Biocatalytic Aromaticity-Breaking Epoxidation of Naphthalene and Nucleophilic Ring-Opening Reactions

Article abstract of DOI:10.1021/acscatal.0c05588

Aromatic hydroxylation reactions catalyzed by heme-thiolate enzymes proceed via an epoxide intermediate. These aromatic epoxides could be valuable building blocks for organic synthesis giving access to a range of chiral trans-disubstituted cyclohexadiene

Full text of DOI:10.1021/acscatal.0c05588

pubs.acs.org/acscatalysis
Research Article
Biocatalytic Aromaticity-Breaking Epoxidation of Naphthalene and
Nucleophilic Ring-Opening Reactions
Wuyuan Zhang,* Huanhuan Li, Sabry H. H. Younes, Patricia Gómez de Santos, Florian Tieves,
Gideon Grogan, Martin Pabst, Miguel Alcalde, Adrian C. Whitwood, and Frank Hollmann*
Cite This: ACS Catal. 2021, 11, 2644−2649
Read Online
ACCESS
Metrics & More
Article Recommendations
*
sı Supporting Information
ABSTRACT: Aromatic hydroxylation reactions catalyzed by heme-thiolate
enzymes proceed via an epoxide intermediate. These aromatic epoxides could
be valuable building blocks for organic synthesis giving access to a range of
chiral trans-disubstituted cyclohexadiene synthons. Here, we show that
naphthalene epoxides generated by fungal peroxygenases can be subjected to
nucleophilic ring opening, yielding non-racemic trans-disubstituted cyclo-
hexadiene derivates, which in turn can be used for further chemical
transformations. This approach may represent a promising shortcut for the
synthesis of natural products and APIs.
KEYWORDS: arene epoxides, arene oxyfunctionalization, biocatalysis, chemoenzymatic reactions, naphthalene epoxides, oxidation,
peroxygenase
romatic systems are thermodynamically and kinetically
very inert. Reactions breaking aromaticity are rare but
evolved peroxygenase from Agrocybe aegerita (rAaeUPO) as a
catalyst; more specifically, we first used a previously evolved
A
1
,2
24
interesting from a preparative point of view. Even less
common are enzymatic aromaticity-breaking reactions. Non-
heme Fe-dioxygenases have been reported for stereoselective
variant (PaDa-I). Naphthalene was used as a starting
11
material. Upon addition of PaDa-I (0.2 μMfinal) to a buffered
solution of naphthalene and H (2 mM each), we could
detect (and quantify) naphthalene-1,2-epoxide (2) via its
O
2
2
3
−5
cis-dihydroxylation of arenes.
More recently, enzymatic
25
6
−8
9
characteristic absorption band at 266 nm. 2 was relatively
stable and rearranged into the corresponding 1-naphthol
within several minutes (Figures S30 and S31). The epoxide
formation rate correlated with the concentration of the
Birch-type reductions
have also been reported.
Aromatic hydroxylation reactions catalyzed by heme-
and dearomatizing arene oxidations
10
dependent enzymes such as P450 monooxygenases and
11−15
biocatalyst (Table 1, entries 1−3). Increasing the H O
related peroxygenases
are well known to proceed via an
2
2
concentration above 2 mM resulted in a decreased
concentration of 2 (Table 1, entries 2, 4, and 5), which is
readily explained by the oxidative inactivation of heme
intermediate, short-lived arene oxide prone to rapid,
spontaneous rearrangement into the corresponding phenol
10
products (known as NIH-shift, Scheme 1b). Interestingly,
capturing the intermediate arene oxides with nucleophiles has
so far not been considered. This is unfortunate as arene oxides
are potentially very useful building blocks for chemical
synthesis. Nucleophilic opening of the reactive epoxide ring
leads to trans-disubstituted cyclohexadiene derivates, which
26
enzymes by excess H O . Further investigations will focus
2
2
on determining the kinetic parameters of the enzyme-catalyzed
epoxidation reaction. No epoxide formation was observed in
the absence of PaDa-I, naphthalene, or H O . Isotope labeling
2
2
1
8
experiments using H2 O confirmed that the oxygen atom
2
16−20
incorporated originated from H O (Figure S27).
2
2
can serve as starting materials for various syntheses.
Their
The decay of the intermediate epoxide accelerated upon
addition of azide, which we interpreted to be the result of
nucleophilic ring opening. Next, we investigated the factors
influencing the overall yield in the desired addition product
synthetic relevance today, however, is limited due to tedious,
21−23
multistep synthesis protocols (Scheme 1a).
