Development of a synthesis method for odor sesquiterpenoid, (?)-rotundone, using non-heme Fe2+-chelate catalyst and ferric-chelate reductase

Article abstract of DOI:10.1080/09168451.2019.1625264

(?)-Rotundone, a sesquiterpenoid that has a characteristic woody and peppery odor, is a key aroma component of spicy foodstuffs, such as black pepper and Australian Shiraz wine. (?)-Rotundone shows the lowest level of odor threshold in natural compounds and remarkably improves the quality of various fruit flavors. To develop a method for the synthesis of (?)-rotundone, we focused on non-heme Fe²⁺-chelates, which are biomimetic catalysts of the active center of oxygenases and enzymatic supply and regeneration of those catalysts. That is, we constructed a unique combination system composed of the oxidative synthesis of (?)-rotundone using the non-heme Fe²⁺-chelate catalyst, Fe(II)-EDTA, and the enzymatic supply and regeneration of Fe²⁺-chelate by ferric-chelate reductase, YqjH, from Escherichia coli. In addition, we improved the yield of (?)- rotundone by the application of cyclodextrin and glucose dehydrogenase to this system, and thus established a platform for efficient (?)-rotundone production.

Full text of DOI:10.1080/09168451.2019.1625264

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: https://www.tandfonline.com/loi/tbbb20
Development of a synthesis method for odor
sesquiterpenoid, (−)-rotundone, using non-heme
2+
Fe -chelate catalyst and ferric-chelate reductase
Satoru Umezawa, Shunsuke Konishi & Kuniki Kino
To cite this article: Satoru Umezawa, Shunsuke Konishi & Kuniki Kino (2019): Development
2+
of a synthesis method for odor sesquiterpenoid, (−)-rotundone, using non-heme Fe -chelate
catalyst and ferric-chelate reductase, Bioscience, Biotechnology, and Biochemistry, DOI:
10.1080/09168451.2019.1625264
To link to this article: https://doi.org/10.1080/09168451.2019.1625264
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BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
https://doi.org/10.1080/09168451.2019.1625264
Development of a synthesis method for odor sesquiterpenoid, (−)-rotundone,
2
+
using non-heme Fe -chelate catalyst and ferric-chelate reductase
a,b
b
a
Satoru Umezawa , Shunsuke Konishi and Kuniki Kino
a
b
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, Tokyo, Japan; Technical Research Institute
R&D Center, T. Hasegawa Co., Ltd., Kanagawa, Japan
ABSTRACT
ARTICLE HISTORY
(
−)-Rotundone, a sesquiterpenoid that has a characteristic woody and peppery odor, is a key aroma
Received 19 April 2019
Accepted 21 May 2019
component of spicy foodstuffs, such as black pepper and Australian Shiraz wine. (−)-Rotundone
shows the lowest level of odor threshold in natural compounds and remarkably improves the
quality of various fruit flavors. To develop a method for the synthesis of (−)-rotundone, we focused
on non-heme Fe -chelates, which are biomimetic catalysts of the active center of oxygenases
and enzymatic supply and regeneration of those catalysts. That is, we constructed a unique
combination system composed of the oxidative synthesis of (−)-rotundone using the non-heme
Fe -chelate catalyst, Fe(II)-EDTA, and the enzymatic supply and regeneration of Fe -chelate by
ferric-chelate reductase, YqjH, from Escherichia coli. In addition, we improved the yield of (−)-
rotundone by the application of cyclodextrin and glucose dehydrogenase to this system, and
thus established a platform for efficient (−)-rotundone production.
KEYWORDS
2+
(
−)-rotundone; Fe -chelate
2+
catalyst; ferric-chelate
reductase; oxygenation
2+
2+
Sesquiterpenoids, which are oxygenated derivatives
of sesquiterpenes, constitute the large diverse class
among terpene compounds and exhibit various
biological activities [1–3]. Among those sesquiterpe-
noids, we focused on (−)-rotundone [(3S,5R,8S)-
and peroxidants, which should be replaced in order
to carry out more sustainable chemistry.
a
Furthermore, there has been an increase in consu-
mer demands for products of “naturalness” in the
food industry, and the European regulation (EC No
1334/2008, article 3.2) defines natural flavoring sub-
stance that leads to the added value of natural pro-
ducts [13–15]. In these situations, biotechnological
methods are a promising alternative. P450s are the
most commonly used approach for the production
of terpenoids, and several sesquiterpenoids, such as
artemisinin and (+)-nootkatone, are already on the
market [2,14,16]. CYP71BE5 from V. vinifera has
been reported to catalyze the oxidation of α-
guaiene to (−)-rotundone [8]. Additionally, the oxi-
dation of α-guaiene with laccase [17] or by aeration
in an organic solvent and on cellulose filter paper
[18] has been reported. However, previous studies
have experienced difficulties due to a low selectivity
or productivity, and an effective method for the
synthesis of (−)-rotundone has not yet been
developed.
