Defining Pathways of Anaerobic Alkane Oxidation: Synthesis of Enantiomers of 4-Methylalkanoic Acids and (2-Methylalkyl)malonic Acids

The coenzyme A (CoA) esters of 4-methylalkanoic acids are pivotal intermediates in metabolism of alkanes by anaerobic bacteria found in O 2 -deprived environments. A generic method for synthesis of either ( R )- or ( S )-acid in high enantiomeric purity from enantiomers of methyl 3-hydroxy-2-meth-ylpropionate is described for ( R )- and ( S )-4-methyloctanoic acid and ( R )-4-methyldodecanoic acid. In a typical procedure silyl-protection of methyl ( S )-3-hydroxy-2-methylpropionate was followed by reduction of the ester to a primary alcohol, which was tosylated. Cu(I)-catalysed cross-coupling of the tosylate with propylmagnesium chloride followed by deprotection, tosylation and base-induced reaction with di-t -butyl malonate, gave di-t -butyl ( R )-(2-methylhexyl)malonate. Microwave heating of the diester in 2,2,2-trifluoroethanol gave a 42% overall yield of ( R )-4-methyloctanoic acid, which was shown to be the enantiomer derived by metabolism of hexane by proteobacte-rium Aromatoleum sp. HxN1. Deprotection of the diester with trifluoroacetic acid gave ( R )-2-(2-methylhexyl)malonic acid, which is the biological precursor of ( R )-4-methyloctanoic acid (via CoA esters


Introduction
Whereas alkane oxidation by cytochrome P450 mono-oxygenases (CYP450s) has been long known and extensively studied, [1][2][3] research on dioxygen-free microbial functionalisa-tion of alkanes is a much more recent oeuvre. [4,5]However, anaerobic 'oxidation' of hydrocarbons is believed to be of ancient provenance and was once widespread, but as dioxygen populated the Earth's atmosphere, it became restricted to subsurface habitats such as petroleum reservoirs and the deep sea. [4]Anaerobic degradation of the representative hydrocarbon hexane by the proteobacterium Aromatoleum sp.HxN1 with nitrate as an electron acceptor requires two radical enzymes, the first of which attacks a CÀ H bond, whilst the second effects a molecular rearrangement reminiscent of coenzyme B 12dependent methylmalonyl-CoA mutase. [6,7]A putative HxN1 enzyme uses a cysteinyl radical to abstract stereospecifically the pro-S hydrogen atom from C-2 of hexane. [8]This step may lead to a discrete hex-2-yl radical, which is trapped by fumarate to afford (2R,1'R)-1'-methylpentylsuccinate (MPS, Scheme 1).Alternatively, the formation of MPS may be a concerted reaction in which hydrogen atom abstraction is coupled with addition of the hex-2-yl moiety to fumarate. [8]MPS is converted into its coenzyme A (CoA) ester (MPS-CoA), which equilibrates with the diastereoisomer (2S,1'R)-1'-methylpentylsuccinyl-CoA.This step is followed by an intramolecular rearrangement to (4R)-(2-methylhexyl)malonyl-CoA (MHM-CoA), which may be coenzyme B 12 -dependent. [9,10]Decarboxylation of MHM-CoA yields (R)-4-methyloctanoyl-CoA, which is further degraded to acetyl-and propionyl-CoA. [6,7]Elucidation of this pathway (summarised in Scheme 1) was aided by the synthesis of reference compounds and stereospecifically dideuterated hexanes as substrates. [8]o explore further mechanistic details of the pathway described, we required efficient syntheses of (R)-4-methyloctanoic acid and (R)-(2'-methylhexyl)malonic acid [(R)-1 and (R)-2, respectively, Figure 1] for conversion into their corresponding CoA esters.In this paper, a versatile route to these compounds is presented and extended to the corresponding (S)-isomers.This methodology is generic and thus applicable to other 4-methylalkanoic acids and (2'-methylalkyl)malonic acids, which are intermediates (as CoA esters) in the anaerobic oxidation of lower and higher alkanes.

