe, and K. J. Wicker Incorporation of Labelled Precursors into A16886B, a Novel P-Lactam Antibiotic Produced by Streptomyces clavuligerus. Antimicrob. Ag. Chemother. 1, 247 (1972). 

Staub I, Sieber SA (2008) P-Lactams as selective chemical probes for the in vivo labelling of bacterial enzymes involved in cell wall biosynthesis, antibiotic resistance, and virulence. J Am Chem Soc 130 13400-13409... 

Another study tested to what extent antibiotics affect the membrane integrity of Escherichia coli and hence inhibit the ChemChrome V6 labelhng. Inhibition of the fluorescent staining was only observed for membrane permeabilizing antibiotics, even at concentrations below the MIC but not for antibiotics with other mechanisms of action e. g. 6-lactams. As an application, colistin could be determined in milk by measuring the decrease in the number of labelled Escherichia coli cells relative to the initial number that had been added to the milk (D Haese and Nelis 2000). 

Penicillins and cephalosporins contain a P-lactam ring, which is fused with a thiazolidine ring. These types of compounds are synthesized by both prokaryotic and eukaryotic microorganisms, including Streptomyces, Penicillium, Aspergillus, and Cephalosporium species (Luckner, 1990). Important precursors are L-2-aminoadipic acid, L-cysteine, and L-valine. An important intermediate is 6-(L-aminoadipyl)-L-cysteinyl-D-valine (Luckner, 1990). The valine unit with a d-configuration found in one portion of the molecule is labeled when radioactively labeled L-valine is added to the culture. The most important antibiotic of this group, penicillin G (3) from Penicillium spp., is derived from similar precursors. 

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