Highly sensitive intramolecularly quenched fluorogenic substrates for renin based on the combination of L-2-amino-3-(7-methoxy-4-coumaryl)propionic acid with 2,4-dinitrophenyl groups at various positions

The development of renin inhibitors for the treatment of hypertension requires highly sensitive substrates to evaluate potency and to characterize the mechanism of tight-binding inhibitors. A series of intramolecularly quenched fluorogenic renin substrates, based on the N-terminal tetradecapeptide sequence of human angiotensinogen (hTDP), was synthesized using a solid-phase technique. Incorporation of the fluorescent amino acid L-Amp [L-2-amino-3-(7-methoxy-4-coumaryl)propionic acid] and the DNP (2,4-dinitrophenyl) group at various positions resulted in >90% quenching efficiency and strong product fluorescence. Shortening the hTDP sequence to an octapeptide from histidine in P5 to histidine in P3′ (substrate 3) resulted in an acceptable k_cat/K_m (41000 M⁻¹·s⁻¹) and further systematic variation gave substrate 9, DNP-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-L-Amp, with a k_cat/K_m value of 350000 M⁻¹·s⁻¹ and 94% quenching efficiency. The free side chain of lysine, replacing the isoleucine residue at P6 position in the angiotensinogen sequence, contributed to the increased value for k_cat. The pH dependence of k_cat/K_m for renin and substrate 9 showed that the optimal pH is at pH 6–7. It also showed two titrating groups on the acidic side of the pH optimum, and one titrating group with a pK_a of 7.8 on the alkaline side. The combination of good kinetic and spectroscopic properties resulted in a >20-fold improvement in the sensitivity of renin assay, compared with the commercial substrate Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys(DABCYL)-Arg {where EDANS is 5-[(2-aminoethyl)amino]naphthalene-1-sulphonic acid and DABCYL is 4-(4-dimethylaminophenylazo)benzoic acid} (k_cat/K_m=268000 M⁻¹· s⁻¹, quenching efficiency <80%). The detection limit in a microplate renin assay was 60 pM, making substrate 9 well suited for the evaluation of inhibitors at picomolar concentrations.