title: Aminonaphthalenesulfonamides, a new class of modifiable fluorescent detecting groups and their use in substrates for serine protease enzymes.
abstract: A series of new compounds, 6-amino-1-naphthalenesulfonamides (ANSN), were used as fluorescent detecting groups for substrates of amidases. These compounds have a high quantum fluorescent yield, and the sulfonyl moiety permits a large range of chemical modification. Fifteen ANSN substrates with the structure (N alpha-Z)Arg-ANSNR1R2 were synthesized and evaluated for their reactivity with 8 proteases involved in blood coagulation and fibrinolysis. Thrombin, activated protein C, and urokinase rapidly hydrolyzed substrates with monosubstituted sulfonamide moieties (R1 = H). The maximum rate of substrate homologue). The hydrolysis rates for substrates with branched substituents were slower than their linear analogues. Monosubstituted (N alpha-Z)Arg-ANSNR1R2 possessing cyclohexyl or benzyl groups in the sulfonamide moiety were hydrolyzed by these three enzymes at rates similar to that of the n-butyl homologue (except the cyclohexyl compound for u-PA). Factor Xa rapidly hydrolyzed substrates with short alkyl chains, especially when R1 = R2 = CH3 or C2H5. Lys-plasmin and rt-PA demonstrated low activity with these compounds, and the best results were accomplished for monosubstituted compounds when R2 = benzyl (for both enzymes). Factor VIIa and factor IXa beta exhibited no activity with these substrates. A series of 14 peptidyl ANSN substrates were synthesized, and their reactivity for the same 8 enzymes was evaluated. Thrombin, factor Xa, APC, and Lys-plasmin hydrolyzed all of the substrates investigated. Urokinase, rt-PA, and factor IXa beta exhibited reactivity with a more limited group of substrates, and factor VIIa hydrolyzed only one compound (MesD-LGR-ANSN(C2H5)2). The substrate ZGGRR-ANSNH (cyclo-C6H11) showed considerable specificity for APC in comparison with other enzymes (kcat/KM = 19,300 M-1 s-1 for APC, 1560 for factor IIa, and 180 for factor Xa). This kinetic advantage in substrate hydrolysis was utilized to evaluate the activation of protein C by thrombin in a continuous assay format. Substrate (D-LPR-ANSNHC3H7) was used to evaluate factor IX activation by the factor VIIa/tissue factor enzymatic complex in a discontinuous assay. A comparison between the commercially available substrate chromozyme TH (p-nitroanilide) and the ANSN substrate with the same peptide sequence (TosGPR) demonstrated that aminonaphthalenesulfonamide increased the specificity (kcat/KM) of substrate hydrolysis by thrombin more than 30 times, with respect to factor Xa substrate hydrolysis.
authors.affiliation: Institute of Biochemistry, Vilnius-MTP, Lithuania.
authors.forename: S
authors.initials: S
authors.lastname: Butenas
authors.forename: T
authors.initials: T
authors.lastname: Orfeo
authors.forename: J H
authors.initials: JH
authors.lastname: Lawson
authors.forename: K G
authors.initials: KG
authors.lastname: Mann
mesh_terms: D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004789:Enzyme Activation; D005164:Factor IX / Q000378:metabolism; D015942:Factor VIIa / Q000378:metabolism; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates / Q000737:chemistry* / Q000378:metabolism; D011486:Protein C / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D011486:Protein C; D005164:Factor IX; D013925:Thromboplastin; D012697:Serine Endopeptidases; D015942:Factor VIIa; D013917:Thrombin