Late-Stage Conversion of Diphenylphosphonate to Fluorophosphonate Probes for the Investigation of Serine Hydrolases

Abstract

SUMMARY Diphenylphosphonates (DPPs) have been used for 50 years to inactivate serine hydrolases, creating adducts representative of tetrahedral intermediates of this important class of enzymes. Failure to react at active site serine residues, however, can thwart their usefulness. Here we describe a facile route and allied mechanistic studies to highly reactive, structurally complex organofluorophosphonates (FPs) by direct fluorinative hydrolysis of DPPs. Advantages over current preparations of FPs are exemplified by the synthesis of a β-lactam containing peptide substrate analogue capable of modifying the C-terminal, dual-function thioesterase involved in nocardicin A biosynthesis. Although this serine hydrolase was found to resist modification by classical DPP inhibitors, active site selective phosphonylation by the corresponding FP occurs rapidly to form a stable adduct. This simple, late-stage method enables the ready preparation of FP probes that retain important structural motifs of native substrates, thus promoting efforts in mechanistic enzymology by accessing biologically relevant enzyme-inhibitor co-structures.

Structure of a bound peptide phosphonate reveals the mechanism of nocardicin bifunctional thioesterase epimerase-hydrolase half-reactions

“…Due to the natural hydrolytic or macrocyclization activity of NRPS thioesterase domains, it has proved challenging to obtain structures of acyl-enzyme intermediates. To best explore the structural and mechanistic features of the unique epimerase/hydrolase chemistry performed by NocTE, we pursued the design of small-molecule inhibitors capable of producing an enzyme-inactivator adduct with maximal native structural fidelity 28 . Biochemical characterization of NocTE had demonstrated its marked selectivity towards β-lactam-bearing, tri- and pentapeptide N -acetylcysteamine (SNAC) thioester substrate analogues 17 .…”

“…Ultimately, the serendipitously discovered ability to convert the DPP group to a much more reactive fluorophosphonate (FP) warhead ( 4 , Fig. 1) using a late-stage, two-step fluorinative hydrolysis and methylation protocol led to a potent and selective inactivator of NocTE that was stable in buffer for prolonged periods of time and conserved the desired structural properties of the enzyme 28 .…”

“…While many TE structures have been determined, few bear bound substrates or structurally accurate inhibitor mimics. The discovery 28 of a mild, two-step method to convert diphenylphosphonate 7 to the more reactive fluorophosphonate 4 enabled an exceptional view into the dual functions of NocTE at high resolution. A complementary approach to characterize the acyl-enzyme intermediate has recently been described that utilizes genetic code expansion to incorporate a 1,3-diaminopropionate non-canonical amino acid in place of the catalytic serine 37 .…”

“…The methylfluorophopshonate derivative of nocardicin G ( 4 ) was prepared as its C-terminal racemate as previously described 28 . Briefly, fluorinative hydrolysis of nocardicin G diphenylphosphonate ( 7 ) using ammonium fluoride in dry acetonitrile produced the free fluorophoshonate salt, which was subsequently dissolved in anhydrous ampoule d 6 -DMSO prior to methylation with freshly distilled ethereal diazomethane.…”

Fluorophosphonates on‐Demand: A General and Simplified Approach toward Fluorophosphonate Synthesis

“…Moreover, isolation and purification of FPs derived from this approach often requires aqueous workup, chromatography, and/or distillation, which can contribute to product degradation and yield reduction. Finally, the isolated FPs must be used immediately or stored under dry or cryogenic conditions to prevent degradation due to their reactive nature and lability to hydrolysis over time [6g] …”