Redox-Regulated Inhibition of T7 RNA Polymerase via Establishment of Disulfide Linkages by Substituted Dppz Dirhodium(II,II) Complexes

Abstract

The series of dirhodium(II) complexes cis-[Rh(2)(O(2)CCH(3))(2)(R(1)R(2)dppz)(2)](2+) 1-6 (R(1) = R(2) = H, MeO, Me, Cl, NO(2) for 1-4, 6, respectively, and R(1)= H, R(2) = CN for 5), coordinated to R(1)R(2)dppz ligands with electron-donating or -withdrawing substituents at positions 7,8 of dppz (dppz = dipyrido[3,2-a:2',3'-c]phenazine), were synthesized and their effect on the transcription process in vitro was monitored. Complexes 1-6 are easily reduced, readily oxidize cysteine, and engage in redox-based reactions with T7-RNA Polymerase (T7-RNAP), which contains accessible thiol groups. Transcription is inhibited in vitro by 1-6 via formation of intra- and inter-T7-RNAP disulfide bonds that affect the enzyme critical sulfhydryl cysteine groups. The progressively increasing electron-withdrawing character of the dppz substituents (MeO < Me < H < Cl < CN < NO(2)) gives rise to the order 2 < 3 < 1 < 4 < 5 < 6 for the measured IC(50) values of 1-6. The ease of reduction for 1-6 is consistent with the energies of the dppz-centered lowest unoccupied molecular orbitals (LUMOs), which decrease with the electron-withdrawing character of the dppz substituents. The ligand-centered reductions for 1-6 are supported by electron paramagnetic resonance (EPR) studies which support the conclusion that reduction of 1-6 leads to the formation of dppz centered radicals [Rh(2)(O(2)CCH(3))(2)(R(1)R(2)dppz)(2)](*+) with isotropic g values approximately 2.003 which are essentially identical to the reported value for the free radical dppz anions. The EPR results are corroborated by density functional theory (DFT) calculations, which indicate that the complexes contain dppz-based LUMOs primarily phenazine (phz) in character; the unpaired electron is completely delocalized in the phenazine orbitals in 4-6. The low IC(50) values for 1-6 lend further support to the fact that they exhibit redox-based activity with the enzyme and lead to the conclusion that the complexes constitute a sensitive redox-regulated series of T7-RNAP inhibitors with the potential to control or inhibit other important biochemical processes.