5
-isopropenyl-3,8-dimethyl-3,4,5,6,7,8-hexahydro-1
(
2H)-azulenone], which has a woody and peppery
odor, with the lowest level of odor threshold in
natural products (8 ng/L in water, 16 ng/L in red
wine), and which is recognized as a key aroma
component of spicy foodstuffs, such as black pepper
and Australian Shiraz wine [4,5]. Furthermore, this
compound has been identified in several fruits (e.g.
grapefruit, orange, mango, and apple) and exhibits
significant effects for the improvement of the overall
qualities of flavors with a subthreshold addition
[
6,7]. However, the amount of (−)-rotundone
found in natural sources is limited (from the ppb
to ppm order); therefore, an effective synthesis
method is required for its use in industrial applica-
tions [4]. It is presumed that (−)-rotundone
is oxidatively synthesized from the precursor sesqui-
terpene, α-guaiene, in Vitis vinifera (grapevine)
On the other hand, in the field of biomimetic
catalysts, studies have been carried out on non-
[
8,9]. And α-guaiene and its derivatives are widely
2
+
distributed in natural sources, including guaiac
wood oil or patchouli oil [10,11]. In terms of con-
ventional chemical methods, allylic oxidation using
chromate (VI) or manganite (VII) are regular can-
didates and advanced methods are under investiga-
tion [12]. However, these methods require
hazardous and non-ecological heavy metal catalysts
heme Fe -chelate catalysts, which mimic the active
center of oxygenases, such as 2-oxoglutarate-
dependent dioxygenase and pterin-dependent
hydroxylase [19,20]. Iron-containing oxygenases
have been found to generate high valent iron(IV)-
oxo intermediates as active species using molecular
oxygen and had catalyzed the oxygenation of
CONTACT Kuniki Kino
kkino@waseda.jp
Supplemental data for this article can be accessed here.
©
2019 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
S. UMEZAWA ET AL.
Screening assay
The screening for (−)-rotundone synthesis was per-
formed with reaction mixtures (500 µL) containing
2
0 mM of each metal salt (e.g. FeSO , FeCl , Fe
4 3 2
(
SO ) , FeCl , CuCl, CuI, CuSO , ZnSO , MnSO ,
4 3 3 4 4 4
MgSO , and CoSO ), 1% (vol/vol) Tween-80, 5 mM
4
4
Figure 1. Catalytic synthesis of odor sesquiterpenoid, (−)-
rotundone, using non-heme Fe -chelate catalyst and ferric-
chelate reductase.
α-guaiene, and 50 mM KH PO -K HPO buffer
2+
2
4
2
4
(
pH 7.5). CuCl and CuI were saturated due to
their low solubility. The reactions were carried
out at 30°C for 1 h in glass vials and extracted
using the double amount of ethyl acetate with
30 µg of (R)-carvone as an internal standard. The
products were analyzed qualitatively with GC-MS
and quantitated with HPLC.
2
+
substrates [21]. Moreover, non-heme Fe -chelate
catalysts are recognized to possess the same func-
tions and the ability to catalyze various oxidative
reactions containing aliphatic hydroxylation and
alkene epoxidation [22]. In particular, as the allylic
oxidation of cyclohexene to cyclohexenol and cyclo-
2
+
Preparation of ferric-chelate reductase, YqjH
hexenone with non-heme Fe -chelate catalysts was
reported [23], we speculated that these methods
could be applied to (−)-rotundone synthesis.
Moreover, we designed a combination system com-
posed of the catalytic synthesis of (−)-rotundone
and the enzymatic supply and regeneration of Fe -
chelate (Figure 1). A vast number of microbes
secrete iron-specific chelators, denoted as sidero-
Purified YqjH was prepared in a similar manner as
described previously [27]. The gene yqjH was ampli-
fied by PCR using E. coli BL21(DE3) genomic DNA
as a template and primers containing Nde I or
Hind III restriction sites, respectively. The amplified
fragments were digested and inserted into a pET-21a
(+) vector (Novagen). E. coli BL21(DE3) cells were
transformed with this plasmid. The recombinant
cells were grown in 200 mL of LB medium contain-
ing 100 µg/ml ampicillin at 37°C until reaching an
OD600 value of 0.7 ~ 1.0. Then, 1 mM IPTG was
added and the cultivation was continued for 15 h at
25°C. The cells were harvested by centrifugation and
resuspended in 50 mM HEPES-KOH buffer (pH
7.0). The cells were disrupted by sonication and
centrifuged to obtain a supernatant. His-tagged
YqjH was purified using a HisTrap HP 5 mL column
equipped with ÄKTA Prime Plus and desalted using
a PD-10 column, then eluted in 50 or 200 mM
HEPES-KOH buffer (pH 7.0). The expression and
purification of YqjH was confirmed by SDS-PAGE
analysis.
