Abstract

One solution to the ever-growing problem of antibiotic resistance is antivirulence, a process which aims to disarm bacteria rather than kill them. A common virulence factor is the formation of biofilms, in which bacteria come together to form a community that protect themselves from antibiotics. Biofilms form through communication between bacteria by a process known as quorum sensing. During quorum sensing, bacteria synthesize and secrete signaling molecules known as autoinducers. Once a certain concentration threshold of autoinducers is reached, the bacteria begin to express genes that lead to biofilm formation. The synthesis of one quorum sensing molecule, autoinducer-2, is catalyzed by the enzyme LuxS, which is conserved throughout many species of bacteria. This reaction involves a cleavage of the natural substrate, S-ribosyl-L-homocysteine, into L-homocysteine and 4,5-dihydroxy-2,3-pentanedione. Inhibition of this reaction has previously succeeded through mimicking the natural substrate. To achieve more effective inhibition, and explore the possibility of a new strategy, a derivative of the natural substrate has been synthesized by combining L-homocysteine with an electrophilic group to promote formation of a covalent bond in the enzyme binding site and act as an irreversible inhibitor. A synthetic route to this derivative commenced by protecting the acid and amine of L- homocystine as a methyl ester and a carbamate, and by protecting the alcohols of D-erythronolactone with tert- butyldimethylsilyl chloride. This lactone was reduced, subjected to a Wittig reaction, and coupled to homocysteine and deprotected to introduce an α,β-unsaturated carbonyl as the electrophile for reaction with LuxS binding site cysteine. The luxs gene has been introduced to a pET-22b(+) plasmid to be expressed in E. coli cells so that the enzyme can be purified for enzyme activity assays.

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