Mechanistic studies of desaturase-mediated oxidations

Abstract

The study of the mechanism of fatty acid desaturases often requires the use of synthetic probes and/or standards. In this work, a general procedure for preparing chiral fluorine-tagged dialkyl sulfoxides was developed using DAG methodology. 18-fluoro-(10S)sulfoxy-1-octadecanol, 18-fluoro-(10i?)-sulfoxy-l-octadecanol, 15-fluoro-(l 15)-sulfoxypentadecane, and 15-fluoro-(lli?)-sulfoxypentadecane were prepared. These standards were used in the determination of the stereochemistry of enzymatically produced sulfoxides arising from the introduction of fluorine-tagged thia fatty acid analogues to a soluble and a membrane-bound A 9 desaturase.In this study, it has been shown that the Pirkle binding model is successful for predicting the direction of induced non-equivalence of fluorine-tagged dialkyl sulfoxide enantiomers by ! H NMR using the chiral solvating agents AMA, MPAA and TFAE. However, the observed non-equivalence of 19 F NMR signals of these sulfoxides under similar conditions is dependent on the nature of the chiral reagent used.The identification of the products of a triple mutant, T117R/G188L/D280K of the castor A 9 desaturase was also undertaken using synthetic standards in combination with GC-MS. (10Z)-and (10£)-9-hydroxy-10-octadecenoate and (9Z)-and (9£)-ll-hydroxy-9octadecenoate were synthesized by known methods. The dienoic fatty acid esters, methyl 9Z,ll£'-octadecadienoate with a minor component of methyl 9is,ll£'-octadecadienoate were prepared in a 9:1 ratio and a mixture of 9Z,llZ-octadecadienoate and methyl 9£',11Zoctadecadienoate were also prepared. in Chemoselectivity 12Chapter 2: Results and Discussion 2.1 Determination of the enantioselectivity of soluble A 9 desaturase-mediated sulfoxidation using fluorine-tagged substrates 16 2.1.1 Introduction 16 V 2.1.1.1 Use of 'H-decoupled 19 F NMR in the study of desaturase-mediated oxidation 16 2.1.1.2 Efforts to determine the enantioselectivity of soluble A 9 desaturasemediated sulfoxidation \j 2.1.2 Project goals 20 2.1.3 Synthesis of fluorine-tagged chiral sulfoxide standards 21 2.1.3.1 Synthesis of (105)-and (107v)-18-fluoro-10-sulfoxy-l-octadecanol 26 2.1.3.2 Synthesis of (US)-and (117?)-15-fluoro-ll-sulfoxypentadecane 36 2.1.4 Synthesis of (R) and (S) (9-anthryl)methoxyacetic acid (AMA) 41 2.1.5 NMR experiments 45 2.1.5.1 Assignment of the absolute configuration of 18-fluoro-10-sulfoxy-loctadecanol produced by a soluble A 9 desaturase 45 2.1.5.2 Investigation of the role of the C1 hydroxyl group on the CSA induced NMR non-equivalences 48 2.1.5.3 Assignment of the absolute configuration of methyl 15-fluoro-l 1sulfoxypentadecanoate produced by growing S. cerevisae cultures 49 2.1.5.4 Investigation of the effect of dilution on the direction of induced non-equivalence 51 2.1.5.5 A study of the direction of induced non-equivalence in 'H and 'H-decoupled 19 F NMR for various chiral solvation reagents 52 2.1.6 Conclusions and future directions 55 2.2 Study of the catalytic diversity of an acyl-ACP desaturase mutant 57 2.2.1 Introduction 57 2.2.2 Project go...