A general method for the preparation of internally quenched fluorogenic protease substrates using solid-phase peptide synthesis.
A general scheme for obtaining a fluorescent donor/acceptor peptide substrate via solid-phase synthesis methodology is presented. The key feature of this method is the design of a glutamic acid derivative that has been modified on the carboxyl side chain with a 5-[(2'-aminoethyl)-amino]naphthelenesulfonic acid (EDANS) to create a fluorescent donor moiety that can be incorporated near the C-terminus of the peptide substrate. The corresponding fluorescent acceptor group containing a 4-[[4-(dimethylamino)phenyl]azo]benzoic acid (DABCYL) can then be attached to the resin-bound peptide at the N-terminus while all side-chain groups are still fully protected. Substrates for renin and HIV proteinase are synthesized as examples.
Maggiora
L L
LL
Biochemistry Unit, Upjohn Laboratories, Kalamazoo, Michigan 49001.
Smith
C W
CW
Zhang
Z Y
ZY
D000595:Amino Acid Sequence; D010450:Endopeptidases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D016333:HIV Protease / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates / Q000138:chemical synthesis / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis*; D012083:Renin / Q000378:metabolism; D013379:Substrate Specificity; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D010455:Peptides; C063126:5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid; C063127:4-(4-dimethylaminophenylazo)benzoic acid; D004124:p-Dimethylaminoazobenzene; D010450:Endopeptidases; D016333:HIV Protease; D012083:Renin