The total synthesis of the biologically active, dextrorotatory enantiomer of 3-methyl-7(beta)-phenylacetamido-(DELTA)('3)-O-2-isocephem-4-carboxylic acid was accomplished. The key step involved the asymmetric cycloaddition of azidoacetyl chloride to the cinnamylidene Schiff base of protected D-threonine to generate the desired monocyclic cis (beta)-lactam diastereomer (9:1). The absolute configuration of the final product was confirmed by comparing its antimicrobial activity with that of the corresponding racemate. / The influence of (a) the (beta)-chiral center in the starting (alpha)-amino acid, (b) the bulk of the carboxylic acid and (c) the distribution of bulk throughout the imine on the stereochemical outcome of the reaction was studied. The absence of racemization during the cycloaddition was demonstrated by the use of deuterated precursors. The great potential of D-glucosamine derivatives as chiral templates in this reaction was clearly illustrated.

No description available.

No description available.

Spermine (Spm), a potent bactericidal polyamine, exerts a strong synergistic effect with β-lactams against methicillin-resistant Staphylococcus aureus (MRSA) in a pH-dependent manner. At high pH (>8) Spm is a potent nucleophile, and able to form Spm-β-lactam adduct. At physiological pH (or lower), Spm carries positive charges, and can bind to DNA through charge interactions. The potential of Spm interfering with cell wall was first investigated. A spontaneous mutant of MRSA Mu50 selected for Spm resistance conferred resistance to Spm/β-lactam synergy. This phenotype was due to the presence of a 7-bp deletion in pbpB as identified by genome resequencing and confirmed by complementation. Analysis of cell wall composition by HPLC revealed the combination of Spm and β-lactam can reduce the cross-linkage of peptidoglycan. These two lines of evidence suggest Spm may perturb cell wall integrity in favor of β-lactam efficacy with PBPs as a promising target. However, from the results of microarray analysis and fluorescent Bocillin labeling, Spm did not appear to suppress the PBPs expression or alter their interactions with β-lactams. Next, transcriptome analyses reveal the genes responsive to the synergy effect overlap extensively with those to high Spm challenge, implying the enhanced detrimental effect of Spm facilitated by β-lactams in inhibition on cell growth. In particular the induction of iron transport and reduction of energy production under synergy were depicted in this study, and high dose Spm was found to turn off the SigB regulon. Of interest, the tetM gene encoding a ribosomal protection protein for tetracycline (Tc) resistance exhibited the most significant fold change and high signals by both high and low dose Spm. Further analysis by qRT-PCR demonstrated the tetM expression was specifically induced by Tc and Spm to a comparable level but not by other polyamines, suggesting a similar mode of action by Spm and Tc in interactions with the ribosome to initiate tetM induction. Collectively, these data indicated the role of Spm could be multifarious with more than one target, and a combination of Spm and β-lactams may inhibit growth of MRSA in a more complicated manner than just potentiating β-lactam inhibition on PBPs.

This thesis is concerned with the synthesis of functionalised piperidines. The piperidine unit is found in numerous alkaloids and other natural products, and has also been incorporated into synthetic compounds of significant therapeutic or strategic synthetic potential. Although numerous syntheses of individual piperidine compounds have been published, there are few general routes to this class of compounds, and there is therefore a need for such routes to be developed. Bicyclic lactams derived from (S)-pyroglutamic acid have been shown to be useful synthons in the synthesis of pyrrolidines. An analogous bicyclic lactam was therefore synthesised from (S)-lysine. Alkylations at the carbon atom a- to the lactam carbonyl group were studied in detail. A number of different groups could be introduced in good to excellent yield, and with moderate to good stereoselectivity. The selectivity of these reactions was believed to depend on the shape of the bicyclic system, and a stereoelectronic effect of the nitrogen lone pair. The bicyclic lactam was further elaborated to an activated enone. This was shown to be relatively unreactive to cycloaddition reactions. It is believed that this is because the activating ester group is not conjugated with the carbon-carbon double bond. However, conjugate addition could be achieved in high yield and excellent stereoselectivity using a zinc enolate Reformatsky reagent. An epoxide could also be formed in excellent yield and with excellent stereoselectivity. The Reformatsky adduct was shown to be a suitable substrate for a Pb(IV)-mediated arylation reaction. Examples of the functionalised lactams were deprotected to give hydroxymethyl piperidinones and cyclic amino acids. This route therefore allows access to the desired novel compounds.

