The effects of metal cofactors on the reactivity of quercetin 2,4-dioxygenase: synthetic model studies with M(ii)-complexes (M = Mn, Co, Ni, Cu, Zn) and assessment of the regulatory factors in catalytic efficacy

Abstract

This paper demonstrates the metal ion effects on the quercetin 2,4-dioxygenase (2,4-QD)-like reactivity. For this purpose, a series of five metal(II)–acetato complexes [M^II(L)(OAc)] {M = Mn (1^OAc), Co (2^OAc), Ni (3^OAc), Cu (4^OAc), Zn (5^OAc); OAc = acetate} supported with a newly designed N₃O-donor carboxylato ligand L⁻ {L⁻ = 2-((benzyl((6′-methyl-[2,2′-bipyridin]-6-yl)methyl)amino)methyl)benzoate} has been synthesised as models for the active sites of M^II-substituted 2,4-QDs. The enzyme–substrate (ES) model complexes [M^II(L)(fla)] {M = Mn (1^fla), Co (2^fla), Ni (3^fla), Cu (4^fla), Zn (5^fla); flaH = flavonol} have been synthesised by reacting flaH with their corresponding acetate-bound complexes in basic conditions. Detailed physicochemical properties of all the compounds are reported. Furthermore, single-crystal X-ray diffractions have been done to determine the structures of the compounds 2^OAc·2H₂O, 3^OAc, 4^OAc·CH₂Cl₂·2H₂O, 5^OAc·2H₂O and 2^fla·MeOH. The enzymatic reactivities of complexes 1^OAc–5^OAc towards the dioxygenation of flavonol have been explored in detail. All the complexes effectively catalyse the oxygenative degradation of flavonol in N,N-dimethylformamide (DMF) medium at 70 °C under multiple-turnover conditions and produce enzyme-type products. Kinetic investigations were performed to see the metal ions’ effects on reactivity. The reaction rates vary with the metal ions, showing the order Co > Ni > Zn > Mn > Cu. The studies reveal that the reactivities of the [M^II(L)(OAc)] complexes are governed primarily by three factors viz the ES adduct formation constant (K_f), the redox potential (E_pa) of the bound fla⁻/fla˙ couple, and the degree of delocalisation of the fla˙ radical with the metal electrons, which are drastically influenced by the M²⁺ ions. In the mechanistic interpretation, a single-electron transfer (SET) from the bound-flavonolate to dioxygen has been proposed to generate the catalytically important “M(II)–fla˙” radical and superoxide ion, which react further to bring about the dioxygenation reaction. The identification of the metal(II)-bound flavonoxy radical intermediate for the case of cobalt using EPR spectroscopy and the detection of superoxide ion by NBT²⁺ test and EPR spin-trapping experiment (DMPO test) are remarkable in envisaging the reaction pathway.