2
+
3
+
phores, which capture ferric ion and form Fe -
3
+
chelates. Fe -chelates are then transported intracel-
lularly and reduced enzymatically by ferric-chelate
reductase to utilize ferrous iron [24,25]. Therefore,
we focused on this ferric-chelate reductase for the
supply and regeneration of Fe -chelate catalyst.
Among ferric-chelate reductases, we selected YqjH
from Escherichia coli since it has been well studied
and is supposed to possess sufficient catalytic activ-
ity for this concept [26,27]. This system is comprised
of the mutual reactions of metal-catalyzed oxygena-
tion and the enzymatic supply and regeneration of
catalyst, which provides a new insight into the
synthesis of sesquiterpenoids.
2
+
Materials and methods
Construction of ferric-chelate reductase system
Reagents
(
−)-Rotundone synthesis using ferric-chelate reduc-
tase, YqjH, was performed with a reaction mixture
500 µL) containing 5 mM α-guaiene, 1% Tween-80,
mM NADH or NADPH, 1 mM of each ferric iron
e.g. FeCl , Fe(III)-dicitrate, or Fe(III)-EDTA) and
+
+
NADH, NADPH, NAD , and NADP were pur-
chased from Nakarai Tesque (Kyoto, Japan).
Catalase from bovine liver, HP-β-CD, glucose dehy-
drogenase from Bacillus sp., and formaldehyde test
Wako were purchased from FUJIFILM Wako Pure
Chemical (Osaka, Japan). Galvinoxyl free radical was
purchased from Tokyo Chemical Industry (Tokyo,
Japan). α-Guaiene, (−)-rotundone, and rotundol
were synthesized and provided by T. Hasegawa
(
5
(
0
3
.3 mg/mL purified YqjH in 50 mM HEPES-KOH
buffer (pH 7.0). Fe(III)-dicitrate was prepared by
mixing FeCl3 and trisodium citrate solution at
a molar ratio of 1:2. Fe(III)-EDTA was prepared by
dissolving EDTA-Na-Fe(III)∙3H O. To verify the
2
(Kanagawa, Japan). All other chemicals were of ana-
prerequisites for (−)-rotundone synthesis, cofactors
lytical grade.
(
NADH, NADPH, and ferric iron) were subtracted

SYNTHESIS OF (−)-ROTUNDONE USING FERRIC-CHELATE REDUCTASE
3
from the reaction mixture one by one. The reactions
were carried out at 30°C for 2 h, then stopped and
extracted by adding the double amount of ethyl
acetate with an internal standard. The consumption
of NAD(P)H was monitored with a wavelength of
FUJIFILM Wako Pure Chemical, Osaka, Japan). The
extracted samples (10 µL) were analyzed with follow-
ing separation program: gradient elution with acetoni-
trile 50–100% against ultrapure water for 10 min,
column washing with acetonitrile 100% for 3 min, re-
equilibration with acetonitrile 50% for 5 min at flow
rate 1 mL/min. α-Guaiene and rotundol were detected
at a wavelength of 220 nm, and (−)-rotundone and
demethylated analog of (−)-rotundone were detected
at a wavelength of 240 nm.
3
40 nm. The products were analyzed with GC-MS
and HPLC.
Validation and improvement of ferric-chelate
reductase system
The GC-MS analysis was performed using a 7890
series GC system, 5977B Network Mass Selective
Detector, with an HP-5ms Ultra Inert (30 m ×
0.25 mm × 0.25 µm; Agilent Technologies, Santa
Clara, CA, USA). The extracted samples (1 µL, split
ratio 10:1, injection temp. 250°C) were analyzed using
the following separation program: column oven initial
temperature of 40°C for 1 min, 15°C/min gradient to
300°C at a flow rate of 1 mL/min He carrier gas.
To verify the reaction mechanism, the reactions
(
1
500 µL) were performed with 5 mM α-guaiene,
% Tween-80, 5 mM NADPH, 1 mM Fe(III)-
EDTA, 0.3 mg/mL purified YqjH in 50 mM
HEPES-KOH buffer (pH 7.0), and 100 U/mL cata-
lase or 1 mM galvinoxyl free radical. The reactions
were carried out at 30°C for 2 h, then stopped and
extracted by adding the double amount of ethyl
acetate with an internal standard. To improve the
efficiency of this system, the reactions (500 µL)
were performed with 5 mM α-guaiene, 1% Tween-
1
13
H and C NMR analysis was performed using
a JNM-ECX400 (JEOL Resonance, Tokyo, Japan).
