Novel inhibitors of procollagen C-proteinase. Part 2: glutamic acid hydroxamates.

Glutamic acid derived hydroxamates were identified as potent and selective inhibitors of procollagen C-proteinase, an essential enzyme for the processing of procollagens to fibrillar collagens. Such compounds have potential therapeutic application in the treatment of fibrosis.

Novel inhibitors of procollagen C-terminal proteinase. Part 1: diamino Acid hydroxamates.

The parallel synthesis of novel inhibitors of procollagen C-terminal proteinase is described. The synthetic strategy allowed for the facile synthesis of a large number of side-chain diversified diamino acid hydroxamates, of which the D-diaminopropionic acid derivatives were shown to be single digit nanomolar PCP inhibitors.

Amino acid derived sulfonamide hydroxamates as inhibitors of procollagen C-proteinase: solid-phase synthesis of ornithine analogues.

A discussion of the solid-phase synthesis of ornithine derived sulfonamide hydroxamic acids is illustrated. These analogues are shown to be potent, non-peptide inhibitors of procollagen C-proteinase (PCP).

Synthesis, biological activity and conformational study of a somatostatin hexapeptide analogue containing a reduced peptide bond.

The cyclic hexapeptide analogue of somatostatin, c[Phe psi(CH2-N)Pro-Phe-D-Trp-Lys-Thr], was prepared by solid-phase synthesis of the linear precursor, followed by cyclization using diphenylphosphoryl azide. The inhibition of GH release, as well as receptor affinity, is greatly decreased. Conformational analysis by NMR in DMSO/H2O (1/1) revealed the presence of a type II' beta-turn in the core tetrapeptide region and a delta-turn over the reduced peptide bond.

Comparative study of methods to couple hindered peptides.

A comparative study of modern coupling reactions involving Boc-protected amino acid derivatives and dipeptides with N-terminal alpha,alpha-dialkylation and N-methylation was carried out. The coupling reactions were run using either equimolar amounts of the amino and activated carboxyl components or an excess of the activated carboxyl component. Yields of the target tripeptide Boc-Phe-Xaa-Phe-OBzl (Xaa = (NMe)Ala, (NMe)Aib, or (NMe) alpha Ac5c) were compared. Less than 10% of the product was obtained from methods utilizing pivaloyl mixed anhydride, pentafluorophenyl ester or acyl fluoride activation when Xaa = (NMe)Aib and (NMe) alpha Ac5c. At room temperature, significant yields of these two products were obtained from reactions which utilized an excess of the HBTU reagent (O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate), the PyBroP reagent (bromo-tris-pyrrolidino-phosphonium hexafluorophosphate) or Boc-Phe-NCA (Boc-protected phenylalanine N-carboxyanhydride). Moreover, the Boc-Phe-NCA method was superior when used over a prolonged reaction time or at elevated temperature.

Morphiceptin and beta-casomorphin-5 analogues containing a reduced peptide bond: selective mu-receptor agonists and a novel mu antagonist, H-Tyr-Pro psi (CH2-NH)Phe-Pro-Gly-OH.

In order to prevent enzymatic degradation of beta-casomorphin-5 (1) and morphiceptin, reduced peptide bonds were incorporated at the 2-3 and 3-4 bonds, respectively. The analogues were synthesized by a combination of solid phase methodology and reductive alkylation of resin-bound peptide amines with Boc-amino acid aldehydes (Boc: tert-butyloxycarbonyl) in the presence of NaBH3CN. During reversed phase high pressure liquid chromatography purification, peak shape distortions could be observed. Epimerization was excluded, based on gas chromatography/mass spectroscopy analysis, which indicated acceptable levels of racemization (less than 3%) in the crude product. Instead, the phenomena could be attributed to slow cis/trans isomerizations originating from the Xxx-Pro bonds in the sequence. The presence of different conformational isomers was also established by 1H-nmr spectroscopy in DMSO-d6. All analogues showed high stability in blood plasma, enhanced binding affinity for the mu receptor, and very low binding to the delta receptor. While the Phe 3 psi(CH2-N)Pro4 analogues (3) and (5) displayed agonist activity, the Pro 2 psi(CH2-NH)Phe3 modified analogue (2) showed antagonist activity comparable to D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2.

Assignment of the 1H-NMR resonances of the four rotamers of beta-casomorphin-5 in DMSO.

We report the complete assignment of the 1H-nmr spectrum of beta-casomorphin-5 in DMSO-d6 solution. With a combination of one-dimensional, double quantum filtered correlated spectroscopy, homonuclear Hartmann-Hahn, and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY) spectra, we were able to differentiate the four conformers originating from two Xxx-Pro bonds present in the sequence. Exchange peaks in the ROESY spectra confirmed the presence of four interchanging conformational isomers. Based on integrations, the relative populations of the four species were estimated, while characteristic sequential nuclear Overhauser enhancements (NOEs) were used to determine the orientation of the Xxx-Pro bonds. This orientation was also shown to correlate with the chemical shift changes for the alpha protons of both the Xxx and Pro residues. Finally, interresidue NOEs indicate conformational preferences for the aromatic side chains, especially in the all-trans conformer.

Cyclic hexapeptides related to somatostatin. Conformational analysis employing 1H-NMR and molecular dynamics.

We report the conformational analysis of a series of cyclic hexapeptides related to the hormone somatostatin utilizing 1H NMR spectroscopy and NOE restrained molecular dynamics. The conformational preferences and results from biological analysis of these analogs (previous paper) allow for refinement of the current understanding of the structure-activity relationship of somatostatin. For most of the molecules examined, a beta II' turn about the D-tryptophan-lysine residues, postulated to be required for biological activity, was present. From the NOE restrained molecular dynamics, it can be seen that the turn structure is important for the maintenance of the proper orientation of the side chains of the adjacent phenylalanine, tryptophan and lysine. The biologically active analogs have the side chains of lysine and D-tryptophan extended away from the 18-membered ring in close proximity to each other for a significant portion of the dynamic simulations. Although other conformations are accessible and monitored during the simulations, we believe this is important for biological recognition. The absence of the beta II' turn at the D-tryptophan-lysine disrupts this side chain array producing inactive molecules. The role of the bridging region, the Phe-Pro dipeptide, is to stabilize the beta II' turn and help maintain the proper orientation of the biologically important side chains.

Determination of the chiral purity of dipeptide isosteres containing a reduced peptide bond by gas chromatographic analysis.

The stereochemical purity of psi (CH2-NH) dipeptides has been determined using gas chromatography-mass spectrometry. Different structures were found due to the derivatization procedures. A selective preparation of the linear bistrifluoroacetylated derivative and the monotrifluoroacetylated lactam makes it possible to monitor the chiral purity of the pseudodipeptides synthesized. Racemization occurring during peptide hydrolysis can be differentiated from racemization during the synthesis by using deuterium labelling. The method allows the optimization of the synthesis protocols and will be useful for further monitoring of the chiral purity of the pseudopeptides synthesized.