A Streamlined Synthesis of Adenosine Vinylsulfonamide Probes for Aminoacyl-AMP Mimicry in NRPS.

Adenosine vinylsulfonamide (AVS) probes provide a powerful means of stabilizing the thioesterification state in nonribosomal peptide synthetases (NRPSs) by mimicking aminoacyl-AMP intermediates. Existing synthetic strategies have enabled broad application of this probe class, yet they often rely on strongly basic conditions for constructing the key CC bond from α-amino aldehydes, substrates that are inherently prone to epimerization. These features limit practical access to AVS derivatives from nonproteinogenic amino acids, many of which are central to NRPS-mediated natural product biosynthesis. To address these constraints, we developed a concise three-step route beginning from N-Boc amino alcohols that proceeds under operationally simple and stereochemically controlled conditions. A preassembled adenosine phosphinyl sulfonamide serves as an effective coupling partner in a Ba(OH)2-mediated Horner reaction performed near 0°C, enabling highly diastereoselective AVS formation with minimal epimerization under mild, open-air conditions. The method tolerates diverse amino acid-derived substrates, including electron-rich L-Tyr derivatives relevant to tetrahydroisoquinoline alkaloid biosynthesis. Functional evaluation by LC-MS/MS confirmed covalent capture of the NRPS SfmC by an L-Tyr-derived probe, demonstrating that AVS constructs prepared by this route retain full biochemical competence. This streamlined synthesis enhances reliable access to stereochemically well-defined AVS probes and supports structural and mechanistic studies across diverse NRPS systems.