Utilizing
heme-enzyme-derived aromatic epoxides, which are formed
directly from the corresponding arenes under mild conditions,
would give a more straightforward access to such trans-
disubstituted cyclohexadiene derivates.
We therefore set out to investigate whether trans-
disubstituted cyclohexadiene derivatives can be obtained in a
chemoenzymatic reaction comprising enzymatic formation of
arene oxides followed by nucleophilic epoxide opening
Received: December 21, 2020
Revised: January 29, 2021
Published: February 12, 2021
(Scheme 1c). As a starting point, we chose the recombinant,
©
2021 The Authors. Published by
American Chemical Society
https://dx.doi.org/10.1021/acscatal.0c05588
2
644
ACS Catal. 2021, 11, 2644−2649

ACS Catalysis
pubs.acs.org/acscatalysis
Research Article
a
Scheme 1. Synthesis of Arene Oxides
a
(a) The established, multistep synthesis of arene oxides at the example of benzene oxide. (b) Heme-enzyme-catalyzed oxyfunctionalisation of
aromatic compounds (e.g., benzene) proceeds via an intermediate arene epoxide spontaneously rearranging into the corresponding phenol; (c) In
this work, we demonstrate that the intermediate arene oxides (e.g., obtained from peroxygenase-catalyzed transformation of naphthalene) can be
reacted with nucleophiles, yielding chiral trans-disubstituted cyclohexadiene derivates.
(
4a) (Table 1). Increasing the nucleophile concentration
COSY, and HMQC-analysis of the products, see Figures S1−
22). The isolated yields ranged between 20 and 73% giving
access to 20−50 mg of the products, thereby correlating with
the above-determined NMR-yields. So far, only the crystal
structure of 4b is available, and we compared the circular
dichroism spectra of some representative products (i.e., 4d, 4g,
and 4i) with the CD spectrum of 4b (Figures S32 and S33).
The congruence of the optical rotatory dispersion curves
suggests an identical (1S, 2S) configuration of these products
as well.
significantly shifted the ratio of the desired (4a) to the
undesired (3) (Table 1, entries 6−9). Prolonging the reaction
time allocated to the enzymatic epoxidation had no significant
influence on the overall conversion of the naphthalene starting
material (ranging between 80 and 85%, Table 1, entries 10−
1
3) but increased the yield in the undesired rearrangement
product 1-naphthol (3). Adding NaN from the beginning of
3
the reaction resulted in the complete recovery of the
naphthalene starting material, which we attribute to the N -
−
3
related inactivation of the heme enzyme. It is worth
mentioning that the azide attacked the epoxide selectively at
the C1 position (yielding the 1-azido-2-ol product). This
selectivity was observed only with epoxide 2 and we are
currently lacking a plausible explanation for this peculiarity.
From a semi-preparative reaction of 1-bromonaphthalene,
approximately 39 mg of essentially pure (1S,2S)-2-azido-5-
bromo-1,2-dihydronaphthalen-1-ol was isolated (73% isolated
yield), crystallized, and analyzed via X-ray crystallography (see
the Supporting Information for further details). The structure
of the ring-opened product 4b showed an excellent Flack
parameter [−0.02(3)], thus allowing the determination of the
absolute configuration ((1S,2S)-2-azido-5-bromo-1,2-dihydro-
naphthalen-1-ol). This corresponds well with the predicted
stereoselectivity of the PaDa-I-catalyzed epoxidation of
naphthalene (Figure 1B). Also, the crystallization of 4a was
successful; however, probably due to the lack of a heavy atom
in the structure, the crystalized product 4a showed a less
convincing configuration (Flack) parameter [−0.3(3)]. As
mentioned above, the ring opening occurred via a nucleophilic
attack in the C2 position.
In an attempt to also broaden the nucleophile scope of the
reaction, we tested formate as an alternative nucleophile
(Figure 2, 4j and 4k). The isolated yields, however, were
significantly lower (9−15%) than observed using azide, which
most likely can be attributed to the poorer nucleophilicity of
formate as compared to azide under the reaction conditions.
Aiming at extending the arene scope of the proposed
chemoenzymatic reaction sequence, we also evaluated benzene
as the starting material. PaDa-I, the rAaeUPO variant used so
far, exhibited poor activity with benzene (Figure 2, 4m, blue).
