Formation of naphthalene hydrates in the enzymatic conversion of 1,2-dihydronaphthalene by two fungal peroxygenases and subsequent naphthalene formation

Article abstract of DOI:10.1016/j.molcatb.2013.08.017

The formation of naphthalene hydrates (i.e. 1- and 2-hydroxy-1,2- dihydronaphthalene) displays a new activity (besides epoxidation) in the enzymatic transformation of 1,2-dihydronaphthalene by two fungal unspecific peroxygenases (UPOs) accounting for 16-19% of the overall turnover. These arene hydrates decayed into naphthalene that in turn was converted by UPOs into naphthols. The oxygen transferred during hydroxylation was shown to derive from hydrogen peroxide proving a true peroxygenation reaction.

Full text of DOI:10.1016/j.molcatb.2013.08.017

Journal of Molecular Catalysis B: Enzymatic 103 (2014) 56–60
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Formation of naphthalene hydrates in the enzymatic conversion of
1
,2-dihydronaphthalene by two fungal peroxygenases and
subsequent naphthalene formation
a,∗
a
b
a
Martin Kluge , René Ullrich , Katrin Scheibner , Martin Hofrichter
a
Department of Bio- and Environmental Sciences, TU Dresden – International Institute Zittau, Markt 23, 02763 Zittau, Germany
Enzyme Technology Group, Faculty of Natural Sciences, Lausitz University of Applied Sciences, Großenhainer Straße 57, 01968 Senftenberg, Germany
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 3 September 2013
The formation of naphthalene hydrates (i.e. 1- and 2-hydroxy-1,2-dihydronaphthalene) displays a new
activity (besides epoxidation) in the enzymatic transformation of 1,2-dihydronaphthalene by two fungal
unspecific peroxygenases (UPOs) accounting for 16–19% of the overall turnover. These arene hydrates
decayed into naphthalene that in turn was converted by UPOs into naphthols. The oxygen transferred dur-
ing hydroxylation was shown to derive from hydrogen peroxide proving a true peroxygenation reaction.
Keywords:
Unspecific peroxygenase
Naphthalene hydrates
Aromatization
©
2013 Elsevier B.V. All rights reserved.
Oxygenation
1
. Introduction
However, products formed in this manner do not include unsubsti-
tuted aromatic hydrocarbons such as naphthalene.
Enzymatic aromatizations play an important role in human and
microbial metabolism. Biological aromatization occurs mainly with
specifically oxyfunctionalized precursors and ˛-unsaturated rings
cyclic enones) like androgens [1] but also with smaller rings such
as 2-cyclohexenone [2]. An example is the vertebral unspecific
monooxygenase (aromatase, EC 1.14.14.1) catalyzing the oxida-
tive deformylation at the 10-position by excessive hydroxylation
of the C-19 in androgens [3]. NADPH dehydrogenase of Saccha-
romyces carlsbergensis (“Old Yellow Enzyme”, EC 1.6.99.1) oxidizes
The agaric fungi Agrocybe aegerita and Coprinellus radians secrete
unspecific peroxygenases (AaeUPO and CraUPO) belonging to
the relatively new, second sub-subclass of peroxide-consuming
enzymes, the peroxygenases (EC 1.11.2.1). These enzymes share
spectral, structural and catalytic properties both with peroxidases
and cytochrome P450-monooxygenases (P450), and catalyze var-
ious peroxygenation reactions, some of which are highly regio-
and stereoselective [6–8]. Among others, it has been shown that
AaeUPO, besides catalyzing benzylic hydroxylation [9], epoxidizes
double bonds like those in styrene and its derivatives [8,10]. Thus,
the conversion of 1,2-dihydronaphthalene by AaeUPO resulted in
the preferential formation of the respective 1,2-epoxide in high
yields but only with an ee of 32% that is far less than the ee-
values observed for other benzylic substrates [8,10]. Surprisingly,
naphthalene and its characteristic oxidation products (naphthols
and naphthoquinone) were also found as products. This finding
prompted the authors to investigate this aromatization more in
detail by searching for an eligible decrescent intermediate.
