Cell-Free Biocatalytic Synthesis of Novel Penicillin Derivatives
Mark Trujillo, Armando Barajas
Department of Chemistry & Biochemistry
Faculty Supervisor: Gassner
β-lactam antibiotics represent one of the most important first line treatments for life threatening gram-positive bacterial infections. Current industrial approaches to synthesize antibiotics are reliant on toxic reagents and organic solvent systems. To improve accessibility and minimize environmental impact, our lab is developing a green-chemical cell-free biocatalytic pathway to produce semisynthetic penicillins. This process uses aldehydes as a starting material and takes advantage of two enzymes: Acylating aldehyde dehydrogenase (AAD) oxidizes the aldehyde with NAD⁺ to form an acyl–enzyme intermediate and then transfers the acyl group to a thiol acceptor substrate to release the corresponding thioester and NADH as reaction products. Isopenicillin N acyltransferase (IAT) forms an acyl–enzyme intermediate with that acyl-thioester product of AAD, then transfers the acyl group onto the amino group of 6-aminopenicillanic acid to release the penicillin derivative. In this study benzaldehydes functionalized at the ortho, meta, or para position were targeted to generate a total of 9 penicillin derivatives. Here we report the results of in silico docking of tolualdehydes as well as nitrobenzaldehydes, and hydroxybenzaldehydes into the expanded active site volume of a double alanine mutant of AAD and report the use of these compounds as substrates for the biocatalytic synthesis of potent new penicillin derivatives called nitrocillins, salicillins, and tolucillins. Each molecule was synthesized in a 96-well plate and quantified through the production of NADH at an absorbance of 340nm recorded with a microplate reader. Thioester intermediates and penicillin products were confirmed by reverse phase HPLC. Potency was evaluated through a biological assay based on the inhibition of growth of gram-positive bacteria showing similar efficacy to Penicillin G.