Acyl derivatives thioester reduction

The loading module comprises three domains. The first (CL) shows homology to ATP-dependent carboxylic acid-CoA ligases, the second is a putative enoyl reductase (ER) and the third an ACP. The probable sequence of operations starts with the enoic acid 74 derived from shikimic acid which is reduced by the ER domain. The first domain will activate the carboxylic acid to an active acyl derivative ready for transfer to the thiol residue of the ACP. The final saturated product will end up attached to the ACP as a thioester derivative ready for transfer to the KS domain of the first chain extension module. The timing of the reduction in this sequence of operations cannot be predicted. 

The aldehyde intermediate can be isolated if 1 equivalent of diisobutvl-aluminum hydride (D1BAH) is used as the reducing agent instead of LiAlH4. The reaction has to be carried out at -78 °C to avoid further reduction to the alcohol. Such partial reductions of carboxylic acid derivatives to aldehydes also occur in numerous biological pathways, although the substrate is either a thioester or acyl phosphate rather than an ester. 

Cysteine sulfhydryls and cystine disulfides may undergo a variety of reactions, including alkylation to form stable thioether derivatives, acylation to form relatively unstable thioesters, and a number of oxidation and reduction processes (Figure 1.10). Derivatization of the side chain sulfhydryl of cysteine is one of the most important reactions of modification and conjugation techniques for proteins. 

This coupling procedure with the thioesters proved sensitive to the substitution pattern of both the amino acid and alkene. In contrast, coupling reactions with the M-acyl oxazolidinone derivatives such as 22 proved to be much more effective (Scheme 14) [20]. Mechanistic studies suggested that an alternative pathway was operating in these cases, where reduction of the al-... 

Despite their enormous structural diversity, polyketide metabolites are related by their common derivation from highly functionalised carbon chains whose assemblies are controlled by multifunctional enzyme complexes, the polyketide synthases (PKSs) which, like the closely related fatty acid synthases, catalyse repetitious sequences of decarboxylative condensation reactions between simple acyl thioesters and malonate, as shown in Fig. 3 [7]. Each condensation is followed by a cycle of modifying reactions ketoreduction, dehydration and enoyl reduction. In contrast to fatty acid biosynthesis where the full cycle of essentially reductive modifications normally follow each condensation reduction, the PKSs can use this sequence in a highly selective and controlled manner to assemble polyketide intermediates with an enormous number of permutations of functionality along the chain. As shown in Fig. 3, the reduction sequence can be largely or entirely omitted to produce the classical polyketide intermediate which bears a carbonyl on every alternate carbon and which normally cyclises to aromatic polyketide metabolites. On the other hand, the reductive sequence can be used fully or partially after each condensation to produce highly functionalised intermediates such as the Reduced polyketide in Fig. 3. Basic questions to be answered are (i) what is the actual polyketide intermediate... 

Camarero et al. [108] used the hydrazine safety-catch linker to prepare peptide thioesters. After assembling the peptide using standard Fmoc protocols, the fully protected peptide resin was activated by mild oxidation with N-bromosuc-cinimide (NB S) in the presence of pyridine, forming a reactive acyl diazene that was then deaved with an a-amino add S-alkyl thioester such as H-AA-SEt, where AA is Gly or Ala. After TFA deprotection, peptide thioesters were obtained in good yields. Although the oxidation step did produce racemization, and other sensitive amino acids such as Tyr(tBu) and Trp(Boc) were not affected, Met and Cys presented some problems. Met was completely oxidized, and a reductive cleavage was required. For Cys, the Cys(Trt) derivative should be avoided and use of Cys(Npys) or Cys(S-StBu) is recommended instead. 

