Tuning activity-based probe selectivity for serine proteases by on-resin ‘click’ construction of peptide diphenyl phosphonates

Abstract

Activity-based probes (ABPs) are powerful tools for functional proteomics studies. Their selectivity can be influenced by modification of a recognition element that interacts with pockets near the active site. For serine proteases there are a limited number of simple and efficient synthetic procedures for the development of selective probes. Here we describe a new synthetic route combining solid and solution phase chemistries to generate a small library of diphenyl phosphonate probes. Building blocks carrying a P1 recognition element and an electrophilic phosphonate warhead were prepared in solution and 'clicked' on-resin onto a tripeptide. We show the ability to modulate the activity and selectivity of diphenyl phosphonate ABPs and demonstrate activity-dependent labeling of endogenous proteases within a tissue proteome. The herein described synthetic approach therefore serves as a valuable method for rapid diversification of serine protease ABPs.

Phosphono Bisbenzguanidines as Irreversible Dipeptidomimetic Inhibitors and Activity‐Based Probes of Matriptase‐2

“…[21,33,41] Other guanidinium/uronium coupling reagents or carbodiimides have also been employed for the preparation of several phosphono dipeptides. [18,20,22,24,33,38,42] The resulting Boc-protected peptides 17-21 occurred as am ixture of two diastereomers after the introduction of asecond chiral center,whichoriginated from the introduced amino acid.…”

“…Deprotec-tion of the amino group was achieved after treatment with as olution of HBr in acetic acid. [20,21,33,37,38,40] The bromide salts 13 were directly subjected to ac oupling reaction with the amino acids Boc-l-Phe-OH (14;B oc: tert-butoxycarbonyl), Bocd-Phe-OH (15), and Boc-l-Lys(Cbz)-OH( 16), respectively,b ya pplying HATU as coupling reagent andD IPEA as base. [21,33,41] Other guanidinium/uronium coupling reagents or carbodiimides have also been employed for the preparation of several phosphono dipeptides.…”

Covalent protein modification: the current landscape of residue-specific electrophiles

“…Most recently, a library of peptide-based diphenyl phosphonates contained members selective for many serine proteases, including chymotrypsin, cathepsin G and urokinase-type plasminogen activator (uPA) [13]. Additionally, diphenyl phophoramidate probes (Figure 1c) retain serine protease reactivity and selectivity while being amenable to strictly solid-phase synthesis techniques [14].…”

Aggregation of poly(acrylic acid)-containing elastin-mimetic copolymers

“…The observed lack of degradation is in agreement with our previous studies, and other reports of CuAAC click chemistry with peptides on resin. 22,50 …”