We therefore also evaluated the SoLo variant (engineered from
PaDa-I) for the transformation of benzene (Figure 2, 4m,
green). The significantly higher activity of SoLo on benzene
(48% yield) compared to PaDa-I (traces) can be rationalized
by its modified active site geometry: SoLo carries two
mutations at the heme access channel (F191S and G241D,
Figure 3). Possibly, the G241D substitution induces a
displacement in the α-helix hosting the catalytic acid−base
pair (R189-E196). This conformational change may favor the
positioning of smaller arenes (see docking simulations, Figure
S34) and therefore facilitate the conversion of benzene.
Finally, we explored the synthetic possibilities of 4a.
Rearomatization proved to be astonishingly difficult as only
concentrated perchloric acid enabled the full conversion of the
intermediate epoxide (2) into the aromatic azide (5) (Figure 4
and see also Table S1). In contrast, catalytic hydrogenation
over Pd/C to form a compound (7, Figures S23 and S24) or
27
To further explore the substrate scope of the proposed
reaction sequence, a range of naphthalene derivates were
evaluated (Figure 2). Using azide as a nucleophile, the yields
ranged between 19 and 75%; products 4a, 4b, 4c, 4d, and 4e
were prepared on a semi-synthetic (0.2 mmol) scale (for full
1
13
1
1
experimental details as well as H NMR, C NMR, H− H
2
645
https://dx.doi.org/10.1021/acscatal.0c05588
ACS Catal. 2021, 11, 2644−2649

ACS Catalysis
pubs.acs.org/acscatalysis
Research Article
Table 1. Influence of Reaction Time and Nucleophile
Concentration on the Proposed Epoxidation Ring-Opening
Reaction
In this study, we have demonstrated that peroxygenases
provide a convenient access to naphthalene epoxide and
possibly further arene epoxides for the synthesis of chiral trans-
disubstituted cyclohexadiene derivates.
EXPERIMENTAL SECTION
■
Biocatalyst Preparation. The expression of the rAaeUPO
variants PaDa-I and SoLo was performed in recombinant
Pichia pastoris following a previously described procedure.
28
entry c(PaDa-I) [nM] c(H O ) [mM] c(2) [mM] TON (PaDa-I)
2
2
The culture broth of P. pastoris cells containing the
peroxygenase in the supernatant was centrifuged at 8000
rpm for 2 h at 4 °C. The supernatant was filtered through a 20
μm filter and kept at −80 °C until further use. A detailed
description of the cultivation procedure can be found in the
Supporting Information. The enzymes used in the study were
essentially pure as confirmed by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) in 12% gels
stained with Coomassie Brilliant Blue R-250 (Figure S34).
Chemoenzymatic Reactions. A typical epoxidation
reaction was performed at a 1 mL scale in NaPi buffer (100
mM, pH 7, containing 30% v/v acetonitrile as a cosolvent) at
PaDa-I-catalyzed Epoxidation of Naphthalene
1
2
3
4
5
100
200
400
200
200
2
2
2
1
4
0.82
1.06
1.29
0.73
0.91
8200
5300
3200
3650
4500
c(NaN )
reaction time
c(4a)
[mM]
c(3)
[mM]
TON
(PaDa-I)
3
b
[
mM]
[min]
c
Chemoenzymatic Reaction
6
7
8
9
1
1
1
1
31.3
62.5
125
250
125
125
125
125
2.5
2.5
2.5
2.5
3
4
6
10
0.61
0.97
1.48
1.36
1.42
1.43
1.34
1.27
0.36
0.30
0.16
0.23
0.19
0.26
0.30
0.32
3050
4850
7400
6800
7100
7250
6700
6350
2
5 °C. The reaction mixture contained an aromatic starting
material (2 mM) and rAaeUPO (PaDa-I or SoLo 200 nM).
Reactions were started by addition of H O (2 mM final) and
0
1
2
3
2
2
gently stirred for 2.5 min after which the nucleophile was
added from a concentrated stock solution. After the
completion of the reaction, the reaction mixture was extracted
with an aliquot dichloromethane. The organic phase was dried
a
Reaction conditions: [naphthalene] = 2 mM, [H O ] = 1−4 mM,
2
2
and [PaDa-I] = 100−400 nM in NaPi buffer pH 7.0 (30% CH CN),
3
over MgSO and evaporated under reduced pressure. The
4
3
0 °C. The spectrum was recorded after diluting the solutions 100
crude product was purified over a silica column using 10%
ethyl acetate in pentane/heptane as the eluent.