(4S,7R)-7-isopropenyl-4-methyl-3,4,5,6,7,8-hexahydro-1
(2H)-azulenone (demethylated analog of (−)-rotun-
8
0
0, 5 mM NADH or NADPH, 1 mM Fe(III)-EDTA,
.3 mg/mL purified YqjH in 200 mM HEPES-KOH
1
done); H NMR (400 MHz, C D , Figure S7): δ = 0.81
6
6
buffer (pH 7.0), and 1% (weight/vol) hydroxypro-
pyl-β-cyclodextrin (HP-β-CD). Then, the NAD(P)
(3H, d, J= 7.2 Hz), 1.27 (1H, m), 1.38 (1H, ddd, J= 4.0,
4.0, 9.6 Hz), 1.45 (1H, m), 1.64 (3H, s), 1.63–1.69
(2H, m), 1.82 (1H, m), 1.98 (1H, m), 2.03–2.07
(3H, m), 2.11 (1H, m), 2.97 (1H, d, J= 14.8 Hz), 4.75
(2H, d, J= 15.6 Hz). C NMR (100 MHz, C D , Figure
6 6
S8): δ = 16.78, 20.70, 28.40, 29.87, 31.21, 32.42, 34.18,
+
+
H was substituted with 1 mM NAD or NADP ,
00 mM glucose, and 0.01–1 U/mL glucose dehy-
1
13
drogenase (GlcDH). The reactions were carried out
at 30°C for 2 h, then stopped and extracted by
adding the double amount of ethyl acetate with an
internal standard. The products were analyzed with
GC-MS and HPLC. For the isolation and charac-
terization of the by-product, preparative-scale reac-
tions (20 ml) were performed with 5 mM
α-guaiene, 1% Tween-80, 1 mM Fe(III)-EDTA, 1%
36.72, 45.48, 109.20, 139.24, 150.68, 177.44, 207.23.
Results
Screening of the catalytic activity for (−)-
rotundone synthesis
+
HP-β-CD, 1 mM NAD , 100 mM glucose, 0.1 U/ml
GlcDH, and recombinant cells expressing YqjH
At the outset of this research, we performed a screening
of microorganisms able to catalyze the allylic oxidation
of α-guaiene to (−)-rotundone. However, contrary to
our expectations, multiple strains including E. coli
BL21(DE3) exhibited slight oxidizing activities against
α-guaiene (data not shown). Therefore, we deduced
that some ubiquitous components of microorganisms
could affect oxidation. As such, we verified the effects
of intracellular factors, such as metal ions and coen-
zymes. As a result of shifting targets, (−)-rotundone
synthesis was confirmed using reduced form metal
(
OD600 = 20, prepared as described above) in 200
HEPES-KOH buffer. The reactions were carried out
at 30°C and extracted by adding the double amount
of ethyl acetate with an internal standard. The
reactions were conducted for several batches to
accumulate the by-product for NMR analysis. To
trace by-products in another means, C1 com-
pounds (e.g. methanol, formaldehyde, and formic
acid) were analyzed with GC-MS and formaldehyde
was quantitated using a formaldehyde test Wako.
2+
ions, in particular, Fe (Figure 2 and Figure S1).
Ferrous iron is susceptible to molecular oxygen and is
able to donate electrons and generate reactive oxygen
Product analysis
2+
HPLC analysis was performed using a Prominence
LC-20 system (Shimadzu, Kyoto, Japan) with
a Wakosil-II 5C18 HG column (4.6 mm × 150 mm,
species. Oxygenases with a Fe -chelate active center or
2+
biomimetic Fe -chelate catalysts utilize these proper-
ties for oxygenation [21,22], such that the formation of

4
S. UMEZAWA ET AL.
Figure 2. Synthesis of (−)-rotundone using intracellular metal
ions. Reaction mixtures contained α-guaiene and 20 mM of
each metal salt. The amount of synthesized (−)-rotundone
using FeSO was defined as 100%.
4
(−)-rotundone could be possible via these mechanisms.
Since E. coli BL21(DE3) exhibited oxidizing activity, we
focused on the ferric-chelate reductase, YqjH, as
2
+
Figure 3. Synthesis of (−)-rotundone using YqjH. (a) Reaction
mixtures contained α-guaiene, YqjH, and each cofactor
described with a + symbol at 5 mM NAD(P)H or 1 mM Fe(III)-
EDTA. (b) Reaction mixtures contained α-guaiene, NADPH,
YqjH, and 1 mM of each ferric iron. Data show the means ±
standard deviations of three independent experiments.
a candidate for supplying the Fe -chelate catalyst in
−)-rotundone synthesis.
