Effect of intramolecular hydrogen bonding on biomimetic reactions by flavins

Introduction

Flavoenzymes are a ubiquitous and diverse class of biological redox catalysts [1, 2]. The active site responsible for the activation of molecular oxygen has been established as the enzyme-bound 4a-hydroperoxyflavin [1–4]. One of the important roles of the proteins has been suggested to stabilize the intermediate 4a-hydroperoxyflavins by hydrogen bonds of the N(5) to the proteins. Otherwise, the 4a-hydroperoxyflavins undergo spontaneous elimination of H₂O₂ to yield the oxidized flavins [5]. Thus, the intramolecular hydrogen bonding of N(5) to the carboxylic acid group at C(6) in the flavin system has been anticipated to stabilize the 4a-hydroperoxide intermediate by suppression of the elimination of H₂O₂ (Scheme 1). The model reactions for flavoenzymes performed so far have used the flavinium derivatives for the purpose of the stabilization of the 4a-hydroperoxyflavins as well as flavin semiquinone radicals [6–9]. Also, the oxidation of thiols to disulfides, which involves nucleophilic attack to C-4a position of isoalloxazine, is stimulated by the activation of C-4a position through hydrogen bonding [10].

Scheme 1

In this work, to obtain an insight into the role of hydrogen bonding to control the reactivities of flavin coenzymes, the oxidation of thioanisole (5) in the presence of carboxyflavins 1–4 has been studied.

Results and discussion

The reaction of thioanisole (5) with H₂O₂ in acetonitrile in the presence of flavin derivatives (1–4, Figure 1) afforded thioanisole S-oxide (6) in varying yields (Table 1). In the absence of the flavin derivatives, only 10% of the sulfide 5 was oxidized. In the presence of flavin derivatives without the carboxyl group (compounds 1 and 2), the conversion was only 9–12%. This shows that the oxidized flavin without the carboxy group does not form 4a-hydroperoxide intermediate and, as such, does not play a role in the oxygenation of sulfides. Further, the reaction of 5 with H₂O₂ in the presence of 10-butyl-6-carboxyflavin (4a) gave the S-oxide 6 in 65% yields, whereas the same reaction in the presence of 10-butyl-7-carboxyflavin (3a) afforded 6 in a much smaller yield of 35% (Table 1). Similar results have been obtained with decyl substituted flavins (Table 1). These results suggest that the intramolecular hydrogen bonding of N(5) to the carboxyl group at C-6 is essential for this type of activation of H₂O₂ by the flavin derivatives. The stabilization of 4a-hydroperoxide intermediate is found to be optimal with carboxylate group at C(6) in comparison with C(7). The C(7) derivative may be involved in a much weaker intermolecular hydrogen bond. Further, the esterification of the carboxyl groups of flavin 4b leads to the loss of reactivity (entry 10, Table 1), again suggesting the role of hydrogen bonding in the activation of H₂O₂. The generation of the intermediate C4a-hydroperoxyflavin is consistent with the appearance of absorption at λ = 384 nm in the UV-visible spectrum [11]. The role of hydrogen bonding in the activation of H₂O₂ was further confirmed in the experiments conducted in the presence of benzoic acid, which may form hydrogen bonding between N(5) and activate the 4a position. The addition of benzoic acid results in the enhancement of yields from 9–12% to 42–45%.

Figure 1

Flavins used in this study.

Experimental