−
1
−1
times; an extinction coefficient of 8850 mM cm at 266.5 nm was
b
used to calculate the concentration. Time allocated for the enzymatic
reaction step (i.e., prior NaN addition). [Naphthalene] = 2 mM,
c
Crystal Structures of 4a and 4b. Diffraction data were
collected at 110 K on an Oxford Diffraction SuperNova
diffractometer with Cu Kα radiation (λ = 1.54184 Å) using an
EOS CCD camera. The crystal was cooled with an Oxford
Instruments Cryojet. Diffractometer control, data collection,
initial unit cell determination, frame integration, and unit-cell
3
[
H O ] = 2 mM, and [PaDa-I] = 200 nM in NaPi buffer pH 7.0 (30%
3
2 2
CH CN), 30 °C. The reactions continued for another 4 h before
further characterizations were performed; products were quantified by
high-performance liquid chromatography (HPLC). TON = [mol
molPaDa‑I ].
×
(4)
−
1
29
refinement was carried out with “Crysalis”. Face-indexed
I
Cu -catalyzed 1,3−dipolar cycloaddition with terminal alkynes
absorption corrections were applied using spherical harmonics,
3
0
(
yielding products 8a and 8b, Figures S25 and S26) proved to
implemented in the SCALE3 ABSPACK scaling algorithm.
31
be straightforward (Figure 4). 5 could be readily reduced into
aromatic amine (6, Figure S28).
OLEX2 was used for the overall structure solution,
refinement, and preparation of computer graphics and
Figure 1. Crystal structure of (1S,2S)-2-azido-5-bromo-1,2-dihydronaphthalen-1-ol (A) obtained from the PaDa-I-catalyzed epoxidation of 1-Br-
naphthalene followed by nucleophilic ring opening with NaN . (B) Active site model of PaDa-I in complex with naphthalene presenting the pro-
3
(
R)-face compound I. Naphthalene is placed between Phe121 and Phe199 in a T-packing mode with strong hydrophobic interaction with Phe69
that maintains a highly conserved orientation. Dark blue: Phe triad orienting the substrate, yellow: acid−base pair, and light blue: Phe pair involved
in the guidance of the substrate to the active site.
2
646
https://dx.doi.org/10.1021/acscatal.0c05588
ACS Catal. 2021, 11, 2644−2649

ACS Catalysis
pubs.acs.org/acscatalysis
Research Article
Figure 2. Preliminary product scope of the rAaeUPO variants PaDa-I and SoLo. Reaction conditions: [substrate] = 2 mM, [H O ] = 2 mM, and
2
2
[
peroxygenase] = 200 nM in NaPi buffer pH 7.0 (30% CH CN), [NaN ] = 125 mM, T = 30 °C. The conversion of benzene derivatives was
3 3
1
determined by H NMR. n.d.: not detected. a: isolated yield and structure confirmed by the crystal structure or 2D NMR; b: NMR-yield;
characteristic peaks after ring opening (between 6.2 and 6.8 ppm) were used for the integration of the substrate, phenol as the side product, and
diene as the product.
Figure 3. Active site models of the rAaeUPO variants used (PaDa-I, left and SoLo, right). The mutations in SoLo with respect to PaDa-I (F191S,
G241D) are highlighted in pink.
publication data. Within OLEX2, the algorithm used for the
Molecular docking simulations were performed using the
32
34
structure solution was dual-space within ShelXT. Refinement
Autodock VINA algorithm included in YASARA Structure
33
35,36
by full-matrix least-squares used the SHELXL algorithm
within OLEX2. All non-hydrogen atoms were refined
anisotropically. Hydrogen atoms were placed by a difference
map and refined.
software.
Docking computations were performed at the
level of the YASARA force field by running a number of 100
docking trials. Models were visualized with PyMOL Molecular
̈
Graphics System, Version 2.0 Schrodinger, LLC.
