title: The synthesis and application of Fmoc-Lys(5-Fam) building blocks.
abstract: Fluorescence resonance energy transfer (FRET) peptide substrates are often utilized for protease activity assays. This study has examined the preparation of FRET triple-helical peptide (THP) substrates using 5-carboxyfluorescein (5-Fam) as the fluorophore and 4,4-dimethylamino-azobenzene-4'-carboxylic acid (Dabcyl) as the quencher. The N(α)-(9-fluorenylmethoxycarbonyl)-N(ε)-(5-carboxyfluorescein)-L-lysine [Fmoc-Lys(5-Fam)] building block was synthesized utilizing two distinct synthetic routes. The first involved copper complexation of Lys while the second utilized Fmoc-Lys with microwave irradiation. Both approaches allowed convenient production of a very pure final product at a reasonable cost. Fmoc-Lys(5-Fam) and Fmoc-Lys(Dabcyl) were incorporated into the sequence of a THP substrate utilizing automated solid-phase peptide synthesis protocols. A second substrate was assembled where (7-methoxycoumarin-4-yl)-acetyl (Mca) was the fluorophore and 2,4-dinitrophenyl (Dnp) was the quencher. Circular dichroism spectroscopy was used to determine the influence of the fluorophore/quencher pair on the stability of the triple-helix. The activity of the two substrates was examined with three matrix metalloproteinases (MMPs), MMP-1, MMP-13, and MT1-MMP. The combination of 5-Fam as fluorophore and Dabcyl as quencher resulted in a triple-helical substrate that, compared with the fluorophore/quencher pair of Mca/Dnp, had a slightly destabilized triple-helix but was hydrolyzed more rapidly by MMP-1 and MMP-13 and had greater sensitivity.
authors: Departments of Chemistry and Biology, Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL, 34987.
authors: Michal
authors: M
authors: Tokmina-Roszyk
authors: Dorota
authors: D
authors: Tokmina-Roszyk
authors: Gregg B
authors: GB
authors: Fields
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis; D008239:Lysine; D020782:Matrix Metalloproteinases; D010455:Peptides; D013379:Substrate Specificity
chemical_list: D010455:Peptides; D020782:Matrix Metalloproteinases; D008239:Lysine
keywords: collagen; fluorescein; fluorescence resonance energy transfer; matrix metalloproteinase; triple-helix