N-Activated β-lactams as versatile reagents for acyl carrier protein labeling

Abstract

Acyl carrier proteins are critical components of fatty acid and polyketide biosynthesis. Their primary function is to shuttle intermediates between active sites via a covalently bound phosphopantetheine arm. Small molecules capable of acylating this prosthetic group will provide a simple and reversible means of introducing novel functionality onto carrier protein domains. A series of N-activated β-lactams are prepared to examine site-specific acylation of the phosphopantetheine-thiol. In general, β-lactams are found to be significantly more reactive than our previously studied β-lactones. Selectivity for the holo over apo-form of acyl carrier proteins is demonstrated indicating that only the phosphopantetheine-thiol is modified. Incorporation of an N-propargyloxycarbonyl group provides an alkyne handle for conjugation to fluorophores and affinity labels. The utility of these groups for mechanistic interrogation of a critical step in polyketide biosynthesis is examined through comparison to traditional probes. In all, we expect the probes described in this study to serve as valuable and versatile tools for mechanistic interrogation of fatty acid, polyketide, and nonribosomal peptide biosynthesis.

A mechanism-based fluorescence transfer assay for examining ketosynthase selectivity

“…24 As a result, we decided to begin with this ACP/KS pair for proof of principle studies. It should be noted that careful choice of the donor ACP is critical for success using this technique.…”

“…We knew from our previous work that the 6-deoxyerythronolide B synthase (DEBS) module 2 ACP (ACP2) could efficiently transfer a β-lactam-derived fluorescent probe to the KS active site of the DEBS module 6 KSAT 25 didomain (KSAT6). 24 As a result, we decided to begin with this ACP/KS pair for proof of principle studies. It should be noted that careful choice of the donor ACP is critical for success using this technique.…”

“…23, 24 Incorporation of an alkyne functionality at various ring positions provides a chemical handle for conjugation with fluorophores (Figure 2). The thioester-ACP resulting from incubation of holo -ACP with a propargyloxycarbonyl (Poc)-activated β-lactam was shown to be competitive with traditional N -acetylcysteamine (SNAc) thioesters for KS acylation.…”

“…Recently, our lab has developed a series of β-lactone and β-lactam probes capable of directly acylating the Ppant-thiol of holo-ACPs. 23,24 Incorporation of an alkyne functionality at various ring positions provides a chemical handle for conjugation with fluorophores (Fig. 2).…”

Enzymatic Reconstitution and Biosynthetic Investigation of the Bacterial Carbazole Neocarazostatin A

“…N-Acetylcysteamine (SNAC) is commonly used as a simplified synthetic mimic of the reactive biosynthetic intermediates, such as acyl-ACP in the biosynthetic studies. 6,7 To isolate new compounds for structural elucidation, we chemically prepared R-3-hydroxybutyryl-SNAC (Figures S2−4). Upon incubation of NzsJ with R-3hydroxybutyryl-SNAC and the enzymatic system of NzsH, the target molecules with identical molecular weights and UV absorption but different retention times (compound 5 and 6) were observed in the HR-ESIMS and HPLC analyses (Figure 2A, Figure S5).…”