2
647
https://dx.doi.org/10.1021/acscatal.0c05588
ACS Catal. 2021, 11, 2644−2649

ACS Catalysis
pubs.acs.org/acscatalysis
Research Article
Figure 4. Exploring the synthetic potential of 4. Reaction conditions: (a) Following the epoxidation and ring-opening reactions, the pH of the
solution was then adjusted to approximately 9.0 using NaOH (5 M). The Pd/C catalyst (10 mol % final concentration) was added. N gas was
2
bubbled through the solution for 10 min, and a balloon of pure H was applied to initiate the reduction. The reduction was carried out for 6 h. (b)
2
Perchloric acid (3 M) was added after the epoxidation and ring-opening reactions and the mixture was stirred for 5 h. (c) Isolated azido alcohol 7
and alkyne (e.g., 1.5 equiv of phenylacetylene) were dissolved in H O and tert-butyl alcohol (2:1, v/v). CuSO ·5H O (5 mol %) and sodium
2
4
2
ascorbate (10 mol %) were added. The reaction mixture was stirred for 8 h at 30 °C. (d) Sequence of reactions (b) and (a).
ASSOCIATED CONTENT
Miguel Alcalde − Department of Biocatalysis, Institute of
Catalysis, CSIC, 28049 Madrid, Spain; orcid.org/0000-
■
*
sı Supporting Information
0001-6780-7616
The Supporting Information is available free of charge at
Adrian C. Whitwood − Department of Chemistry, University
of York, YO10 5DD York, U.K.; orcid.org/0000-0002-
https://pubs.acs.org/doi/10.1021/acscatal.0c05588.
5132-5468
Experimental procedures; analytical data; methods and
synthetic procedures; X-ray crystallography; and deter-
mination of the absolute configuration (PDF)
Complete contact information is available at:
https://pubs.acs.org/10.1021/acscatal.0c05588
Funding
Financial support by the National Key Research and
Development Program of China (No. 2019YFA0905100),
the European Research Council (ERC Consolidator Grant No.
648026), and the Netherlands Organization for Scientific
Research (VICI grant, No. 724.014.003) is gratefully acknowl-
edged. The authors also thank the Ministry of Science,
Innovation and Universities (Spain) for the FPI contract (BES-
2017-080040, PGS) and the Comunidad de Madrid Synergy
CAM Project Y2018/BIO-4738-EVOCHIMERA-CM.
AUTHOR INFORMATION
■
Corresponding Authors
Wuyuan Zhang − Department of Biotechnology, Delft
University of Technology, 2629HZ Delft, The Netherlands;
Tianjin Institute of Industrial Biotechnology, Chinese
Academy of Sciences, Tianjin 300308, China; orcid.org/
0
000-0002-3182-5107; Email: zhangwy@tib.cas.cn
Frank Hollmann − Department of Biotechnology, Delft
University of Technology, 2629HZ Delft, The Netherlands;
orcid.org/0000-0003-4821-756X; Email: f.hollmann@
tudelft.nl
Notes
The authors declare no competing financial interest.
REFERENCES
■
Authors
(
1) Wertjes, W. C.; Southgate, E. H.; Sarlah, D. Recent advances in
chemical dearomatization of nonactivated arenes. Chem. Soc. Rev.
018, 47, 7996−8017.
2) Hicks, J.; Vasko, P.; Goicoechea, J. M.; Aldridge, S. Synthesis,
structure and reaction chemistry of a nucleophilic aluminyl anion.
Nature 2018, 557, 92−95.
(3) Varghese, V.; Hudlicky, T. Short Chemoenzymatic Total
Synthesis of ent-Hydromorphone: An Oxidative Dearomatization/
Intramolecular [4+2] Cycloaddition/Amination Sequence. Angew.
Chem., Int. Ed. 2014, 53, 4355−4358.
4) Hudlicky, T.; Reed, J. W. Celebrating 20 Years of SYNLETT-
Special Account On the Merits of Biocatalysis and the Impact of
Arene cis-Dihydrodiols on Enantioselective Synthesis. Synlett 2009,
Huanhuan Li − School of Chemical Engineering and
Technology, Xi’an Jiaotong University, Xi’an 710049, China
Sabry H. H. Younes − Department of Biotechnology, Delft
University of Technology, 2629HZ Delft, The Netherlands;
Department of Chemistry, Faculty of Sciences, Sohag
University, Sohag 82524, Egypt
Patricia Gómez de Santos − Department of Biocatalysis,
Institute of Catalysis, CSIC, 28049 Madrid, Spain
Florian Tieves − Department of Biotechnology, Delft
University of Technology, 2629HZ Delft, The Netherlands
Gideon Grogan − York Structural Biology Laboratory,
Department of Chemistry, University of York, YO10 5DD
York, U.K.; orcid.org/0000-0003-1383-7056
2
(
(
2
(
009, 685−703.
