Regioselective hydroxylation of diverse flavonoids by an aromatic peroxygenase
Graphical abstract
Introduction
Flavonoids are the most incident antioxidants in higher plants with a large structural diversity.1 The precondition to their radical scavenging effect is the number and location of phenolic groups.2 For example the ortho-dihydroxy (catechol) substitution raise the radical scavenging activity.3 However, the synthesis of these complex compounds is complicated given that the selective transfer of oxygen atoms to non- or little activated carbons is still a challenging reaction in chemical synthesis.4 Therefore, multi-step syntheses are predominantly used in industrial processes of chemical hydroxylation.5, 6 Though progress has been reported in using hydrogen peroxide and metal catalyst for the oxidation of benzene and toluene derivatives, the number of direct hydroxylations as well as their selectivity is still limited.7, 8 More recently, a paper reported an efficient one-step oxidative modification of hydroxylated flavonoids with 2-iodoxybenzoic acid (IBX) in order to obtain catecholic flavonoids but not C-6 hydroxylation was observed.9 Another approach would be to use biocatalysts, such as cytochrome P450 monooxygenases (P450s) for highly selective one-step reactions under environmentally sound conditions.10 Nowadays this type of biotransformation is rarely used in chemical industry and restricted to whole cell processes, since P450s are poorly stable, catalytically slow, and require expensive cofactors as well as associated proteins.11 Another approach, the use of laboratory-evolved and engineered P450s for the H2O2-dependent monooxygenation via the so-called peroxide ‘shunt’ pathway, has been demonstrated but needs further optimization.12 Thus, biotransformations based on the activity of stable extracellular oxidoreductases would offer an elegant alternative. Possible candidates are found within the fungal proteomes including oxidases, peroxidases, as well as aromatic peroxygenases (APOs, EC 1.11.2.1).13 Enzymes of the latter group have been found in agaric basidiomycetes, and act as functional hybrids of heme thiolate peroxidases and P450s.14 The best-characterized fungal aromatic peroxygenase, from Agrocybe aegerita (AaeAPO), is involved in the H2O2-dependent hydroxylation/epoxidation of aromatic rings and benzylic compounds,14, 15, 16, 17, 18, 19, 20 phenol oxidation,15, 17 sulfoxidation of tricyclic heterocycles,16 N-oxidation of pyridine derivatives,21 and cleavage of diverse ethers.22 Here we demonstrate the catalytic potential of APOs for the H2O2-dependent regioselective hydroxylation of diverse flavonoids by the use of AaeAPO.
Section snippets
Hydroxylation of flavonoids
In qualitative experiments done with continuous H2O2 supply, we found that AaeAPO monooxygenated diverse flavonoids including flavones, flavanones, flavonols, isoflavons, and anthocyans (Table 1). The products were ring-hydroxylated compounds, which were identified by HPLC/MS based on authentic standards or via NMR. Notably, quercetin (2), daidzein (4), apigenin (5), kaempferol (6), and luteolin (8) yielded only one monohydroxylated flavonoid (MHF), whereas genistein (1) gave two MHFs. The
Discussion
AaeAPO selectively hydroxylated a variety of flavonoids in the presence of hydrogen peroxide, predominantly at the C6-position. We could show by 18O-labeling studies that the AaeAPO-catalyzed hydroxylation of flavonoids is a true peroxygenation, i.e., the transferred oxygen comes from the cosubstrate, H2O2. The results further indicate that the ring-hydroxylation of flavonoids proceeds via epoxide intermediates (Fig. 5). This picture is consistent with previous results, which proved the initial
Conclusion
The aromatic peroxygenase of A. aegerita monooxygenated a variety of flavonoids. According to the molecular structure of identified hydroxylated metabolites, the enzyme regioselectively hydroxylates the C6-position of flavonoids. We could show by 18O-labeling studies that the AaeAPO-catalyzed hydroxylation of flavonoids is a true peroxygenase reaction that proceeds via initially formed epoxide intermediates. These results raise the possibility that fungal peroxygenases may be useful for
Reactants
Flavonoids were purchased from Extrasynthese (Genay, France) except tangeretin and flavone, which were obtained from TCI (Zwijndrecht, Belgium) and Alfa Aesar (Karlsruhe, Germany), respectively. Organic solvents and H2O2 were purchased from Merck (Darmstadt, Germany) and from J. T. Baker (Mallinckrodt Baker B.V., AA Deventer, Holland). H218O2 (90 atom %, 2% wt/vol), was obtained from Icon Isotopes (New York, USA). All other chemicals were purchased from Sigma–Aldrich (Schnelldorf, Germany).
Acknowledgements
We thank M. Brandt and U. Schneider for technical assistance. Financial support of the German Environmental Foundation (DBU, project numbers 20008/959 and 13225–32) is gratefully acknowledged.
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