(
Construction of the catalytic system with non-heme
2+
Fe -chelate and ferric-chelate reductase
2
+
Fe -chelate supply and regeneration by YqjH was
applied to (−)-rotundone synthesis. We selected
FeCl3 as non-chelated iron, Fe(III)-dicitrate and
Fe(III)-EDTA as chelated iron, which were previously
reported to be reduced by YqjH [26,27]. As a result,
Evaluation of the catalytic system with
Fe(III)-EDTA and YqjH
2
+
This catalytic system is composed of the Fe -chelate
supply and regeneration by YqjH and the oxygena-
(
−)-rotundone was significantly synthesized with
2
+
NAD(P)H, Fe(III)-EDTA, and YqjH, which indicates
tion by the Fe -chelate catalyst. In this regard, the
Fenton-type reaction which causes oxidizing reac-
tions using ferrous iron has been well investigated
2
+
that the catalytic system with non-heme Fe -chelate
and ferric-chelate reductase functioned properly
(Figure 3(a)). YqjH could utilize by both NADH
[28]. Since the Fenton reaction requires H
O as an
2 2
and NADPH to reduce Fe(III)-EDTA. And a little
amount of NADPH was consumed with FeCl3,
and a little amount of (−)-rotundone was detected
oxygen donor, we evaluated this catalytic system in
2
+
the presence of catalase. Moreover, non-heme Fe -
chelate catalysts have been suggested to generate
alkyl radical intermediates during the catalytic
cycle of oxygenation, and a radical scavenger, galvi-
noxyl free radical, could inhibit this cycle [19,23,29].
Thus, we also determined the effect of this scaven-
ger. As shown in Figure 4(a–c), even though an
excess of catalase did not affect this system, the
galvinoxyl free radical quenched the whole
reaction. These results indicate that this oxygenation
(Figure 3(b) and Figure S2). This result indicates
the substrate specificity of YqjH is restricted to che-
lated form ferric iron. On the other hand, NADPH
was consumed with Fe(III)-dicitrate, indicating
the generation of Fe(II)-dicitrate by YqjH despite
a low (−)-rotundone productivity (Figure 3(b) and
Figure S2). This result suggests that the ligand struc-
2
+
ture of Fe -chelate is critical for catalytic
oxygenation.
requires not H
O but presumably O as an oxygen
2 2 2

SYNTHESIS OF (−)-ROTUNDONE USING FERRIC-CHELATE REDUCTASE
5
Figure 4. GC-MS analysis of the reaction products containing α-guaiene, NADPH, Fe(III)-EDTA, and YqjH (a), with 100 U/mL
catalase (b), or 1 mM galvinoxyl free radical (c). MS spectra of each peak (d); α-guaiene (peak 1), (−)-rotundone (peak 2), and
demethylated analog (peak 3).
2
+
donor and that Fe(II)-EDTA functions as the Fe -
chelate catalyst via the generation of radical
intermediates.
product mixture which contained both (−)-
rotundone and demethylated analog varied in accor-
dance with those concentrations. The odor was earthy
and musty at a high concentration around ppm
order, and that was woody and peppery at a low
concentration around ppt order. This profile indi-
cates that there exist appropriate concentrations of
these compounds for flavor and fragrance utilization.
GC-MS analysis also showed that there existed
a by-product with a similar retention time and of
a similar amount to (−)-rotundone (Figure 4, peak
3
). According to the GC-MS fragment patterns and
the UV absorption attributed to α,β-unsaturated car-
bonyl structure, we deduced that this by-product was
an analog of (−)-rotundone. As such, this compound
was isolated by silica gel column chromatography and
Improvement of the efficiency of (−)-rotundone
synthesis using cyclodextrin and glucose
dehydrogenase
1
preparative thin layer chromatography. Using H and
1
3
C NMR spectroscopy, the structure was determined
to be a demethylated analog of (−)-rotundone
Figure 4(d)). This compound is a novel terpenoid,
not currently registered on the CAS database, and has
an earthy, musty odor that differs from (−)-
rotundone. In addition, the odor profile of the
To improve the efficiency of (−)-rotundone synth-
esis, we addressed the stabilization of α-guaiene
and the enhanced supply of NAD(P)H. The hydro-
phobicity and volatility of sesquiterpene often leads
to low efficiencies in conversion reactions.
(

6
S. UMEZAWA ET AL.
Figure 4. (Continued)
2
+
Cyclodextrin is known to overcome this problem,
and HP-β-CD, a hydroxypropylated derivative of
β-cyclodextrin, is particularly suitable for sesquiter-
pene [30]. The electron source of this catalytic sys-
tem is NAD(P)H. The regeneration of these
coenzymes could avoid the accumulation of
this would be due to membrane permeability of Fe -
chelate or substrate and the margin of further improve-
ment. Through these investigations, the demethylated
by-product was generated at the same level as (−)-
rotundone. The same amount of formaldehyde as
demethylated by-products was also found to be gener-
ated (Figure S6). In addition to the structure of the by-
+
NAD(P) and enhance the energy supply to facilitate
the catalytic cycle. We selected glucose dehydrogen-
2+
product, this result also indicates that the Fe -chelate
catalyst (this time Fe(II)-EDTA) possessed the catalytic
activity for dissociating a methyl group from a substrate
compound at an allylic position.
ase from Bacillus sp., which was capable of reducing
+
+
both NAD and NADP .