5) Hudlicky, T.; Gonzalez, D.; Gibson, D. T. Enzymatic
Martin Pabst − Department of Biotechnology, Delft University
of Technology, 2629HZ Delft, The Netherlands
dihydroxylation of aromatics in enantioselective synthesis: Expanding
asymmetric methodology. Aldrichimica Acta 1999, 32, 35−62.
2
648
https://dx.doi.org/10.1021/acscatal.0c05588
ACS Catal. 2021, 11, 2644−2649

ACS Catalysis
pubs.acs.org/acscatalysis
Research Article
(
6) Kung, J. W.; Baumann, S.; von Bergen, M.; Muller, M.;
intermediary epoxidation. Appl. Microbiol. Biotechnol. 2009, 81, 1071−
1076.
Hagedoorn, P.-L.; Hagen, W. R.; Boll, M. Reversible Biological Birch
Reduction at an Extremely Low Redox Potential. J. Am. Chem. Soc.
(26) Burek, B. O. O.; Bormann, S.; Hollmann, F.; Bloh, J.;
Holtmann, D. Hydrogen peroxide driven biocatalysis. Green Chem.
2019, 21, 3232−3249.
2
(
010, 132, 9850−9856.
7) Kung, J. W.; Loffler, C.; Dorner, K.; Heintz, D.; Gallien, S.; Van
Dorsselaer, A.; Friedrich, T.; Boll, M. Identification and character-
ization of the tungsten-containing class of benzoyl-coenzyme A
reductases. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 17687−17692.
(27) Gomez de Santos, P.; Canellas, M.; Tieves, F.; Younes, S. H.
H.; Molina-Espeja, P.; Hofrichter, M.; Hollmann, F.; Guallar, V.;
Alcalde, M. Selective synthesis of the human drug metabolite 5′-
hydroxypropranolol by an evolved self-sufficient peroxygenase. ACS
Catal. 2018, 8, 4789−4799.
(8) Thiele, B.; Rieder, O.; Golding, B. T.; Muller, M.; Boll, M.
Mechanism of Enzymatic Birch Reduction: Stereochemical Course
and Exchange Reactions of Benzoyl-CoA Reductase. J. Am. Chem. Soc.
(28) Molina-Espeja, P.; Ma, S.; Mate, D. M.; Ludwig, R.; Alcalde, M.
Tandem-yeast expression system for engineering and producing
2
(
008, 130, 14050−14051.
unspecific peroxygenase. Enzyme Microb. Technol. 2015, 73−74, 29−
9) Baker Dockrey, S. A.; Lukowski, A. L.; Becker, M. R.; Narayan,
3
(
3.
A. R. H. Biocatalytic site- and enantioselective oxidative dearomatiza-
29) CrysAlisPro. Oxford Diffraction Ltd., Version 1.171.34.41.
tion of phenols. Nat. Chem. 2018, 10, 119−125.
(30) Empirical Absorption Correction using Spherical Harmonics,
(10) Boyd, D. R.; Hamilton, J. T. G.; Sharma, N. D.; Harrison, J. S.;
Implemented in SCALE3 ABSPACK Scaling Algorithm within
CrysAlisPro Software. Oxford Diffraction Ltd., Version 1.171.34.40.
McRoberts, W. C.; Harper, D. B. Isolation of a stable benzene oxide
from a fungal biotransformation and evidence for an ’NIH shift’ of the
carbomethoxy group during hydroxylation of methyl benzoates. Chem.
Commun. 2000, 1481−1482.
(31) Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A.
K.; Puschmann, H. OLEX2: a complete structure solution, refinement
and analysis program. J. Appl. Crystallogr. 2009, 42, 339−341.
(11) Molina-Espeja, P.; Canellas, M.; Plou, F. J.; Hofrichter, M.;
(32) Sheldrick, G. SHELXT - Integrated space-group and crystal-
Lucas, F.; Guallar, V.; Alcalde, M. Synthesis of 1-Naphthol by a
Natural Peroxygenase Engineered by Directed Evolution. Chem-
BioChem 2016, 17, 341−349.
structure determination. Acta Crystallogr., Sect. A: Found. Adv. 2015,
1, 3−8.
33) Sheldrick, G. A short history of SHELX. Acta Crystallogr., Sect.
A: Found. Adv. 2008, 64, 112−122.
