Synthesis and Structural/Functional Characterization of Selective M14 Metallocarboxypeptidase Inhibitors Based on Phosphinic Pseudopeptide Scaffold: Implications on the Design of Specific Optical Probes

Abstract

Metallocarboxypeptidases (MCPs) of the M14 family are Zn 2+ -dependent exoproteases present in almost every tissue or fluid in mammals. These enzymes perform a large variety of physiological functions and are involved in several pathologies, such as pancreatic diseases, inflammation, fibrinolysis, and cancer. Here, we describe the synthesis and functional/structural characterization of a series of reversible tight-binding phosphinic pseudopeptide inhibitors that show high specificity and potency toward these proteases. Characterization of their inhibitory potential against a large variety of MCPs, combined with high-resolution crystal structures of three selected candidates in complex with human carboxypeptidase A (CPA)1, allowed to decipher the structural determinants governing selectivity for type-A of the M14A MCP family. Further, the phosphinic pseudopeptide framework was exploited to generate an optical probe selectively targeting human CPAs. The phosphinic pseudopeptides presented here constitute the first example of chemical probes useful to selectively report on type-A MCPs activity in complex media.

Practical Synthesis of Phosphinic Dipeptides by Tandem Esterification of Aminophosphinic and Acrylic Acids under Silylating Conditions

“…Melting points (measured on an Electrothermal apparatus) are uncorrected. 1 H, 13 C and 31 P NMR spectra were recorded on a Varian 200 MHz Mercury spectrometer and on a Bruker Avance Neo 400 MHz at 25 • C. 1 H and 13 C spectra are referenced according to the residual peak of the solvent based on literature data [36]. 31 P NMR chemical shifts are reported in ppm downfield from 85% H 3 PO 4 (external standard).…”

“…31 P NMR chemical shifts are reported in ppm downfield from 85% H 3 PO 4 (external standard). 13 C and 31 P NMR spectra are fully proton-decoupled. Phosphinic diacids of type 5 tend to aggregate, causing peak broadening in the NMR spectra.…”

“…190-195 • C; TLC R f (CHCl 3 /MeOH/AcOH: 7/2/1) = 0.58. 1 H NMR (500 MHz, d 6 -DMSO + 4%TFA) δ 7.68 (d, J = 9.5 Hz, 1H), 7.37-6.94 (m, 15H), 4.95 (d, J = 13 Hz, 1H), 4.88 (d, J = 13 Hz, 1H), 3.14-2.84 (m, 4H), 2.78-2.68 (m, 1H), 2.10-1.92 (m, 1H), 1.84-1.74 (m, 1H); 13…”

“…153-156 • C; [a] D 25 = −32 (c = 0.31, DMSO); TLC R f (CHCl 3 /MeOH/AcOH: 7/2/1) = 0.6. 1 H NMR (200 MHz, d 6 -DMSO) δ 7.66 (d, J = 9.5 Hz, 1H), 7.39-6.88 (m, 10H), 5.02-4.78 (m, 2H), 4.02-3.73 (m, 1H), 3.17-2.97 (m, 1H), 2.82-2.57 (m, 2H), 2.11-1.86 (m, 1H), 1.78-1.26 (m, 4H), 0.86 (app t, J = 6.3 Hz, 6H); 13 C NMR (50 MHz, d 6 -DMSO) δ 176.5 (d, 3 J PC = 7.6 Hz), 156.1 (d, 3…”

“…By acting as transition state analogues of natural peptides during proteolysis, phosphinic peptides have provided a large number of potent inhibitors of zinc and aspartyl proteases over previous decades [2,5,6]. In addition, their propensity to bind tightly to their target has fueled efforts towards the development of enzymatic probes for imaging applications [10][11][12][13][14]. Their synthesis is typically based on building block strategies where side chains of the phosphinopeptidic scaffold are either preinstalled or introduced at a later stage of the synthetic plan by post-modification protocols [3,4].…”