Thioesters / oxoesters

In contrast to oxoesters, the a-protons of thioesters are sufficiently acidic to permit continuous racemization of the substrate by base-catalyzed deprotonation at the a-carbon. Drueckhammer et al. first demonstrated the feasibility of this approach by performing DKR of a propionate thioester bearing a phenylthiogroup, which also contributes to the acidity of the a-proton (Figure 4.14) [39a]. The enzymatic hydrolysis of the thioester was coupled with a racemization catalyzed by trioctylamine. Owing to the insolubility of the substrate and base in water, they employed a biphasic system (toluene/H2O). Using P. cepacia (Amano PS-30) as the enzyme and a catalytic amount of trioctylamine, they obtained a quantitative yield of the corresponding... 

The reduced electron delocalization in the thioester moiety, compared to oxoesters, results in a higher reactivity towards conjugate addition reactions see Reference 41. 

In contrast to oxoesters, the a-protons of thioesters are sufficiently acidic to permit continuous racemization of the substrate by base-catalyzed deprotonation at the... 

DKR of thioesters (Scheme 21.2) with a chiral center at the a-carbon has been achieved in a water/acetonitrile biphasic system by racemization with mild organic bases, such as trioctylamine, coupled to enantioselective hydrolysis of the thioester with subtilisin Carlsberg.28 Such an approach can be applied to a wide variety of thioesters but not oxoesters, which have less acidic a-protons. 

Beside the well-known polyoxoesters, structurally related polythioesters (PTE) also occur. They are even synthesized by the same enzyme as the polyoxoesters, that is, by the PhaCs [13]. In PTEs the oxygen atom of the oxoester bond in the polymer backbone is replaced by a sulfur atom, thereby constituting thioester bonds. 3-Mercaptopropionic acid, 3-mercaptobutyric acid and 3-mercaptovaleric acid are currently known to occur in PTEs [14]. 

The chemoselectivity of the reaction stems from the combination of a Cys specific, reversible thioester exchange (any Cys residue in either peptide can participate in this equilibrium) with an essentially irreversible intramolecular reaction that is specific to N-terminal Cys residues. Under typical ligation conditions (pH 6.5-7.5,1 mM peptide) the intermoleculartransthioesterification is rate limiting and no thioester intermediate is observed because of rapid rearrangement [47]. The reaction also utilizes the unique reactivity profile of the thioester as an activated acyl group. Compared to oxoesters with identical substituents, thioesters are much more reactive toward thiol nucleophiles [49] (and to a lesser extent toward amine nucleophiles [50]), facilitating rapid... 

W. Yang, D.G. Drueckhammer, Understanding the relative acyl-transfer reactivity of oxoesters and thioesters computational analysis of transition state delocalization effects,... 

Thioesters have the general formula RCSR. They resemble their oxygen counterparts RCOR (oxoesters) in structure and reactivity more than other carboxylic acid derivatives such as acyl chlorides, acid anhydrides, and amides. Thioesters can be prepared from thiols by reaction with acyl chlorides or acid anhydrides in much the same way as oxoesters are prepared from alcohols. 

Thioesters and oxoesters are similar in their rates of nucleophilic acyl substitution, except with amine nucleophiles for which thioesters are much more reactive. Many biological reactions involve nucleophilic acyl substitutions referred to as acyl transfer reactions. The thioester acetyl coenzyme A is an acetyl group donor to alcohols, amines, and assorted other biological nucleophiles. 

A new family of bacterial biopolymers (thioester-containing PHAs) was reported by Lutke-Eversloh et al. (2001a, b). These authors showed that C. necator was able to synthesize and polymerize (R)-3-mercaptopropionic acid, thus generating a novel type of PHA that contains sulphur in the polymer backbone. Whereas in other PHAs the monomers are covalently linked by oxoester bonds, in these bioplastics they are linked by thioester bonds. For this reason, these polymers are referred to as polythioesters (Lutke-Eversloh et al. 2001a, b). 

One surprising characteristic of polythioesters is that, in contrast to those polymers containing oxoester bonds, the thioester linkages are not susceptible to PHA depolymerases (Elbanna et al. 2003, 2004), thus constituting the only known kind of non-biodegradable biopolymers (Kim et al. 2005a). 

Scheme 5.6, Equation 5.1) [19]. Although the acidity of the a-proton is much greater than that of the oxoesters, the presence of a tertiary amine has been shown to be indispensable for quantitative conversion. Biochemical transesterification has also been reported for the DKR of a variety of chiral trifluoroethyl thioesters [20]. The DKR of trifluoroethyl thioesters, using either lipase-catalysed hydrolysis [20b,d,e] or transesteriflcation with 4-morpholine ethanol [20c], has allowed the isolation of enantiomerically pure profen esters based on (S)-naproxen, (S)-fenoprofen and (S)-suprofen in >75% yield with up to 95% ee. Some oxoester with relatively higher... 

On the other hand, various biocatalytic hydrolysis methods have been developed on the basis of DKR. The a-hydrogens of thioesters are more acidic than those of (non-activated) oxoesters, although the rates of base-catalysed hydrolysis of thioesters and oxoesters are very similar. Several thioesters have been subjected to enzymatic resolution by lipases, but their exceptional a-H-acidity has more rarely been exploited for a dynamic resolution process this concept having been verified with several thioesters of a-(phenylthio)pro-pionate (Scheme 3.36). 

Nonribosomal peptide synthesis means that the peptide is not produced by the tRNA-mRNA mechanism described in Chapter 28, Section 28.6. Each amino acid found in 224 is directly selected for incorporation into the growing peptide chain by one of the domains of surfactin synthetase, shown with the pendant SH groups. Substrate activation occurs after binding the amino acid, and the enzyme catalyzes the formation of an aminoacyl adenylate intermediate using Mg2+-ATP and release of a cofactor. Subsequently, the amino acid-O-AMP oxoester is converted into a thioester by a nucleophilic attack of the free thiol-bound cofactor of an adjacent PCP domain. (Note that ATP is adenosine triphosphate and AMP is adenosine monophosphate see Chapter 28, Section 28.5.)... 

The aim of this overview is to focus the attention toward the racemization conditions of two specific acyl donors commonly used for enzyme-catalyzed synthesis, namely the oxoesters and the thioesters. The former are often the substrates of choice because their mild reactivity (in contrast to anhydrides, for instance) limits their susceptibility to parasitic reactions such as spontaneous transacylation or condensation reactions, while at the same time they are easily transformed by enzymes. Thioesters, though being activated substrates from a thermodynamic point of view, possess a peculiar relative kinetic stability against spontaneous hydrolysis [10], which permits their use in biocatalysis moreover, they are far more prone to base-catalyzed racemization as compared to their oxygenated analogs [llj. This combination of features makes them attractive as candidates to be employed in DKRs as alternatives to oxoesters, and examples will be discussed to support this concept. 

It is generally accepted that a set of desaturases which is located in the plastids introduces double bonds into the acyl moieties bound as thioesters to acyl carrier protein or as oxoesters in monogalactosyl diacylglycerols. Both saturated and unsaturated fatty acids are translocated as acyl-CoAs from the plastids to the endoplasmic reticulum. Acyl chains of these activated fatty acids are elongated or incorporated into phosphatidylchohnes and other polar lipids II (cf. Fig. 1), and partly modified, for example, by desaturation. Further modification of acyl moieties leads to hydroxy-, epoxy-, and cyclic fatty acids. 

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