Use of ¹⁹F NMR spectroscopy to probe enzymatic oxidation of fluorine‐tagged sulfides

Abstract

A novel19 F NMR-based method for monitoring the enzymatic oxidation of thia fatty acid analogues is presented. Our approach is based on the observation that methyl !-monofluorinated 9-thia-and 10-thiaoctadecanoates and their S-oxide and S-dioxide derivatives are easily distinguishable via their 1 H-decoupled 19 F spectra. These long-range substituent effects were used to probe the regio-and chemoselectivity of stearoyl ACP (acyl carrier protein) 1 9 desaturase-mediated sulfoxidation. The results clearly demonstrate that mono-oxygenation of a 10-thia analogue ACP 1 9 desaturase was more efficient than that of a 9-thia substrate. A product previously undetected by TLC was observed for the first time in the product mixture obtained from 18-fluoro-9-thiaoctadecanoyl-ACP.

In vitro enzymatic oxidation of a fluorine‐tagged sulfido substrate analogue: a 19F NMR investigation

“…Interestingly, in the case of the plant  9 desaturase, we were able to detect an unknown fluorinated product (Compound U) produced from 1-ACP in addition to the sulfoxide product. 6 We proposed that this compound was a chain-cleavage product 6,7 and in this paper we confirm this hypothesis.…”

“…An aliquot of the organic extract was allowed to evaporate and the residue so obtained examined by 1 H-decoupled 19 F NMR after the addition of CDCl 3 (570 µl). The 19 F NMR spectrum of the product mixture was similar to that obtained previously 6 and is presented in Fig. 1(A).…”

“…Our fluorine-based approach was tested recently using an in vivo membrane-bound yeast  9 desaturating system 5 and a purified in vitro soluble plant  9 desaturase enzyme. 6 In both cases, regioselective sulfoxidation could be easily monitored by 19 F NMR owing to long-range substituent effects on a remote fluorine reporter group. Interestingly, in the case of the plant  9 desaturase, we were able to detect an unknown fluorinated product (Compound U) produced from 1-ACP in addition to the sulfoxide product.…”

ω‐Fluoro Thiafatty Acids: New Mechanistic Probes of Desaturase‐Mediated Reactions

“…The ratio of 9-to 10-sulfoxidized products (1.6 : 1) obtained in this work compares favorably with the values obtained from both direct competition (2.1 : 1 [6]) and noncompetitive incubation of thiastearates (1.7 [5]). Good correlation between sulfoxidation ratios derived from experiments with nonfluorinated and w-fluorinated thiastearates have also been obtained for a soluble, plant D9-desaturase system [9] [15].…”

“…As a first step in this direction, we report here the validation of a 19 F-NMR-based method for monitoring D9-desaturase-mediated sulfoxidation of F-tagged thiasubstrates. A preliminary publication of portions of this work has appeared [9].…”