The aromatization of 1,2-dihydronaphthalene to form naphtha-
lene was described for the aromatic metabolism of Pseudomonas
putida and attributed to a dioxygenase activity [11–13], while in
rat liver, microsomal P450 monooxygenases were proposed to
be responsible for this reaction [14]. In both cases, naphthalene
hydrates emerged but naphthalene formation could not alone be
attributed to their decay and rather a radical mediated dehydro-
genation mechanism was assumed.
(
2
-cyclohexenone via dehydrogenation, which yields hydroxyben-
zene (phenol) and subsequent reduction of a second substrate
molecule cyclohexanone (considered as dismutation) [2]. Forma-
tion of 4-hydroxybenzoic acid from 4-oxocyclohexanecarboxylic
acid by stepwise desaturation was reported for Corynebacterium
cyclohexanicum, in which the final aromatization is accomplished
by a spontaneous rearrangement [4].
Main source of aromatic metabolites in plants and microor-
ganisms is the shikimic acid pathway, in the course of which
the intermediate chorismate (cyclohexa-1,3-diene derivative) can
be converted by a synthase into (aromatic) anthranilate or, after
isomerization to prephenate (cyclohexa-1,4-diene derivative), by a
specific dehydrogenase to 4-hydroxyphenylpyruvate. Beyond that,
chorismate serves as the precursor for diverse aromatic substances,
in particular for secondary metabolites with manifold functions [5].
∗ Corresponding author. Tel.: +49 03583 6127 69; fax: +49 03583 6127 34.
E-mail address: kluge@ihi-zittau.de (M. Kluge).
We describe here the first aromatization reaction catalyzed by
a peroxygenase, starting from an unsaturated cyclic hydrocarbon,
1
381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.08.017

M. Kluge et al. / Journal of Molecular Catalysis B: Enzymatic 103 (2014) 56–60
57
which is enzymatically hydroxylated adjacent to the double bond
2.5. Enzymatic conversions
resulting in arene hydrates that spontaneously decay under water
elimination.
Enzymatic conversions were carried out using 2 mM 1,2- or
1
,4-dihydronaphthalene and 2 mM H O2 in 20 mM potassium
2
phosphate buffer (pH 7) with 20% acetonitrile as co-solvent in
2
. Experimental
a total volume of 0.5 mL at room temperature. 90 nM AaeUPO
−
1
−1
assayed with veratryl alcohol, 120 U mg , 46 kDa)
(
0.5 U mL
2.1. Commercially available chemicals
−
1
or 440 nM CraUPO (0.5 U mL
assayed with veratryl alcohol,
5.8 U mg , 44 kDa) purified with FPLC [7,18] were used in all
−
1
2
All chemicals were purchased at the highest purity avail-
experiments.
able. 1,2-Dihydronaphthalene, 1,4-dihydronaphthalene, solvents
and reagents were obtained from Sigma–Aldrich (Taufkirchen,
Initial enzymatic conversion was followed by HPLC after 2 min
reaction time; after 30 min, the reaction was assumed to be fin-
ished and the reaction mixture acidified (5 ␮L 6 M HCl, pH < 2) to
completely convert the naphthalene hydrates to naphthalene.
Germany), 1,4-epoxy-1,4-dihydronaphthalene from TCI Europe
Zwijndrecht, Belgium) and 18O-labeled H2 O from ICON Isotopes
NJ, USA).
