Biomimetic Thioesters as Probes for Enzymatic Assembly Lines: Synthesis, Applications, and Challenges

Abstract

Thioesters play essential roles in many biosynthetic pathways to fatty acids, esters, polyketides, and non-ribosomal peptides. Coenzyme A (CoA) and related phosphopantetheine thioesters are typically employed as activated acyl units for diverse C-C, C-O, and C-N coupling reactions. To study and control these enzymatic assembly lines in vitro and in vivo structurally simplified analogs such as N-acetylcysteamine (NAC) thioesters have been developed. This review gives an overview on experimental strategies enabled by synthetic NAC thioesters, such as the elucidation of complex biosynthetic pathways and enzyme mechanisms as well as precursor-directed biosynthesis and mutasynthesis. The review also summarizes synthetic protocols and protection group strategies to access these versatile synthetic tools, which are reactive and often unstable compounds. In addition, alternative phosphopantetheine thioester mimics are presented that can be used as protein tags or suicide inhibitors for protein crosslinking and off-loading probes to elucidate polyketide intermediates.

NosN, a Radical S-Adenosylmethionine Methylase, Catalyzes Both C1 Transfer and Formation of the Ester Linkage of the Side-Ring System during the Biosynthesis of Nosiheptide

“…8 SNAC analogs have been employed to imitate the structure of the 4′-phosphopanthetheine prosthetic group of acyl carrier proteins during polyketide biosynthesis. 13 Given its ability to recognize MIA-SNAC, it seemed likely that the true substrate for NosN might be MIA attached to NosJ or MIA attached to Cys8 of the structural peptide at some stage of nosiheptide maturation. 6 As shown in Figure S3, NosN is unable to convert MIA-NosJ to the DMIA derivative in our hands, although this ability has recently been reported by Ding et al 6 Although 5′-deoxyadenosine (5′-dA) is produced, neither MTA nor SAH is produced as a co-product, indicating that the transfer of a C1 unit has not taken place.…”

From chemical metabolism to life: the origin of the genetic coding process

“…2). It could well have been involved in its own synthesis as well as that of diverse compounds involving acyl groups (lipids, essential for compartmentalisation [29]), a variety of (iso)peptides as in the synthesis of fatty acids today, non-ribosomal peptides and polyketides [30]. The involvement of thioesters in a primitive metabolism, predating the systematic input of phosphate has been documented by Segré and co-workers in a convincing way [31].…”

A Polyketide Synthase Component for Oxygen Insertion into Polyketide Backbones

“…To test this hypothesis,w es ynthesized bketo thioesters 7-10 ( Table 1) using various routes.T hese compounds harbor an N-acetylcysteamine (SNAC)u nits as simplified surrogates of the ACP-bound 4'-phosphopantetheinyl intermediates. [16] Assay mixtures with test substrate, OocK, and NADPH were incubated for 90 min at room temperature and then extracted with ethyl acetate.F or substrates 7 and 8 with medium or long aliphatic chains, UHPLC-HRMS ( Figure 3A and Figures S7,S8) analysis showed the formation of anew product with amass difference of + +16 Da (Figure 3a nd Figure S7), which is consistent with substrate oxygenation. No such product was detected for assays with the boiled enzyme or with test compounds 9-17.…”