Modelling the site of bromide binding in vanadate-dependent bromoperoxidases

Abstract

Treatment of Boc-protected (S)-serine (Ser) methyl ester with triphenylphosphine bromide Ph₃PBr (intermittently generated from PPh₃ and N-bromosuccinimide) yields Boc-3-bromoalanine (R)-Boc–BrAlaMe and, after deprotection, bromoalanine methyl ester (R)-BrAlaMe in the form of its hydrobromide. Boc–BrAlaMe and BrAlaMe have been structurally characterised. The reaction between BrAlaMe, salicylaldehyde (sal) and VO²⁺ results in the formation of Schiff base complexes of composition [VO(sal–BrAlaMe)solv]⁺ (solv = CH₃OH: 3, THF: 5) and [VO(sal–BrAla)THF] 4. DFT calculations of the structures of 3, 4 and 5, based on the B3LYP functional and employing the triple zeta basis set 6-311++g(d,p), provide distances Br⋯V = 4.0 ± 0.1 Å, if some distortion of the dihedral angle ∠N–C–C–Br is allowed (affording a maximum energy of ca. 45 kJ mol⁻¹), and thus model Br⋯V distances detected by X-ray methods in bromoperoxidases from the marine algae Ascophyllum nodosum and Corallina pilulifera. The DFT calculations have been validated by comparing calculated and found structures, including the new complex [V^VO(Amp–sal)OMe(MeOH)] (1, Amp is the aminophenol moiety) and the known complex [VO(L-Ser–van)H₂O] (van = vanillin). Additional validation has been undertaken by checking experimental against calculated (BHandHLYP) EPR spectroscopic hyperfine coupling constants. Complexes containing bromine as a substituent at the phenyl moiety of a Schiff base ligand do not allow for an appropriate simulation of the Br⋯V distance in peroxidases. The closest agreement, d(Br⋯V) = 4.87 Å, is achieved with [VO(3Br–salSer)THF] (6), where 3Brsal–Ser is the dianionic Schiff base formed between 3-Br-5-NO₂-salicylaldehyde and serine.