AcetoacetylCoA thiolase (E.C. 2.3.1.9), acetoacetylCoA reductase (E.C. 1.1.1.36), and polyhydroxybutyrate synthetase12471 are the enzymes involved in polyester synthesis. AcetoacetylCoA thiolase catalyzes the head-to-tail Claisen condensation of two acetylCoA molecules. In this reaction, the active site cysteine attacks acetylCoA to form a thioester enzyme intermediate, which then reacts with the enolate derived from enzymatic deprotonation of the other acetylCoA. Mechanistic studies have been performed on this enzyme from Zooglea ramigera, which has been cloned and overexpressed12471. It has been established that the thiolase will form acyl enzyme intermediates with a number of acylCoA substrates, but will only accept acetylCoA as the nucleophile. After subsequent reduction, this results in all polymer units possessing a P-hydroxy group. These polymers are also useful sources of (R)-P-hydroxy acids[2481. 

Woodward s Phosphorane Route. The first penem synthesis, shown in Figure 2, utilized an intramolecular Wittig reaction to form the [2,3] double bond of the thiazoline ring (84). Reductive acylation of the penicillin derived disulfide (44) gave the thioester (45). Ozonolysis of the latter provided the oxalimide (46) which on mild methanolysis gave the azetidinone (47). Well established methods were applied to convert (47) to the phosphorane (48) which underwent thermal cydization to the penem ester (49). Catalytic hydrogenation gave the penem acid [64370-39-4] (50) which was shown to possess antibacterial activity in spite of its rather limited stability. 

The oxidation-reduction method, developed initially by Mukaiyama et al. [133] and related to the previously described organophosphorus methods, has permitted a variety of important solid-phase applications. The mechanism of the activation is complex and involves the oxidation of the triaryl/ alkyl-phosphine to the oxide as well as reduction of the disulfide to the mercapto derivative. However, different active species, such as 81 (Fig. 11), the 2-pyridyl thioester, or even the symmetrical anhydride, have been postulated to form. For the intermediate 81, the peptide bond formation may proceed through a (cyclic transition state. The method has been used for conventional stepwise synthesis [134], acylation of the first protected amino acid to a hydroxymethyl resin, and to achieve segment condensation on a solid support in the opposite direction (N C) [135,136]. Lastly, it has been used for efficient grafting of a polyethylene glycol (molecular weight 2000) derivative to an aminomethyl resin to prepare PEG-PS resins [137]. 

The malonic ester synthesis might seem like an arcane technique that only an organic chemist would use. Still, it is much like the method that cells use to synthesize the long-chain fatty acids found in fats, oils, waxes, and cell membranes. Figure 22-4 outlines the steps that take place in the lengthening of a fatty acid chain by two carbon atoms at a time. The growing acid derivative (acyl-CoA) is activated as its thioester with coenzyme A (structure on page 1027). A malonic ester acylation adds two of the three carbons of malonic acid (as malonyl-CoA), with the third carbon lost in the decarboxylation. A )8-ketoester results. Reduction of the ketone, followed by dehydration and reduction of... 

In a different approach, the reactivity of thiazolium salts derived acyl anion equivalents (biological active aldehyde ) toward sulfur electrophiles has been examined recently (329) and provides a model for the thioester-forming step catalyzed by the lipoic acid containing enzymes. The results suggest that the biological generation of thioesters of coenzyme A from a-keto acids occurs via the direct reductive acylation of enzyme-bound lipoic acid by the active aldehyde, as already shown on page 453. 

Incubation of SNAC 32 yielded no observable acylation of KSl, suggesting that the amide substrate should be derived from an a-amino acid. Equally, no acylation was detected with SNAC 33 indicating that the distal carbonyl is not a factor in substrate viability. Previous work by Claderone et al. revealed that the reduction of the 2-keto-4-methylpentanoyl intermediate to the 2-hydroxy-analogue is performed by the KR domain of module 3 (Scheme 4.1) [6]. This unorthodox reduction step is believed to occur post-KSl elongation, therefore suggesting that the oxidation level of SNAC 27 is correct, and therefore a substrate mimic for KSl. To probe this issue further, a 2-hydroxy-4-methyl-pentanoyl SNAC (34) was synthesised. Upon incubation of SNAC 34 with KSl, a slight reduction in acylation rate was observed ( 20 %) compared to SNAC 27. The reduction in acylation rate may be due to unfavourable loss of planarity in the substrate, or decreased intrinsic reactivity of the thioester (Fig. 4.4). 

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