Hydrogen bond-free flavin redox properties: managing flavins in extreme aprotic solvents

Abstract

We report a simple, single step reaction that transforms riboflavin, which is insoluble in benzene, to tetraphenylacetyl riboflavin (TPARF), which is soluble in this solvent to over 250 mM. Electrochemical analysis of TPARF both alone and in complexes with two benzene-soluble flavin receptors: dibenzylamidopyridine (DBAP) and monobenzylamidopyridine (MBAP), prove that this model system behaves similarly to other previously studied flavin model systems which were soluble only in more polar solvents such as dichloromethane. Binding titrations in both benzene and dichloromethane show that the association constants of TPARF with its ligands are over an order of magnitude higher in benzene than dichloromethane, a consequence of the fact that benzene does not compete for H-bonds, but dichloromethane does. The alteration induced in the visible spectrum of TPARF in benzene upon the addition of DBAP is very similar to the difference produced by transfer to dichloromethane, further indicating that the flavin head group engages in a similar degree of hydrogen bonding with dichloromethane as with its ligands. This work underlines the importance of using a truly nonpolar solvent for the analysis of the effects of hydrogen bonding on the energetics of any biomimetic host-guest model system.

15N Solid-State NMR as a Probe of Flavin H-Bonding

“…80 Indeed, our spectroscopically-validated calculations find that the redox-active diazabutadiene system becomes less net-negative, consistent with the observed increase in reduction midpoint potential, even though electron density is little changed at the actual sites of H-bonding. Thus our calculations are consistent with experimental findings that complexation with DBAP increases the midpoint potential observed for one-electron reduction of TPARF in benzene by 100 mV, 46 similar to the 150 mV increase observed for analogous model systems 44 and the 170 mV increase in the one-electron reduction potential of FMN upon binding to flavodoxin. 81,82 This suggests that NMR-validated calculations may prove useful for understanding reactivity, and possibly even predicting it.…”

“…These results are consistent with the measured 100 mV increase in TPARF’s reduction midpoint potential upon complexation with DBAP. 46 …”

“…44 However it is clear that the commonly-used solvent CHCl 3 significantly attenuated the effect of added H-bonding partners, 45 and recent work shows that this solvent interacts with the flavin, acting as a H-bond donor itself. 46 Thus, many studies likely document the effect of displacement of solvent by the H-bonding partner. In order to be able to work in a non-H-bonding solvent, we designed and synthesized a new flavin model: tetraphenylacetylriboflavin (TPARF), wherein the ribityl side chain is decorated with four benzyl groups that dissolve readily in benzene.…”

Contrasting roles for two conserved arginines: Stabilizing flavin semiquinone or quaternary structure, in bifurcating electron transfer flavoproteins

“…Indeed, the vibronic structure characterizing the Bf flavin's band I was lost in the R165K-RpaETF variant, but not variants perturbed in the ET site (see Figure 2 and caption). Because the vibrational structure is a signature of Hbonding (35,36), its loss in R165K-RpaETF, specifically, suggests that in WT-RpaETF the flavin H-bonds with Arg-165. Bf FAD's greater sensitivity to local perturbation than displayed by ET flavin is consistent with the lower solvent exposure of the Bf flavin (see Table 2).…”

Manipulating reduction potentials in an artificial safranin cofactor

“…Despite the fact that flavoenzymes form more than a tenth of known cofactor-containing enzymes [15], there have been no reports thus far of artificial flavin enzymes which utilize artificial flavin-like cofactors, and likewise no reports of the synthesis of flavin-like cofactors created for use in such a manner. We have reported the synthesis and characterization of riboflavin derivatives with differing hydrophobicity [16, 17], but these lack desirable properties such as changes in reduction potential.…”