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Synthesis and properties of Cbz-Phe-Arg-CHN2 (benzyloxycarbonylphenylalanylarginyldiazomethane) as a proteinase inhibitor.

ABSTRACT: The preparation of peptides terminating in -Arg-CHN2 has been attempted because of their potential value as proteinase inactivators. We have succeeded in one case, converting Cbz-Phe-ArgOH to the diazomethane without blocking the guanidino group. As expected from previous results with such reagents, the new derivative was extremely effective in inactivating a cysteine proteinase specific for cleaving at arginyl bonds, that is, clostripain. However, in contrast with the inertness of serine proteinases to reagents of this type in the cases examined previously, plasma kallikrein was inactivated by Cbz-Phe-Arg-CHN2, although with a considerably lower rate constant than with clostripain. Trypsin, however, was not inactivated, but gradually destroyed the reagent, as had been observed previously with chymotrypsin and Cbz-Phe-CHN2. This has now been re-examined with rho-nitro-Cbz-Ala-Phe-CHN2 and shown to involve a cleavage to rho-nitro-Cbz-Ala-PheOH, probably with liberation of diazomethane.

SUBMITTER: Zumbrunn A

PROVIDER: S-EPMC1148900 | biostudies-other | 1988 Mar

REPOSITORIES: biostudies-other

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Project description:The tremendous social and economic impact of thrombotic disorders, together with the considerable risks associated to the currently available therapies, prompt for the development of more efficient and safer anticoagulants. Novel peptide-based thrombin inhibitors were identified using in silico structure-based design and further validated in vitro. The best candidate compounds contained both L- and D-amino acids, with the general sequence D-Phe(P3)-Pro(P2)-D-Arg(P1)-P1'-CONH₂. The P1' position was scanned with L- and D-isomers of natural or unnatural amino acids, covering the major chemical classes. The most potent non-covalent and proteolysis-resistant inhibitors contain small hydrophobic or polar amino acids (Gly, Ala, Ser, Cys, Thr) at the P1' position. The lead tetrapeptide, D-Phe-Pro-D-Arg-D-Thr-CONH₂, competitively inhibits α-thrombin's cleavage of the S2238 chromogenic substrate with a K(i) of 0.92 µM. In order to understand the molecular details of their inhibitory action, the three-dimensional structure of three peptides (with P1' L-isoleucine (fPrI), L-cysteine (fPrC) or D-threonine (fPrt)) in complex with human α-thrombin were determined by X-ray crystallography. All the inhibitors bind in a substrate-like orientation to the active site of the enzyme. The contacts established between the D-Arg residue in position P1 and thrombin are similar to those observed for the L-isomer in other substrates and inhibitors. However, fPrC and fPrt disrupt the active site His57-Ser195 hydrogen bond, while the combination of a P1 D-Arg and a bulkier P1' residue in fPrI induce an unfavorable geometry for the nucleophilic attack of the scissile bond by the catalytic serine. The experimental models explain the observed relative potency of the inhibitors, as well as their stability to proteolysis. Moreover, the newly identified direct thrombin inhibitors provide a novel pharmacophore platform for developing antithrombotic agents by exploring the conformational constrains imposed by the D-stereochemistry of the residues at positions P1 and P1'.

Project description:The crystal structure of l-phenylalanyl l-phenylalanine (Phe-Phe, FF, a.k.a. diphenylalanine) is not merely noncentrosymmetric, but it is highly dipole parallel aligned. It is for this reason that FF is a nonlinear optical (NLO) material and exhibits strong second harmonic generation (SHG). Enhancement of the SHG response by ortho fluorination was demonstrated. Crystallization is nontrivial, and learning about the zwitterion structures in solution is important for the rational improvement of the crystallization process. Here, we present an NMR study of di-fluorinated FF (Phe(2-F)-Phe(2-F)) and mono-fluorinated FF isomers (Phe(2-F)-Phe and Phe-Phe(2-F)). The dipeptides were prepared by solid-phase synthesis and purified by high-performance liquid chromatography (HPLC). Their 1H and 13C NMR spectra were recorded in partially deuterated water (10% D2O), and two-dimensional (2D) NMR techniques were employed for signal assignments. The unambiguous assignments are reported of all chemical shifts for the aliphatic H and C atoms and of the C atoms of the carboxylate, the amide carbonyl, the CF carbons, and of every arene C atom in each phenyl ring. The dipeptides are trans amides and intramolecular hydrogen bonding between the ammonium group and the amide carbonyl restricts the H3N-CH-C(O) geometry. We explored the rotational profile of the diphenylalanines as a function of the τ = ∠(C-N-C-CO2) dihedral angle at the SMD(B3LYP/6-31G*) level without and with specific hydration and report the associated Karplus curves J(θ) vs θ = ∠(H-N-C-H). The rotational profiles show a maximum of three stationary structures, and relative conformer stabilities of the free diphenylalanines show that the conformation found in the crystal M1 is the least stable among the three, M3 > M2 ≫ M1. Specific water solvation makes all of the difference and adds a large competitive advantage to the water-bridged ion pair M1a. In fact, M1a becomes the most stable and dominant conformation for the parent diphenylalanine and mono1 F-FF and M1a becomes competitive with M3c for mono2 F-FF and di F-FF. Implications are discussed regarding the importance of the conformational preorganization of diphenylalanines in solution and the facility for their crystallization.

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Synthesis and properties of Cbz-Phe-Arg-CHN2 (benzyloxycarbonylphenylalanylarginyldiazomethane) as a proteinase inhibitor.

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