18
(
(
2
2.2. 1,4-Dihydronaphthalene oxide and 1,2-dihydronaphthalene
2.6. High performance liquid chromatography (HPLC)
oxide
HPLC analysis (Agilent 1200 Series, DAD) was performed on
1
,4-Dihydronaphthalene oxide and 1,2-dihydronaphthalene
a Gemini-NX(2) (3 ␮, 150 mm × 2 mm) column in gradient mode
◦
−1
oxide were synthesized by epoxidation of 1,4-dihydronaphthalene
and 1,2-dihydronaphthalene, respectively, using mCPBA in
dichloromethane as oxidant (at 0 C, 12 h) according to Hibbert
and Burt [15]. 1,4-Dihydronaphthalene oxide GC/MS (EI, m/z) 146
72), 128 (83), 117 (79), 115 (100), 1,4-dihydronaphthalene oxide
(0.01% HCOOH, pH 3.5 (A)/MeCN (B)) at 40 C and 0.3 mL min . The
gradient was raised from 15% B at 2 min to 95% B at 11 min and held
for further 4 min. To obtain highly pure samples of naphthalene
hydrates, semi preparative HPLC on a Merck Lichrospher (5 ␮m,
◦
−
1
(
100 mm × 4.6 mm) was run isocratically in 30% B at 1 mL min
−
1
GC/MS (EI, m/z) 146 (63), 117 (29), 115 (21), 104 (100).
and fractions were collected (0.2 mL min ) right after the detector,
re-chromatographed and extracted with dichloromethane for sub-
sequent GC/MS-analysis. Quantification of individual compounds
was based on the peak area at 258 nm. Spectra were recorded in the
range from 210 to 500 nm (0.5 nm steps) at a frequency of 10 Hz.
Isomeric naphthalene hydrates were estimated as the sum derived
from conversion balancing. 1,2-dihydronaphthalene oxide con-
centration was additionally confirmed by 1,2-dihydronaphthalene
oxidation with a recombinant UPO from Novozymes (rNovo) that
exclusively catalyzes epoxidation.
2
.3. 2-Hydroxy-1,2-dihydronaphthalene
2
-Hydroxy-1,2-dihydronaphthalene was prepared from 1,4-
dihydronaphthalene oxide by rearrangement (1 M KOH/EtOH abs.,
C, 4 d) [16]. GC/MS (EI, m/z) 146 (100), 128 (84), 145 (64), 115
58).
◦
5
(
2
.4. 1-Hydroxy-1,2-dihydronaphthalene
1
-Hydroxy-1,2-dihydronaphthalene was prepared from 1,4-
epoxy-1,4-dihydronaphthalene by hydroboration with 9-BBN (RT,
0 h, THF anhydr. under nitrogen) and subsequent oxidation
H O /3 N NaOH, RT, 5 h) according to a method of Brown and
2
.7. Gas chromatography/mass spectroscopy (GC/MS)
2
(
GC/MS analysis (Agilent 6890 GC/5790 MSD) of the extracts was
2
2
accomplished using a ZB-5MS (Zebron, 30 m × 0.25 mm ID, 0.25 ␮m
Prasad [17] (GC/MS (EI, m/z) 146 (100), 128 (68), 131 (65), 145 (55)
15 (47)). Due to their instability, naphthalene hydrates could not
be isolated and therefore not directly be quantified.
−1
FT) capillary column at a helium flow of 1.5 mL min . Injection
1
◦
was done at 190 C in the split mode. The initial oven temperature
of 65 C was held for one minute and raised with 45 C min to
◦
◦
−1
O
OH
1
,2-dihydronaphthalene
naphthalene
oxide
2-h yy dd rr oo xx yy -- 11 ,, 22 -- dd ii hh yy dd rr oo nn aa pp hh tt hh aa ll ee nn ee
OH
1
-hydrox yy -1,2-d ii hh ydronap hh thalene
1
,2-dihydronaphthalene
2
3
4
5
Retention time (min)
Fig. 1. GC-TIC analysis of dichloromethane extracts of the enzymatic conversion of 1,2-dihydronaphthalene by AaeUPO (black) and the decay of chemically synthesized
-hydroxy-1,2-dihydronaphthalene (4.27 min) containing naphthalene (3.63 min) formed by interim spontaneous water elimination (blue). Note: 2-hydroxy-1,2-
dihydronaphthalene co-elutes at 4.27 min. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
1

5
8
M. Kluge et al. / Journal of Molecular Catalysis B: Enzymatic 103 (2014) 56–60
◦
1
90 C and held for further 5 min. 70 eV spectra were recorded in
100
148
the range from 15 to 300 m/z at a frequency of 3.6 Hz.
