Facile synthesis of borofragments and their evaluation in activity-based protein profiling

Abstract

The discovery of enzyme inhibitors relies on synthetic methods that enable rapid and modular construction of small molecules. Heterocyclic fragments designed to maximize enthalpic interactions with their protein targets represent a particularly desirable class of molecules. Here we describe a reagent that enables straightforward construction of "borofragments", in which a heterocycle is separated from the boron center by two or three rotatable bonds. The stability of these molecules depends on the MIDA group which likely acts as a slow-release element under biological conditions. Borofragments can be used to discover inhibitors of enzymes that use catalytic oxygen nucleophiles. We have employed this method to identify inhibitors of ABHD10 and the predicted carboxypeptidase CPVL. This technique should be applicable to other classes of targets.

Figure 1

Competitive ABPP of (MIDA)boronates in PC3 cell lysates. A, Gel-based ABPP analysis of (MIDA)boronates in the soluble proteome of PC3 cells. The proteome was treated with either DMSO or compounds 7a–j (20 μM) for 2 h, then treated with FP-Rh (1.0 μM) for 30 min. Labeled serine hydrolases were resolved by SDS-PAGE and analyzed by fluorescence gel imaging. B, Gel-based ABPP analysis of 7j (1.0 nM–100 μM) and DMSO in the soluble fraction of PC3 cells showing dose-dependent inhibition of ABHD10 and ACOT1/2. C, ABPP-SILAC analysis of 7j at 25 μM (heavy) versus DMSO (light) in whole PC3 cell proteomic lysates showing inhibition of ABHD10, CPVL and, to a lesser extent, ACOT1/2. Data are presented as the mean ± standard deviations of heavy/light ratios for multiple unique peptides from each serine hydrolase.

Scheme 1

Preparation of hydroxymethyl(MIDA)boronate

Scheme 2

Synthesis of azidomethyl(MIDA)boronate

Scheme 3

In situ hydrolysis of 7j to the boronic acid 11 and its covalent reaction with the active size nucleophile of ABHD 10