Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathways that comprise iterative chain elongation, reduction, and dehydration reactions. The bacterial FASII pathway differs significantly from human FAS pathways and is a long-standing target for antibiotic development against Gram-negative bacteria due to differences from the human FAS, and several existing antibacterial agents are known to inhibit FASII enzymes. N-Acetylcysteamine (NAC) fatty acid thioesters have been used as mimics of the natural acyl carrier protein pathway intermediates to assay FASII enzymes, and we now report an assay of FabV from Pseudomonas aeruginosa using (E)-2-decenoyl-NAC. In addition, we have converted an existing UV absorbance assay for FabA, the bifunctional dehydration/epimerization enzyme and key target in the FASII pathway, into a high-throughput enzyme coupled fluorescence assay that has been employed to screen a library of diverse small molecules. With this approach, N-(4-chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC50 = 5.7 ± 0.2) the processing of 3-hydroxydecanoyl-NAC by P. aeruginosa FabA. N42FTA was shown to be potent in blocking crosslinking of Escherichia coli acyl carrier protein and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalises affinity and suggests future design opportunities. Employing NAC fatty acid mimics to develop further high-throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials.

Fatty acid biosynthesis is an essential component of metabolism in both eukaryotes and prokaryotes. The fatty acid biosynthetic pathway of Gram-negative bacteria is an established therapeutic target. Two homologous enzymes FabA and FabZ catalyze a key step in fatty acid biosynthesis; both dehydrate hydroxyacyl fatty acids that are coupled via a phosphopantetheine to an acyl carrier protein (ACP). The resulting trans-2-enoyl-ACP is further polymerized in a processive manner. FabA, however, carries out a second reaction involving isomerization of trans-2-enoyl fatty acid to cis-3-enoyl fatty acid. We have solved the structure of Pseudomonas aeruginosa FabA with a substrate allowing detailed molecular insight into the interactions of the active site. This has allowed a detailed examination of the factors governing the second catalytic step. We have also determined the structure of FabA in complex with small molecules (so-called fragments). These small molecules occupy distinct regions of the active site and form the basis for a rational inhibitor design program.

Isothiourea HBTM-2.1 catalyzes the asymmetric Michael addition/lactonization of aryl-and alkenylacetic acids using -keto-,-unsaturated-phosphonates as ,-unsaturated ester surrogates, giving access to a diverse range of stereodefined lactones or enantioenriched functionalized diesters upon ring-opening.Lewis base organocatalysis has developed as a powerful tool for the enantioselective construction of carbon-carbon bonds. 1 Within this area, the asymmetric addition of enolates and their derivatives via the use of cinchona alkaloids, 2 enamines 3 and azolium enolates 4 generated with Nheterocyclic carbenes (NHCs), 5 to electron-deficient alkenes has received wide-spread attention in recent years. Catalytic asymmetric conjugate additions employing enones and enals is well established, 6 although the use of ,-unsaturated esters and amides remains challenging due to the intrinsic decreased reactivity of these motifs. Efforts to circumvent this issue have used N-acylpyrroles, 7 2-acyl imidazoles 8 and activated imides 9 as ester surrogates, while Evans 10 and Jørgensen 11 have pioneered the use of -keto--unsaturated phosphonates as ester equivalents. Using transition metal and organocatalysts respectively, these methods activate the -ketophosphonate for nucleophilic attack via bidentate coordination of a Lewis acid or hydrogen-bonding to a thiourea catalyst architecture (Scheme 1).Building on Romo's pioneering nucleophilecatalyzed aldol lactonization (NCAL) strategy, 12 we have previously studied the isothiourea 13 catalyzed asymmetric functionalization of carboxylic acids 14 via ammonium enolates. 15 This process requires highly electron deficient alkene components in Michael-lactonisation reactions, with ,-unsaturated esters inert to typical reaction conditions. This manuscript explores -ketophosphonates as ,-unsaturated ester equivalents, 16 affording stereodefined diesters upon ring-opening that are suitable for further synthetic manipulations. Scheme 1. Initial conceptInitial investigations employed phenylacetic acid 1 and -ketophosphonate 2 in a model system and assessed a range of isothiourea Lewis base catalysts (5-7, Table 1). In situ formation of the mixed anhydride with pivaloyl chloride and i-Pr 2 NEt, followed by treatment with isothiourea 5 gave anti-lactone 3 in 66% isolated yield with modest ee (entry 1). A screen of isothioureas revealed HBTM-2.1 7 as the optimum catalyst, providing lactone 3 in 86% ee (entry 3). This catalyst was then examined using toluene and THF as the solvent, affording decreased isolated yields but with high diastereocontrol (entries 4-5). Lowering the temperature to 78 °C (entry 6) led to improved isolated yield, dr and ee. Gratifyingly, a catalyst loading of only 1 mol % at 78 °C gave the product in good yield with excellent stere-

Abstract:The reactivity of N-tosyl-3-pyrroline is significantly lower than that of mono-substituted alkenes in Pd catalysed methoxycarbonylation reactions. For example, most bulky diphosphine/ Pd catalysts, including the well-known Pd catalyst derived from 1,2-bis(Di-TertButylPhosphinoXylene (DTBPX), were found to give no product at all in the methoxycarbonylation of N-tosyl-3-pyrroline. The competing pathways in methoxycarbonylation of N-methane-sulfonyl-3-pyrroline using Pd/ DTBPX were studied using DFT calculations; these show that the coordination of the alkene is unfavourable, and once coordinated, isomerisation is a lower energy pathway that ultimately leads to an alternative product. Experimentally a side product resulting from alkene isomerisation and addition of methanol is formed slowly (if CO is present), and rapidly if CO is not. A less bulky derivative of DTBPX forms the required alkene complex with much lower barriers. A study has been made of the enantioselective carbonylation of N-tosyl-3-pyrroline using water, methanol or aniline as nucleophile. This revealed that there is a range of possible products with most of these initiated by a Pd-catalysed isomerisation of the alkene. Using less bulky members of the Pd/Phanephos family of catalysts, it is possible to produce the methoxycarbonylation product from this poorly reactive alkene with reasonably good chemoselectivity and around 80% e.e. at higher pressures of CO. IntroductionPalladium catalysed alkene carbonylation is a very important reaction in industrial synthesis, since it uses very cheap reagents in a very atom efficient manner. [1][2][3][4][5][6] Applications that have been practiced at commercial scale include: polymer synthesis by co-polymerisation, methyl propionate synthesis by ethylene methoxycarbonylation and formation of racemic fine chemicals by hydroxycarbonylation of vinyl arenes (sometimes generated in situ from alcohols).1,2 A stand-out catalyst is the Pd complex formed from 1,2-bis(Di-Tert-ButylPhosphinoXylene (DTBPX from this point forward), which gives very high rates in ethylene methoxycarbonylation, 1b, 1l, 1o and has also been used for some other applications. 5 The potential for enantioselective hydroxycarbonylation and alkoxycarbonylation to be a useful method for large scale asymmetric synthesis has been appreciated for a long time.2-4 However, this is a challenging reaction and despite many important contributions, high enantioselectivity is rare especially for the intermolecular reaction; further research is needed. Catalysts which do give good enantioselectivity, combined with high regioselectivity in the methoxycarbonylation of styrene are Pd catalysts derived from the bulkier members of the Phanephos ligand family (shown in Scheme 1), 3,4 providing impetus to seek to evaluate and increase substrate scope for alkene carbonylation. In the literature, there are very sparse examples, even using achiral catalysts. Most examples of methoxycarbonylation of internal alkenes have led to tandem isomerisation-linear select...

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