Synthesis of reference standards for elucidating mechanisms of anaerobic alkane degradation

Abstract

Hydrocarbons are the second most prolific compound class found naturally and are used by bacteria as a source of energy in remarkable ways. Until quite recently it was believed that bacteria could only catabolise hydrocarbons in the presence of dioxygen. It has now been established that with the assistance of an electron acceptor, bacteria can break down these relatively inert compounds in anoxic environments using fumarate as a surrogate for O2 in a process mediated by a glycyl radical enzyme (Scheme 1). Scheme 1-1: General scheme for alkane activation by fumarate addition (Enz-Cys. = glycyl radical enzyme; CoA = coenzyme A). The β-proteobacteria HxN1 is an anaerobic bacterium capable of this process and can break down hydrocarbons in the range C6-C8. Previous studies using (2R,5R)-, (2S,5S)- and (2R,5S)-hexane-2,5-d2 showed that the initial step performed by the bacterium was the stereospecific abstraction of the pro-S hydrogen atom by a cysteine thiyl radical from C-2 of the alkane. The resulting hexan-2-yl radical adds to fumarate to give a stabilised radical that is quenched by re-abstraction of the hydrogen from cysteine-SH to form 2-(hexan-2-yl)succinic acid. This thesis describes an extension of the HxN1 study to the anaerobic bacterium OcN1, which degrades hydrocarbons in the range C8-C12. Efficient methods were developed for the synthesis of (2R,9R)-, (2S,9S)- and (2R,9S)-decane-2,5-d2 for microbiological studies. Naphthalene degradation occurs under anaerobic conditions either by carboxylation to 2-naphthoic acid or by methylation followed by addition of fumarate to give 2- ii (naphthalen-2-yl)succinic acid. A route has been developed which yields both 13C10-naphthalene and 13C11-2-naphthoic acid to aid in metabolite analyses. A method by which a variety of alkylsuccinates could be synthesised was initially developed [1] and optimised in the present study. This route involved the addition of HI across an alkene double bond to form an alkan-2-yl iodide. The iodide was converted into an alkyl radical that was reacted with dimethyl fumarate to afford an alkylsuccinate in a biomimetic transformation. The method was employed for the synthesis from henicos-1-ene of the C21 alkylsuccinate, dimethyl 2-(henicosan-2-yl)succinate, which was required as a reference standard for studies of pasteurised control micrososms from mud taken from the River Tyne. It was found through 2D-GC-MS analysis that a significant amount of the C-3 alkylsuccinate was formed as well as other isomers, which can be ascribed to the reversibility of the HI addition to henicos-1-ene. An alternative synthesis was developed that circumvented the problematic step. This synthesis involved the O-methanesulfonate from henicosan-2-ol, which was reacted with malonate anion and further alkylated to form a tricarboxylate that was subjected to Krapcho decarboxylation to yield the alkylsuccinate in good overall yield without any regioisomers. This method also allowed for control of stereochemistry. It has been proposed that the initial hydrogen abstraction by HxN1 from hexane and fumarate addition is a concerted process rather than a stepwise pathway with a distinct hexan-2-yl radical formed. To investigate this question a six cyclopropane substrate analogues were synthesised via Simmons-Smith reactions of the corresponding alkenes. Unfortunately, these potential substrates proved to be toxic to the organism.