Fluorescent derivatives of diphenyl [1-(N-peptidylamino)alkyl]phosphonate esters: synthesis and use in the inhibition and cellular localization of serine proteases.
Three fluorescein- and one Texas Red-labeled derivatives of [1-(N-dipeptidylamino)alkyl]phosphonate diphenyl esters were synthesized and evaluated as inhibitors of serine proteases. The two fluorophores, FITC and TXR, were attached to the peptide phosphonates via an epsilon-aminocaproyl unit that acts as a spacer group and facilitates the binding of the phosphonate inhibitor to the targeted enzymes. These derivatives are potent and specific inhibitors of chymotrypsin, porcine pancreatic elastase (PPE), and human leukocyte elastase (HLE). FTC-Aca-Phe-Leu-PheP(OPh)2 (3) inhibited chymotrypsin very potently (k(obsd)/[I] = 9500 M-1 s-1) and 600-fold better than it did PPE (k(obsd)/[I] = 16 M-1 s-1). FTC-Aca-Ala-Ala-MetP(OPh)2 (1) was a more effective inhibitor of chymotrypsin (k(obsd)/[I] = 190 M-1 s-1) than PPE and HLE (k(obsd)/[I] = 13 and 22 M-1 s-1, respectively). Only HLE and PPE were inhibited by FTC-Aca-Ala-Ala-AlaP(OPh)2 (2) (k(obsd)/[I] = 41 and 22 M-1 s-1, respectively). The specificity of these inhibitors toward the targeted serine proteases depends on the sequence of the tripeptide portion and was not affected by the presence of the fluorescent label. Trypsin, for instance, was not inhibited by any of these compounds. In some cases, the inhibitory potency was increased by the fluorescent label. For example, chymotrypsin was inhibited by the fluorescent compounds, FTC-Aca-Ala-Ala-MetP(OPh)2 (1) and FTC-Aca-Phe-Leu-PheP(OPh)2 (3), more potently than by the nonfluorescent compounds, Boc-Ala-Ala-MetP(OPh)2 (5) and Z-Phe-Leu-PheP(OPh)2 (7).(ABSTRACT TRUNCATED AT 250 WORDS)
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400.
A S
AS
Abuelyaman
D
D
Hudig
S L
SL
Woodard
J C
JC
Powers
D000595:Amino Acid Sequence; D002460:Cell Line; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes / Q000138:chemical synthesis; D006801:Humans; D007694:Killer Cells, Natural / Q000201:enzymology / Q000648:ultrastructure; D007700:Kinetics; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000138:chemical synthesis* / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology; D013379:Substrate Specificity; D014966:Xanthenes
D005456:Fluorescent Dyes; D009943:Organophosphorus Compounds; D015842:Serine Proteinase Inhibitors; D014966:Xanthenes; C034657:Texas red; D012697:Serine Endopeptidases; D016650:Fluorescein-5-isothiocyanate