8
6
0
0
3
. Results and discussion
The enzymatic formation of naphthalene hydrates (i.e.
1
-hydroxy-1,2-dihydronaphthalene
and
2-hydroxy-1,2-
dihydronaphthalene) was evidenced by comparing their UV
and mass spectral data as well as their chromatographic prop-
erties (GC, HPLC) with those of the prepared standards (Fig. 1).
A separation of the hydrates by HPLC could be achieved and the
individual isomers were obtained in a semi-preparative manner
as shown in Fig. 2. Their UV-spectra are almost indistinguishable
from each other indicating some high level of structural similarity.
The isomeric naphthalene hydrates could not be separated by
GC but their 70 eV mass spectra showed vast identity and thus
1
33
40
2
0
0
30
50
70
90
110
130
150
1
8
could be used for O incorporation studies. Fig. 3 shows extensive
1
00
80
146
1
8
18
incorporation of
O from H2 O2 indicated by a +2 m/z shift
into the 146 m/z molecular ion and the 131 m/z ion (indicative
for a methyl loss). Altogether, these data strongly indicate a true
peroxygenation mechanism.
1
31
1
,2-Dihydronaphthalene was converted by both enzymes
6
4
2
0
0
0
0
(
AaeUPO, CraUPO) mainly via epoxidation; in addition, hydroxyl-
ation was observed leading to aromatization and further oxidation
as shown in Scheme 1. No aromatization was observed for 1,4-
dihydronaphthalene, which was exclusively epoxidized by both
enzymes. Although applied with the same activity (according to
the oxidation of veratryl alcohol [7]), enzymes turned out to dif-
fer in the turnover of dihydronaphthalenes as shown in Table 1. A
total turnover of 22,000 cycles (which might be estimated even
higher when considering peroxide shortage) was calculated for
AaeUPO, which is adversely to CraUPO that reached only one
sixth of the cycle numbers (and this with a nearly 4-fold higher
total amount of enzyme). Aromatization of the second ring in 1,2-
dihydronaphthalene accounted for approximately 19% (AaeUPO)
and 16% (CraUPO) of the overall 1,2-dihydronaphthalene turnover
30
50
70
90
110
130
150
Fig. 3. Top: 70 eV mass spectrum of co-eluting naphthalene hydrates showing
extensive O-incorporation (red peaks indicate O-containing fragments shifted
by +2 m/z, intense 104 m/z is caused by an impurity). Bottom: mass spectrum of
-hydroxy-1,2-dihydronaphthalene for comparison. (For interpretation of the ref-
erences to color in this figure legend, the reader is referred to the web version of the
article.)
1
8
18
1
(
the term aromatization refers to the sum of naphthalene hydrates,
naphthalene and naphthols). While AaeUPO converted the sub-
strate almost completely already in the course of the first two
minutes, CraUPO was slower but continued substrate conversion
until the end of the experiment after 30 min (reaching 81% total
conversion); however, the percentage of aromatization remained
constant.
Peak area of 1-naphthalene hydrate detected at 258 nm relates
to that of 2-naphthalene hydrate like 7.4:1 for AaeUPO and 6.4:1 for
CraUPO. Given similar molar extinction coefficients and decay rates,
OH
Table 1
OH
Specific data/values of the conversion of 1,2-dihydronaphthalene by AaeUPO
and CraUPO, and concentration of the metabolites formed: related to 1,2-
dihydronaphthalene, related to total turnover, estimated.