34) Trott, O.; Olson, A. J. AutoDock Vina: Improving the speed
7
(
(12) Kluge, M.; Ullrich, R.; Scheibner, K.; Hofrichter, M. Formation
of naphthalene hydrates in the enzymatic conversion of 1,2-
dihydronaphthalene by two fungal peroxygenases and subsequent
naphthalene formation. J. Mol. Catal. B: Enzym. 2014, 103, 56−60.
(
and accuracy of docking with a new scoring function, efficient
optimization, and multithreading. J. Comput. Chem. 2010, 31, 455−
(13) Hofrichter, M.; Ullrich, R. Oxidations catalyzed by fungal
4
61.
peroxygenases. Curr. Opin. Chem. Biol. 2014, 19, 116−125.
(
35) Krieger, E.; Vriend, G. YASARA Viewmolecular graphics for
all devicesfrom smartphones to workstations. Bioinformatics 2014,
0, 2981−2982.
36) Krieger, E.; Koraimann, G.; Vriend, G. Increasing the precision
(
14) Barkova, K.; Kinne, M.; Ullrich, R.; Hennig, L.; Fuchs, A.;
́
Hofrichter, M. Regioselective hydroxylation of diverse flavonoids by
3
(
an aromatic peroxygenase. Tetrahedron 2011, 67, 4874−4878.
(15) Pecyna, M. J.; Ullrich, R.; Bittner, B.; Clemens, A.; Scheibner,
of comparative models with YASARA NOVAa self-parameterizing
K.; Schubert, R.; Hofrichter, M. Molecular characterization of
aromatic peroxygenase from Agrocybe aegerita. Appl. Microbiol.
Biotechnol. 2009, 84, 885−897.
force field. Proteins 2002, 47, 393−402.
(16) McManus, M. J.; Berchtold, G. A.; Jerina, D. M. Nucleophilic
addition of azide ion to benzene oxide: a reinvestigation. J. Am. Chem.
Soc. 1985, 107, 2977−2978.
(17) Jeffrey, A. M.; Yeh, H. J. C.; Jerina, D. M.; DeMarinis, R. M.;
Foster, C. H.; Piccolo, D. E.; Berchtold, G. A. Stereochemical course
in reactions between nucleophiles and arene oxides. J. Am. Chem. Soc.
1
(
974, 96, 6929−6937.
18) Bertozzi, F.; Crotti, P.; Del Moro, F.; Feringa, B. L.; Macchia,
F.; Pineschi, M. Unprecedented catalytic enantioselective trapping of
arene oxides with dialkylzinc reagents. Chem. Commun. 2001, 2606−
2
(
607.
19) Rastetter, W. H.; Chancellor, T.; Richard, T. J. Biogenesis of
epidithiadioxopiperazines. Nucleophilic additions to benzene oxide
and sym-oxepin oxide. J. Org. Chem. 1982, 47, 1509−1512.
(
20) Vogel, E.; Gunther, H. Benzene Oxide-Oxepin Valence
̈
Tautomerism. Angew. Chem., Int. Ed. 1967, 6, 385−401.
21) Yagi, H.; Jerina, D. M. General synthetic method for non-K-
region arene oxides. J. Am. Chem. Soc. 1975, 97, 3185−3192.
22) Gillard, J. R.; Newlands, M. J.; Bridson, J. N.; Burnell, D. J. Pi-
(
(
facial Stereoselectivity in the Diels-Alder Reactions of Benzene
Oxides. Can. J. Chem. 1991, 69, 1337−1343.
(23) Siddiqi, Z.; Wertjes, W. C.; Sarlah, D. Chemical Equivalent of
Arene Monooxygenases: Dearomative Synthesis of Arene Oxides and
Oxepines. J. Am. Chem. Soc. 2020, 142, 10125−10131.
(24) Molina-Espeja, P.; Garcia-Ruiz, E.; Gonzalez-Perez, D.; Ullrich,
R.; Hofrichter, M.; Alcalde, M. Directed Evolution of Unspecific
Peroxygenase from Agrocybe aegerita. Appl. Environ. Microbiol. 2014,
8
(
0, 3496−3507.
25) Kluge, M.; Ullrich, R.; Dolge, C.; Scheibner, K.; Hofrichter, M.
Hydroxylation of naphthalene by aromatic peroxygenase from
Agrocybe aegerita proceeds via oxygen transfer from H2O2 and
2
649
https://dx.doi.org/10.1021/acscatal.0c05588
ACS Catal. 2021, 11, 2644−2649