Raymond J. Bowers

Enzymatic synthesis of the penicillin and cephalosporin nuclei from an acyclic peptide containing carboxymethylcysteine.

The tripeptide delta-(L- carboxymethylcysteinyl )-L-cysteinyl-D-valine (L-CMC-CV) is converted sequentially into the CMC analog of isopenicillin N, the CMC analog of penicillin N, and the CMC analog of desacetoxycephalosporin C by, respectively, isopenicillin N synthetase, isopenicillin N epimerase, and desacetoxycephalosporin C synthetase, all isolated from the beta-lactam producing prokaryote Streptomyces clavuligerus.

Synthesis and characterization of N, N-dichlorinated amino acids: Taurine, homotaurine, GABA and L-leucine

Epilepsy, trauma and other circumstances leading to hyperexcitable conditions in the CNS tend neurochemically to be associated with excessive stimulated release of glutamic acid and/or a failure of GABA modulated inhibition. Somewhat to a lesser extent, taurine and its homologue homotaurine, have also been shown to antagonize the excitatory actions of glutamic acid. Here we report the successful synthesis and isolation in pure form of N,N-dichlorinated GABA, taurine, homotaurine and leucine. These compounds are much more lipophilic than their parent compounds and may therefore more readily penetrate the blood-brain barrier systems into the neural tissue, where they can be easily dechlorinated. Very preliminary biological testing shows that this may indeed occur. The synthesis and purification methodology will likely also be applicable to a number of other amino acids as well as certain peptides or selected proteins.

Oxidation of vinyl carbamate and formation of 1,N6-ethenodeoxyadenosine in murine lung.

Vinyl carbamate (VC) is derived from ethyl carbamate, a carcinogen formed in fermentation of food and alcoholic products. We have undertaken studies to test the hypothesis that an epoxide generated from VC oxidation leads to formation of 1,N6-ethenodeoxyadenosine (ϵdAS). We have developed approaches using liquid chromatography-mass spectrometry and liquid chromatographytandem mass spectrometry for identification and quantitation of ϵdAS. Scanning and fragment ion analyses confirmed the identity of ϵdAS based on the molecular ion [M + H]+ m/z 276 and the specific fragment ion m/z 160. Chemical oxidation of VC in reactions containing 2′-deoxyadenosine produced ϵdAS with 1H NMR, chromatographic, and mass spectral characteristics identical to those of the authentic ϵdAS, suggesting DNA alkylation by the VC epoxide. Subsequent studies evaluated formation of ϵdAS in incubations of murine lung microsomes or recombinant CYP2E1 with VC. The formation of ϵdAS in incubations of lung microsomes or recombinant CYP2E1 with VC was dependent on protein concentrations, CYP2E1 enzyme levels, and incubation time. The rates of ϵdAS formation were highly correlated with VC concentrations. Peak rates were produced by lung microsomes and recombinant CYP2E1 at 3.0 and 2.5 mM VC, respectively. In inhibitory studies, incubations of VC were performed using lung microsomes from mice treated with the CYP2E1 inhibitor diallyl sulfone (100 mg/kg, p.o.). Results from these studies showed significantly decreased ϵdAS formation in microsomes incubated with VC, with an inhibition of 70% at 3.0 mM. These findings suggested that CYP2E1 is a major enzyme mediating VC oxidation, leading to the formation of a metabolite that alkylates DNA to form the ϵdAS adduct.

Non-radioactive assay and HPLC analysis of cephalosporin C 7α-methoxylation by cell-free extracts of Streptomyces clavuligerus

SummaryEscherichia coli W3110 was found to be 50 times more sensitive to cephamycin C than to cephalosporin C, and also markedly more sensitive to 7α-methoxycephalosporin C than to 7α-hydroxycephalosporin C. Accordingly, this organism could be used to establish a bioassay of cephalosporin C 7α-methoxylation by a Streptomyces clavuligerus cell-free extract. The bioassay results were complemented by HPLC analysis. Modification of the mobile phase from 100 mm NaH2PO4, pH 4.2, to 200 mm, pH 4.0, improved HPLC resolution such that an unidentified peak could be separated from the 7α-hydroxycephalosporin C peak, and the formation of this intermediate as well as the product, 7α-methoxycephalosporin C, could be directly measured in the cell-free reaction system.