Stabilization of Collagen-Model, Triple-Helical Peptides for In Vitro and In Vivo Applications

Abstract

The triple-helical structure of collagen has been accurately reproduced in numerous chemical and recombinant model systems. Triple-helical peptides and proteins have found application for dissecting collagen-stabilizing forces, isolating receptor- and protein-binding sites in collagen, mechanistic examination of collagenolytic proteases, and development of novel biomaterials. Introduction of native-like sequences into triple-helical constructs can reduce the thermal stability of the triple-helix to below that of the physiological environment. In turn, incorporation of nonnative amino acids and/or templates can enhance triple-helix stability. We presently describe approaches by which triple-helical structure can be modulated for use under physiological or near-physiological conditions.

Second Generation Triple-Helical Peptide Inhibitors of Matrix Metalloproteinases

“…To efficiently incorporate Flp and mep into a peptide sequence, convenient synthetic routes for the preparation of Fmoc-Flp (Figure 1, 4 ) and Fmoc-mep (Figure 1, 5 ) were required. We recently reported a convenient synthetic route for the preparation of Fmoc-Flp from readily available, inexpensive trans -(4 R )-hydroxy- l -proline 96. The fluorination step was modified herein.…”

Development of a Förster resonance energy transfer assay for monitoring bacterial collagenase triple-helical peptidase activity

“…The Mca fluorophore/Dnp quencher pair is widely used in protease research, and both Mca and Dnp are commercially available as Fmoc-Lys-modified building blocks. Designed substrates had four Gly-Pro-Hyp tripeptide units on both sides of the putative cleavage site sequence, as Gly-Pro-Hyp repeating tripeptides stabilize the triple-helix [26]. …”

“…Baker; Fmoc-amino acids, 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU), and N , N -dimethylformamide (DMF) (AR grade) from AGTC Bioproducts (Wilmington, MA); 2,5-dihydroxybenzoic acid (DHB) and trifluoroacetic acid (TFA) from Thermo Fisher Scientific (Waltham, MA); N α -(9-fluorenylmethoxycarbonyl)-4 R -fluoro-L-proline (Fmoc-Flp) from Bachem (Torrance, CA); decanoic acid (designated C 10 ) and dithiothreitol (DTT) from Acros Organics (Geel, Belgium); and NovaPEG Rink amide resin from EMD Millipore (San Diego, CA). N α -(9-fluorenylmethoxycarbonyl)-4 R -methyl-L-proline (Fmoc-mep) and fTHP-15 [(Gly-Pro-Hyp) 5 -Gly-Pro-Lys(Mca)-Gly-Gln-Pro-Gly-Leu-Arg-Gly-Gln-Lys(Dnp)-Gly-Val-Arg-(Gly-Pro-Hyp) 5 -NH 2 ] were synthesized as described [19, 22, 26]. …”

Matrix Metalloproteinase Inhibition by Heterotrimeric Triple‐Helical Peptide Transition State Analogues

“…Fmoc-4 R -methyl-L-proline (Fmoc-mep-OH), Knight SSP [Lys(Mca)-Pro-Leu-Gly-Leu-Lys(Dnp)-Ala-Arg-NH 2 ], fluorogenic triple-helical peptide (fTHP)-15 [(Gly-Pro-Hyp) 5 -Gly-Pro-Lys(Mca)-Gly-Pro-Gln-Gly~Leu-Arg-Gly-Gln-Lys(Dnp)-Gly-Val-Arg-(Gly-Pro-Hyp) 5 -NH 2 ], and α1(I-III)GlyΨ{PO 2 H-CH 2 }Leu THPI [C 6 -Gly-Pro-Flp-(Gly-Pro-Hyp) 4 -Gly-Pro-Gln-GlyΨ{PO 2 H-CH 2 }( R , S )Leu-Ala-Gly-Gln-Arg-Gly-Ile-Arg-(Gly-Pro-Hyp) 4 -Gly-Pro-Flp-NH 2 ] were synthesized by methods described previously. [8a, 23] Phosphinate pseudodipeptide Fmoc-GlyΨ{P(OAd)(OH)-CH 2 }Ile-OH [O-adamantyl-P-(9-fluorenylmethyloxycarbonyl-aminomethyl)-P-{2-(2-butyl)propionic acid-3-yl)-phosphinate] was synthesized as described. [24] …”