The addition of HP-β-CD to this system increased the
amount of (−)-rotundone by 1.5-fold (Figure 5(a,b)).
This effect was observed identically with both NADH
and NADPH (Figure S3). Upon applying GlcDH to
the (−)-rotundone synthesis, we optimized each para-
meter, such as GlcDH activity, and the types of coenzyme
Discussion
The structure of over 80,000 terpenoids has been
elucidated, and various useful functions have been
identified [1–3]. However, many of these terpenoids
are found in limited amounts in natural sources,
such that effective synthesis methods are required
for industrial applications. Here, we discovered that
(−)-rotundone could be synthesized from precursor
α-guaiene using the reduced form ferrous iron.
Furthermore, we developed a unique catalytic sys-
+
+
(
0
i.e. NAD or NADP ). For an efficient catalytic cycle,
+
+
.1 U/mL GlcDH and 1 mM of either NAD or NADP
was sufficient (Figure S4). Then, we applied NADH
+
regeneration with 0.1 U/mL GlcDH, 1 mM NAD , and
1
00 mM glucose to the (−)-rotundone synthesis and
found that the amount of (−)-rotundone improved to
.2 mM, which corresponded to a 24% molar conversion
1
2
+
rate against 5 mM α-guaiene (Figure 5(c)). And this
catalytic system functioned even when purified YqjH
was replaced with recombinant cells expressing YqjH,
and was scaled up to the flask size (Figure S5). Although
the synthesis rate and yield of (−)-rotundone were
slightly lowered compared to purified YqjH reaction,
tem using non-heme Fe -chelate and ferric-chelate
reductase, and we improved this system with the
addition of glucose dehydrogenase and cyclodextrin.
It is worth noting that this system is not restricted to
(−)-rotundone synthesis but can also be applied to
the synthesis of other terpenoids. Allylic oxidation

SYNTHESIS OF (−)-ROTUNDONE USING FERRIC-CHELATE REDUCTASE
7
On the other hand, the yield of (−)-rotundone
remained at a molar conversion rate of 24% com-
pared to α-guaiene. This low productivity could
result from the generation of demethylated analog;
however, the total (−)-rotundone and demethylated
analog produced was approximately 2.5 mM, equal
to 50% of the α-guaiene consumed. The remaining
5
0% was not traceable with either GC-MS or HPLC.
As shown in Figure 4, this catalytic cycle was
quenched with a galvinoxyl free radical, which indi-
cated the formation of alkyl radical intermediates
during the oxidative reaction. Therefore, the con-
densation between radical-activated α-guaiene and
another component of the reaction mixture, or
among activated α-guaiene, may have caused the
loss of substrate. The exclusion of unintentional
loss of substrate is necessary for the further
improvement of this method. In this regard,
2
+
a more efficient Fe -chelate catalyst should be
found to improve the efficiency of oxygenation. As
shown in Figure 3, the synthesis of (−)-rotundone
2
+
was dependent on the ligand structure of Fe -
chelate, which suggests that the optimization of the
ligand could lead to a higher efficiency. Bleomycin,
a glycopeptide-type anticancer drug isolated from
Streptomyces verticillus, is a natural potent non-
2
+
heme Fe -chelate catalyst [31,32]. Bleomycin pos-
sesses the activity to activate molecular oxygen and
oxidatively cleave single and double strands of DNA
2
+
with an intramolecular Fe -chelate structure which
consists of penta-coordinate nitrogen ligands. It has
2
+
been reported that N-ligand Fe -chelate catalysts,
which are bleomycin mimics, also exhibit a high
efficiency in oxygenation [19,20,33]. Therefore, we
infer that the yield of (−)-rotundone could be
Figure 5. Time courses of (−)-rotundone synthesis using
α-guaiene, NADPH, Fe(III)-EDTA, and YqjH (a), with 1% HP-β-CD
+
(
b) and 1 mM NAD , 100 mM glucose, and 0.1 U/mL GlcDH as
substitutes for NADPH (c). Circle, (-)-rotundone; square, demethy-
lated analog; diamond, rotundol; triangle, α-guaiene. Data are
shown as the means ± standard deviations of three independent
experiments.
2
+
improved with those N-ligand Fe -chelate catalysts.