a
b
c
2
00
240
280
320
AaeUPO
CraUPO
λ (nm)
Turnovera 2 min (final)
Total turnover number
Proportion of
100% (100%)
42% (81%)
3
3
22 × 10
3.7 × 10
19% (19%)
15% (16%)
b
aromatization after
2
min (final)
Substance concentrations
in HPLC elution order)
6
8
10
12
After 2 min (final)/␮M
1520 (1580)
200
After 2 min (final)/␮M
(
Retention time (min)
1,2-Dihydronaphthalene
676 (1210)
90
oxide
Fig. 2. HPLC elution profiles (recorded at 258 nm) of the AaeAPO-catalyzed
conversion of 1,2-dihydronaphthalene reaction (black line) and the re-
chromatographed hydrate fractions obtained after semi-preparative HPLC:
-hydroxy-1,2-dihydronaphthalene (red) and 1-hydroxy-1,2-dihydronaphthalene
blue). Inset: UV-spectra of the isomeric naphthalene hydrates. (For interpretation
Naphthalene hydrates as
sumc
2
1
-Naphthol
-Naphthol
5 (7)
2 (3)
2
(
210 (270)
16 (150)
7 (7)
13 (45)
69 (256)
1150 (370)
Naphthalene
,2-Dihydronaphthalene
of the references to color in this figure legend, the reader is referred to the web
version of the article.)
1

M. Kluge et al. / Journal of Molecular Catalysis B: Enzymatic 103 (2014) 56–60
59
UPOs
H218O
aromatization
side
reaction
2
+
2
+
1
1
- H O
2
18
2
OH
18
OH
naphthalene
AaeUPO
H2 O2
naphthalene hydrates
18
1
,2-dihydronaphthalene
18
18
O
O
rearrangement
products:
UPOs
main
reaction + H 18O
2
2
1
-naphthol (95%),
2-naphthol (5%)
1
,2-dihydronaphthalene
oxide (32% e.e.)
naphthalene
oxide
Scheme 1. Reaction scheme of the enzymatic conversion of 1,2-dihydronaphthalene by AaeUPO comprising epoxidation and hydroxylation as well as oxygen transfer from
18
O-labeled hydrogen peroxide to the peroxygenation products.
this implies the preferential hydroxylation of the benzylic carbon
C-1-position in the second ring). This finding is in accordance to
4. Conclusions
(
previous results on benzylic hydroxylation of ethyl benzene or
tetralin by AaeUPO [10]. The high stereoselectivity observed for
these hydroxylations (with ee-values > 99% for the (R)-1-ol prod-
ucts) makes it conceivable that this may apply for 1-naphthalene
hydrate as well; however, more detailed investigations on the
stereochemistry of these arene hydrates would be quite chal-
lenging due to their chemical instability. Reported half-lives of
some ten days derived from second order rate constants, in which
the carbocation formation was rate determining [19,20], could
not be confirmed in our experiments. The unstable naphthalene
hydrates rapidly decomposed under water elimination and sub-
sequent aromatization to form naphthalene. Naphthalene in turn
was further epoxidized and rearranged mainly to 1-naphthol (see
Scheme 1) as described earlier [21]. While AaeUPO further con-
verted two thirds of the temporarily formed naphthalene (430 ␮M),
CraUPO left behind 84% of the compound (300 ␮M).
In summary, a new way to produce naphthalene hydrates is pre-
sented, in which the oxygen entirely originates from hydrogen per-
oxide. The enzymatic transformation of 1,2-dihydronaphthalene
by two fungal unspecific peroxidases results in the formation of
1,2-dihydronaphthalene epoxide, naphthalene hydrates, naphtha-
lene and its conversion products (i.e. naphthols) in different yields.
Search for alternative substrates that undergo similar reaction
sequences and for options that allow useful coupling reactions of
emerging arene hydrates are currently under investigation.
Acknowledgements
The authors would gratefully acknowledge financial support
provided by the Deutsche Bundesstiftung Umwelt (projects AZ
1
3225 and AZ 13270 within the ChemBioTec network). Part of the
work was funded by the European Union (KBBE-2010-4-265397).
Further we thank L. Kalum and Dr. H. Lund from Novozymes for pro-
viding recombinant UPO and Dr. S. Fränzle for inspiring discussions.
The initial conversion rate
dihydronaphthalene turnover over 2 min – was at least 180 s for
–
calculated from 1,2-
−
1
AaeUPO, which is in the same range as the kcat for other benzylic
−
1
hydrocarbons and naphthalene [10]. For CraUPO, still 16 s was
calculated, which is nearly the kcat determined for naphthalene
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−
1
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