Call number: LD2668 .T4 1984 V47 / Master of Science

The penicillin tolerance of ammo acid-deprived re/A+ Escherichia coli is attributed to the stringent response. The β-lactam-induced lysis of amino acid-deprived bacteria resulting from relaxation of the stringent response was inhibited by cerulenin, or by glycerol deprivation in the case of a gpsA mutant (defective in the biosynthetic snglycerol 3-phosphate dehydrogenase). Therefore, β-lactam-induced lysis of amino acid-deprived cells was dependent on phospholipid synthesis. Both the priming and the lysis induction stages of β-lactam-induced lysis were shown to require phospholipid synthesis. It has been known for some time that phospholipid synthesis is inhibited by the stringent reponse. These results indicate that the inhibition of peptidoglycan synthesis and the induction of penicillin tolerance during the stringent response are both secondary consequences of the inhibition of phospholipid synthesis. Direct experimental evidence is presented for the first time indicating that the penicillin tolerance of amino acid-deprived E coli was directly attributable to action of guanosine 3',5'-bispyrophosphate (ppGpp) and not to some other effect of amino acid deprivation. The overproduction of ppGpp resulted in the inhibition of peptidoglycan and phospholipid synthesis and in penicillin tolerance. Penicillin tolerance and the inhibition of peptidoglycan synthesis were both suppressed when ppGpp accumulation was prevented by treatment of the bacteria with chloramphenicol, an inhibitor of ppGpp synthetase I activation. Glycerol-3-phosphate acyltransferase, the product of plsB gene, was recently identified as the main site of ppGpp inhibition in phospholipid synthesis. The overexpression of the cloned plsB gene reversed the penicillin tolerance conferred by ppGpp accumulation. This also indicates that the membrane-associated events in peptidoglycan metabolism were dependent on ongoing phospholipid synthesis. Interestingly , treatment with β-lactam antibiotics by itself induced re/A-dependent ppGpp accumulation, but the maximum levels attained were insufficient to confer penicillin tolerance. It was al so demonstrated that penicillin tolerance was induced when phospholipid synthesis was inhibited in normal growing E. coli. This penicillin tolerance was not the result a simple inhibition of growth or a decrease in the membrane levels of individual phospholipids (e.g., acidic phospholipids), but rather the direct result of the inhibition of net phospholipid synthesis. A number of factors that interfere with β-lactam-induced lysis were investigated. (i) It was demonstrated that de-energization of the E. coli cytoplasmic membrane resulted in penicillin tolerance due to the inhibition of both the priming and the lysis induction stages. (ii) Inhibition of protein synthesis in the absence of the stringent response promoted both the priming and the lysis induction stages resulting in a faster onset of β-lactam-induced lysis. (iii) The temperature sensitivity of β-lactam-induced lysis in amino acid-deprived E. coli was re-investigated. Penicillin tolerance resulting from a temperature up-shift was not due to the induction of the heat-shock response, as previously reported, but from a reversible inhibition of unidentified thermosensitive enzyme(s) involved in the lysis induction stage. / Graduate

Typescript Includes 1 report by the author Includes bibliographical references The scope of the Reformatsky reaction of imines with a_-bromoesters and zinc has been investigated. The stereochemistry 2-azetidinones (β_-lactams) formed has been determined by nuclear magnetic resonace spectroscopy.

We have successfully developed an efficient approach to 3,4-disubstituted £\,£]-unsaturated £_-lactams from glutarimide, and proved to be applicable for the synthesis of hydrocinchonine and hydrocinchonidine.

Ph.D.