This is the potential of this catalytic system to select
2
+
the type of Fe -chelate catalysts according to objec-
tive reactions. Regarding the structure of demethy-
lated analog of (−)-rotundone, several studies exist
on the O-demethylation [34], N-demethylation
could be applied to the synthesis of various terpe-
noids, and this research topic is currently being
investigated. Moreover, another property of this cat-
alytic system is that peroxide compounds are not
required as an oxygen donor. This was confirmed
through the use of catalase and galvinoxyl free radi-
cal, whereby we estimated that molecular oxygen
functioned as an oxygen donor. Molecular oxygen
is the most abundant natural oxygen donor, and the
utilization of this donor plays a significant role in
sustainable chemistry. Moreover, the application of
GlcDH and HP-β-CD allowed for this catalytic cycle
to be carried out without the costly addition of
[
22,35], or C-demethylation coupled with decarbox-
ylase [36] by non-heme iron enzymes or non-heme
2
+
Fe -chelate catalysts. However, to the best of
our knowledge, research on successive
C-demethylation and carbonylation is uncommon.
Further investigations on the reaction mechanism
will be necessary.
Author contributions
K.K. and S.U. designed the experiment; S.U. and S.
K. performed the experiments and analyzed the data; S.
U. and K.K. wrote the manuscript.
+
NAD(P)H and with a catalytic amount of NAD(P)
and Fe -chelate.
3
+

8
S. UMEZAWA ET AL.
Acknowledgments
[13] Felipe LO, Oliveira AM, Bicas JL. Bioaromas -
Perspectives for sustainable development. Trends
Food Sci Tech. 2017;62:153–414.
This research was supported in part by a Waseda
University Grant for Special Research Projects (project
number 2016A-032).
[
14] Sales A, Paulino BN, Pastore GM, et al.
Biogeneration of aroma compounds. Curr Opin
Food Sci. 2018;19:77–84.
[15] Román S, Sánchez-Siles LM, Siegrist M. The impor-
tance of food naturalness for consumers: results of
Disclosure statement
a
systematic review. Trends Food Sci Tech.
No potential conflict of interest was reported by the authors.
2017;67:44–57.
[16] Janocha S, Schmitz D, Bernhardt R. Terpene hydro-
xylation with microbial cytochrome P450 monoox-
ygenases. In: schrader J, Bohlmann
Biotechnology of Isoprenoids. Adv Biochem Eng
Biotechnol. 2015;148:215–250.
J
(eds)
References
[
1] Gershenzon J, Dudareva N. The function of ter-
pene natural products in the natural world.
Nature Chem Biol. 2007;3:408–414.
[
[
17] Schilling B, Granier T, Locher E, 1-Hydroxy-
octahydroazulenes as fragrances. Patent WO2012/
001018 A1. 2012
[
2] Schempp FM, Drummond L, Buchhaupt M, et al.
Microbial cell factories for the production of
terpenoid flavor and fragrance compounds.
J Agric Food Chem. 2018;66:2247–2258.
18] Huang A-C, Burrett S, Sefton MA, et al. Production
of the pepper aroma compound, (−)-rotundone, by
aerial oxidation of α-guaiene. J Agric Food Chem.
2014;62:10809–10815.
[
3] Bian G, Deng Z, Liu T. Strategies for terpenoid
overproduction and new terpenoid discovery.
Curr Opin Biotechnol. 2017;48:234–241.
[
[
19] Hong S, Lee Y-M, Ray K, et al. Dioxygen activation
chemistry by synthetic mononuclear nonheme iron,
copper and chromium complexes. Coordin Cem
Rev. 2017;334:25–42.
20] Lindhorst AC, Haslinger S, Kühn FE. Molecular
iron complexes as catalysts for selective C–H bond
[
4] Wood C, Siebert TE, Parker M, et al. From wine to
pepper: rotundone, an obscure sesquiterpene, is
a potent spicy aroma compound. J Agric Food
Chem. 2008;56:3738–3744.
oxygenation
reactions.
Chem
Commun.
[
[
[
[
5] Cincotta F, Verzera A, Tripodi G, et al.
Determination of sesquiterpenes in wines by
HS-SPME coupled with GC-MS. Chromatography.
2
015;51:17193–17212.
[
[
21] Abu-Omar MM, Loaiza A, Hontzeas N. Reaction
mechanisms of mononuclear non-heme iron
oxygenases. Chem Rev. 2005;105:2227–2252.
22] Nam W, Lee Y-M FS. Tuning reactivity and mechan-
ism in oxidation reactions by mononuclear nonheme
2
015;2:410–421.
6] Nakanishi A, Fukushima Y, Miyazawa N, et al.
Identification of rotundone as potent
a
odor-active compound of several kinds of fruits.
J Agric Food Chem. 2017;65:4464–4471.
iron(IV)-oxo
complexes.
