title: Synthesis, kinetic evaluation, and utilization of a biotinylated dipeptide proline diphenyl phosphonate for the disclosure of dipeptidyl peptidase IV-like serine proteases.
abstract: In this study, we report on the synthesis, kinetic characterisation, and application of a novel biotinylated and active site-directed inactivator of dipeptidyl peptidase IV (DPP-IV). Thus, the dipeptide-derived proline diphenyl phosphonate NH(2)-Glu(biotinyl-PEG)-Pro(P)(OPh)(2) has been prepared by a combination of classical solution- and solid-phase methodologies and has been shown to be an irreversible inhibitor of porcine DPP-IV, exhibiting an over all second-order rate constant (k(i)/K(i)) for inhibition of 1.57 x 10(3) M(-1) min(-1). This value compares favourably with previously reported rates of inactivation of DPP-IV by dipeptides containing a P(1) proline diphenyl phosphonate grouping [B. Boduszek, J. Oleksyszyn, C.M. Kam, J. Selzler, R.E. Smith, J.C. Powers, Dipeptide phophonates as inhibitors of dipeptidyl peptidase IV, J. Med. Chem. 37 (1994) 3969-3976; B.F. Gilmore, J.F. Lynas, C.J. Scott, C. McGoohan, L. Martin, B. Walker, Dipeptide proline diphenyl phosphonates are potent, irreversible inhibitors of seprase (FAPalpha), Biochem, Biophys. Res. Commun. 346 (2006) 436-446.], thus demonstrating that the incorporation of the side-chain modified (N-biotinyl-3-(2-(2-(3-aminopropyloxy)-ethoxy)-ethoxy)-propyl) glutamic acid residue at the P(2) position is compatible with inhibitor efficacy. The utilisation of this probe for the detection of both purified dipeptidyl peptidase IV and the disclosure of a dipeptidyl peptidase IV-like activity from a clinical isolate of Porphyromonas gingivalis, using established electrophoretic and Western blotting techniques previously developed by our group, is also demonstrated.
authors.affiliation: School of Pharmacy, Queens University Belfast, Medical Biology Centre, UK. b.gilmore@qub.ac.uk
authors.forename: Brendan F
authors.initials: BF
authors.lastname: Gilmore
authors.forename: Louise
authors.initials: L
authors.lastname: Carson
authors.forename: Laura L
authors.initials: LL
authors.lastname: McShane
authors.forename: Derek
authors.initials: D
authors.lastname: Quinn
authors.forename: Wilson A
authors.initials: WA
authors.lastname: Coulter
authors.forename: Brian
authors.initials: B
authors.lastname: Walker
mesh_terms: D000345:Affinity Labels; D019921:Biotinylation; D002846:Chromatography, Affinity / Q000379:methods*; D004151:Dipeptides; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D063065:Organophosphonates / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D016966:Porphyromonas gingivalis / Q000737:chemistry* / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D004151:Dipeptides; D063065:Organophosphonates; D018819:Dipeptidyl Peptidase 4; D012697:Serine Endopeptidases