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The synthesis and application of Fmoc‐Lys(5‐Fam) building blocks
Author(s) -
TokminaRoszyk Michal,
TokminaRoszyk Dorota,
Fields Gregg B.
Publication year - 2013
Publication title -
peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.22222
Subject(s) - chemistry , fluorophore , substrate (aquarium) , förster resonance energy transfer , circular dichroism , peptide , triple helix , fluorescence , combinatorial chemistry , stereochemistry , biochemistry , oceanography , physics , quantum mechanics , geology
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. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 347–355, 2013.