Acc
Chem
Res.
2014;47:1146–1154.
7] Nakanishi A, Fukushima Y, Miyazawa N, et al.
Quantitation of rotundone in grapefruit (Citrus
paradisi) peel and juice by stable isotope dilution
assay. J Agric Food Chem. 2017;65:5026–5033.
8] Takase H, Sasaki K, Shinmori H, et al.
Cytochrome P450 CYP71BE5 in grapevine (Vitis
vinifera) catalyzes the formation of the spicy
aroma compound (−)-rotundone. J Exp Bot.
[
23] Lee Y-M, Hong S, Morimoto Y, et al. Dioxygen
activation by a non-heme iron(II) complex: forma-
tion of an iron(IV)-oxo complex via C-H activation
by a putative iron(III)-superoxo species. J Am Chem
Soc. 2010;132:10668–10670.
24] Miethke M. Molecular strategies of microbial iron
assimilation: from high-affinity complexes to cofac-
tor assembly systems. Metallomics. 2013;5:15–28.
25] Li K, Chen W-H, Bruner SD. Microbial
siderophore-based iron assimilation and therapeutic
applications. Biometals. 2016;29:377–388.
26] Miethke M, Hou J, Marahiel MA. The
siderophore-interacting protein YqjH acts as a ferric
reductase in different iron assimilation pathways of
Escherichia coli. Biochem. 2011;50:10951–10964.
27] Wang S, Wu Y, Outten FW. Fur and the novel
regulator YqjI control transcription of the ferric
reductase gene yqjH in Escherichia coli. J Bacteriol.
[
[
[
2
016;67:787–798.
[
9] Drew DP, Andersen TB, Sweetman C, et al. Two key
polymorphisms in a newly discovered allele of the
Vitis vinifera TPS24 gene are responsible for the
production of the rotundone precursor α-guaiene.
J Exp Bot. 2016;67:799–808.
[
10] Sundaresan V, Singh SP, Mishra AN, et al.
Composition and comparison of essential oils of
Pogostemon cablin (Blanco) Benth. (Patchouli) and
Pogostemon travancoricus Bedd. var. travancoricus.
J Essent Oil Res. 2009;21:220–222.
[
[
2011;193:563–574.
[
11] Tissandié L, Gaysinski M, Brévard H, et al. Revisiting
the chemistry of guaiacwood oil: identification and
28] Barbusiński K. Fenton reaction - controversy con-
cerning the chemistry. Ecol Chem Eng.
formation
pathways
of
5,11-
and
2009;16:347–358.
10,11-epoxyguaianes. J Nat Prod. 2017;80:526–537.
VI
[29] Morimoto Y, Lee Y-M, Nam W, et al. An autocata-
lytic radical chain pathway in formation of an
iron(IV)-oxo complex by oxidation of an iron(II)
[
12] Muzart J. Homogeneous Cr -catalyzed benzylic,
allylic and propargylic oxidations by tert-butyl
hydroperoxide. Mini-Rev Org Chem. 2009;6:9–20.

SYNTHESIS OF (−)-ROTUNDONE USING FERRIC-CHELATE REDUCTASE
9
complex with dioxygen and isopropanol. Chem
Commun. 2013;49:2500–2502.
[33] Geersing A, Ségaud N, van der Wijst MGP, et al.
Importance of metal-ion exchange for the biologi-
cal activity of coordination complexes of the bio-
[
[
30] Lima PSS, Lucchese AM, Araújo-Filho HG, et al.
Inclusion of terpenes in cyclodextrins: prepara-
tion, characterization and pharmacological
approaches. Carbohyd Polym. 2016;151:965–987.
31] Liu LV, Bell CB, Wong SD, et al. Definition of
the intermediates and mechanism of the antic-
ancer drug bleomycin using nuclear resonance
vibrational spectroscopy and related methods.
Proc Natl Acad Sci USA. 2010;107:22419–22424.
32] Murray V, Chen JK, Chung LH. The interaction
of the metallo-glycopeptide anti-tumour drug
mimetic
ligand
N4Py.
Inorg
Chem.
2018;57:7748–7756.
[34] Hagel JM, Facchini PJ. Biochemistry and occurrence
of O-demethylation in plant metabolism. Front
Physiol. 2010;1:14.
[35] Yi C, Yang C-G, He C. A non-heme iron-mediated
chemical demethylation in DNA and RNA. Acc
Chem Res. 2009;42:519–529.
[36] Lee AK, Banta AB, Wei JH, et al. C-4 sterol
demethylation enzymes distinguish bacterial and
eukaryotic sterol synthesis. Proc Natl Acad Sci
USA. 2018;115:5884–5889.
[
bleomycin with DNA. Int
018;19:1372–1400.
J
Mol Sci.
2