Phosphinic Peptide Matrix Metalloproteinase-9 Inhibitors by Solid-Phase Synthesis Using a Building Block Approach

Abstract

The solid-phase synthesis of an array of different pseudopeptides containing a phosphinic glycine ± leucine moiety (-GY{P(O)OH-CH 2 }L-) [1] is described. The resulting pseudopeptides were shown to act as matrix metalloproteinase-9 (MMP-9) inhibitors. Starting from available materials, the protected amino acid isosters benzyloxycarbonyl aminomethylphosphinic acid (glycine analogue) and ethyl a-isobutylacrylate (leucine analogue) were synthesized and coupled with the bis(trimethylsilyl)phosphonite. Protective group interchange yielded a protected phosphinic dipeptide building block 1 ready for use in solid-phase peptide synthesis. Solid-phase peptide synthesis was performed with 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry on a polyethylene glycol polyamide (PEGA) support and the coupling of 1 (1.5 equiv) was carried out with standard activation. The P 4 ± P 2 and P 2 ' ± P 4 ' positions of the pseudopeptides were designed by analogy of the cleavage sequences of different natural extracellular matrix protein substrates with a synthetic peptide substrate of MMP-9. The crude peptides were obtained in high yield and purity as determined by RP-HPLC, and were characterized by electrospray mass spectrometry and amino acid analysis after purification. Enzyme kinetic investigations with MMP-9 of the purified peptide inhibitors showed K i values in the range from mm to nm.

Phosphinic Peptide Inhibitors of Macrophage Metalloelastase (MMP-12). Selectivity and Mechanism of Binding

“…Recombinant mouse full length pro-MMP-9 was expressed in BHK cells and subsequently purified from the conditioned medium [22]. Recombinant rabbit MMP-14 catalytic domain was cloned from rabbit osteoclasts [29] and purified from the supernatant of transfected E. coli cells (Ovejero, M.C, Lenhardt, T. and Ferreras, M. unpublished data).…”

Crystal Structures of Active LytM

“…mp, 95-96 8C. 24 The second component, N-acryloylglycinate 2 was obtained using the mixed anhydride procedure. This accomplished the activation of acrylic acid (1.44 g, 0.02 mol) to the corresponding anhydride by means of isobutyl chloroformate (2.72 ml, 0.021 mol) in the presence of N-methylmorpholine (2.33 ml, 0.021 mol) in anhydrous THF (20 ml) at 0 8C.…”

“…21,22 Then, the adduct was hydrolysed in acidic conditions and the obtained amino acid was N-protected with benzyl chloroformate in basic conditions as described in the literature. 23,24 The overall yield of the procedure was 45%. Mp, 90-91 8C (colourless crystals recrystallized from ethyl acetate/toluene), lit.…”

Efficient synthesis of Fmoc‐protected phosphinic pseudodipeptides: Building blocks for the synthesis of matrix metalloproteinase inhibitors

“…8–10 But our initial attempt of Michael-type addition of Fmoc-protected aminomethyl phosphinic acid and electron deficient acrylate was not successful. 8,11 Thus, Cbz-protected aminomethyl phosphinic acid 3 was converted to its trivalent state by the action of HMDS and subsequently reacted with an α -isopropyl- α , β -unsaturated ester 12 to give product 4 in good yield (Scheme 1). However, as will be described subsequently, the Cbz group is very efficiently exchanged for Fmoc in the final step of the synthesis.…”

“…Meldal and coworkers, Yiotakis et al, and Yokomatsu and coworkers reported the synthesis of different phosphinate dipeptide building blocks by using a procedure involving the Michael addition of variously protected aminomethyl phosphinic acids to an acrylic acid ester followed by protecting group manipulation to afford the desired product. 8 Using a creative variation of these methods, FmocNHCH 2 PO(OAd)-CH 2 CH(Pr i )CO 2 H ( 1a , Scheme 2) was prepared and subsequently incorporated into a triple-helical sequence, resulting in a construct, which exhibited potent and selective inhibition of MMP-2 and MMP-9. 7 However, this previously described synthetic route to 1a required a low yielding (35%) and problematic final step involving a Ru-catalyzed deprotection of an allyl ester.…”