Synthesis of (R)-and (S)-4-methyloctanoic acid via (2'-methylhexyl)malonic acids
To access (R)-and (S)-4-methyloctanoic acid 1 and other 4methylalkanoic acids we considered either using a prochiral starting material or a readily available single enantiomer of a chiral compound.Cleavage of 5-methyl-1,3-dioxan-2-one with a chiral amine to separable diastereoisomeric carbamates could only be achieved on an analytical scale (data not shown).We therefore focussed on the enantiomers of methyl 3-hydroxy-2-methylpropionate 3 as starting materials.The advantage of these precursors is that they are commercially available at relative low cost, have a defined methylsubstituted stereogenic centre and contain two functional groups that can be appropriately modified in turn to afford, in principle, any 4-methylalkanoic acid or (2'-methylalkyl)malonic acid.
The enantiomeric purities of the 4-methyloctanoic acids (S)-1 and (R)-1 were analysed using chiral gas chromatography (CGC) using (rac)-4-methyloctanoic acid for reference.The formation of solely (R)-1 in the degradation of hexane by the anaerobe HxN1 was also confirmed by CGC (see Figure 2 for chromatograms), in agreement with this assignment made using samples of 4-methyloctanoic acid synthesised by a different method that gave optically impure materials.
(S)-4-Methyloctanoic acid [(S)-1] was also synthesised from (S)-3 via conversion of (R)-5 into the corresponding 4-tertbutylbenzyl ether (R)-12 by reaction with O-4-tert-butylbenzyl trichloroacetimidate 13 in dichloromethane (DCM)cyclohexane solution at room temperature in the presence of a catalytic amount of triflic acid (Scheme 5 and Supporting Information Scheme S2).[46] The required 13 was prepared from 4-tert-butylbenzyl alcohol 14 and trichloroacetonitrile in the presence of potassium tert-butoxide. [47]Standard base (NaH)-induced benzylation was not possible because this gave racemic 12 and ultimately (rac)-4-methyloctanoic acid owing to migration of the TBDMS group on formation of the intermediate alkoxide (see Supporting Information, Figure S1).(R)-12 was subjected to desilylation, followed by tosylation of the liberated hydroxyl group and finally cross-coupling with propylmagnesium chloride to give 4-tert-butylbenzyl  ether (S)-15.The sequence of reactions shown in Scheme 5 was used to convert (S)-15 into (S)-4-methyloctanoic acid (S)-1.The final product was analysed by the CGC method (Supporting Information, Figures S1 and S2) under the same conditions as used for the previous analyses of all 4-meth-yloctanoic acids.The ee of the newly synthesised (S)-4methyloctanoic acid (S)-1 was 83 % (see Figure S2).To minimise silyl migration during protection of alcohol 5, the O-4-tert-butylbenzylation was then performed at À 15 °C.The enantiomeric purity of the resulting ether (R)-12 was determined as 98 % by 1 H NMR spectroscopy using the chiral shift reagent europium(III) tris[3-(heptafluoropropyl hydroxy-methylene)-d-camphorate] with alcohol 16 derived by removal of the TBDPS group from 12 (Figure S3).
Application of the method described to the synthesis of 4methylalkanoic acids of increased chain length was investigated.Starting from (S)-7 and using heptylmagnesium chloride in the cross-coupling step enabled the addition of a C 7 alkyl chain and afforded the (R) enantiomer of 4-methyldodecanoic acid (R)-18 in an overall yield of 49 %.
By using this versatile synthetic approach, the number of carbon atoms in the chain can be easily varied and a vast range of 4-methylalkanoic acids of high enantiomeric purity can be readily synthesised.Deprotection with concomitant decarboxylation of the di-tert-butyl malonic ester derivative to produce a monocarboxylic acid was facilitated in one pot using microwave heating in TFE and the final compound did not require any further purification.Deprotection in trifluoroacetic acid afforded an alkylmalonic acid.