Transthioesterification, reversible

The ability of DNA to serve as a template for the reversible formation of a PNA oligomer was reported by the Ghadiri group in 2009 [44]. The overall framework here relied on the use of a transthioesterification reaction as the exchange mechanism, operating between derivatives of DNA bases (for example, the adenine... 

In 2004, Ramstrom and coworkers reported the first prototype DCLs based on a reversible transthioesterification reaction [47]. DCLs of potentially ten different thioesters were generated from a series of five thioesters - all prepared from 3-sulfanylpropionic acid - and one thiol (thiocholine). Transthioesterification (exchange) reactions were observed by simply mixing the different DCL components in aqueous solution. Interestingly, when added to the equilibrated mixture of thioesters, the enzyme acetylcholinesterase was able to recognize and subsequently hydrolyze its best substrate selectively. This selective hydrolysis... 

The results presented thus far show that substrate specificity at the second elongation step exceeds that of acyl loading also highlight the potential problem of KS enzymatic stalling. In order to restore enzymatic activity either the acyl chain must hydrolyse off the active site Cys, or the upstream ACP must de-acylate the KS in a reversal of the initial acylation step. Once on the upstream ACP, the acyl chain may hydrolyse off the PPant arm, possibly catalysed by an acyl hydrolase (AH) domain (Scheme 6.2) [13]. In theory, both mechanisms are plausible, but there is a lack of any clear evidence in the literature for reversibility of the KS acylation step, other than the not unsurprising observation that a large excess of SNAC thiol was able to remove an acyl group from the KS active site by transthioesterification [6]. 

Mechanistically, NCL is a two-step process, which is initiated by a reversible transthioesterification step followed by a spontaneous, irreversible S- to N-acyl transfer (Fig. 1). The reactivity is affected by several factors such as concentration of the reactants, temperature, pH, and choice of an appropriate thiol. Also the C-terminally thioes-terified amino acid at the ligation site has to be considered carefully [3]. In feet, NCL relies basically on an observation ofWieland et al., who first demonstrated the transfer of a valyl residue from a reactive thioester onto a cysteine under slightly basic conditions followed by a rearrangement and yielding a Val-Cys dipeptide [4]. 

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