Elsevier

Tetrahedron

Volume 67, Issue 26, 1 July 2011, Pages 4874-4878
Tetrahedron

Regioselective hydroxylation of diverse flavonoids by an aromatic peroxygenase

https://doi.org/10.1016/j.tet.2011.05.008Get rights and content

Abstract

Aromatic peroxygenases are extracellular fungal biocatalysts that selectively oxidize a variety of organic compounds. We found that the peroxygenase of the fungus Agrocybe aegerita (AaeAPO) catalyzes the H2O2-dependent hydroxylation of diverse flavonoids. The reactions proceeded rapidly and regioselectively yielding preferentially monohydroxylated products, e.g., from flavanone, apigenin, luteolin, flavone as well as daidzein, quercetin, kaempferol, and genistein. In addition to hydroxylation, O-demethylation of fully methoxylated tangeretin was catalyzed by AaeAPO. The enzyme was merely lacking activity on the quercetin glycoside rutin, maybe due to sterical hindrance by the bulky sugar substituents. Mechanistic studies indicated the presence of epoxide intermediates during hydroxylation and incorporation of H2O2-derived oxygen into the reaction products. Our results raise the possibility that fungal peroxygenases may be useful for versatile, cost-effective, and scalable syntheses of flavonoid metabolites.

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.

References and notes (35)

  • S.A.B.E. Van Acker et al.

    Free Radical Biol. Med.

    (1996)
  • A. Seyoum et al.

    Phytochemistry

    (2006)
  • G.D. Yadav et al.

    Appl. Catal., A

    (2003)
  • V.B. Urlacher et al.

    Trends Biotechnol.

    (2006)
  • M. Kinne et al.

    Tetrahedron Lett.

    (2008)
  • M. Kinne et al.

    Bioorg. Med. Chem. Lett.

    (2009)
  • M. Kinne et al.

    Biochem. Biophys. Res. Commun.

    (2010)
  • R. Ullrich et al.

    FEBS Lett.

    (2008)
  • M. Kinne et al.

    J. Biol. Chem.

    (2009)
  • A.O. Latunde-Dada et al.

    J. Biol. Chem.

    (2001)
  • E. Kostrzewa-Susłow et al.

    J. Mol. Catal. B: Enzym.

    (2007)
  • E. Kostrzewa-Susłow et al.

    J. Mol. Catal. B: Enzym.

    (2008)
  • M. Foti et al.

    J. Agric. Food Chem.

    (1996)
  • R. Ullrich et al.

    Cell. Mol. Life Sci.

    (2007)
  • A. Cybulski et al.
    (2001)
  • B. Lücke et al.

    Adv. Synth. Catal.

    (2004)
  • M. Tani et al.

    Angew. Chem., Int. Ed.

    (2005)
  • Cited by (36)

    • Total synthesis of scutellarin and apigenin 7-O-β-d-glucuronide

      2019, Carbohydrate Research
      Citation Excerpt :

      With 34 in hand, the substitution of iodide with hydroxyl group or its surrogates was investigated extensively. Impeded by the severe steric hindrance of the iodide atom imposed by the two flanked substituents, all attempts in copper- or palladium-catalyzed coupling with allyl alcohol, 1-trimethylsilyethanol, benzyl alcohol, and benzaldoxime under different conditions [26] uniformly met with failure, except for the try of coupling with sodium methoxide under the effect of CuBr, which indeed afforded the methyl protected scutellarein derivative [9b]. However, the methylated scutellarein can not serve as intermediate in the novel synthetic route, since the demethylation entails harsh reaction conditions.

    • Exploring the catalase activity of unspecific peroxygenases and the mechanism of peroxide-dependent heme destruction

      2016, Journal of Molecular Catalysis B: Enzymatic
      Citation Excerpt :

      The so far mostly studied UPO is secreted by the agaric basidiomycete Agrocybe aegerita (AaeUPO) [5]. AaeUPO has been shown to convert over 100 substrates via peroxygenation and beyond that, it catalyzes one-electron oxidations of a broad spectrum of phenolic compounds [1,6–11]. Despite the broad substrate spectrum, reactions catalyzed by UPOs can be highly regio- and enatio-selective [9,10].

    • Biocatalytic portfolio of Basidiomycota

      2016, Current Opinion in Chemical Biology
    View all citing articles on Scopus
    View full text