S-Adenosylhomocysteine Analogue of a Fairy Chemical, Imidazole-4-carboxamide, as its Metabolite in Rice and Yeast and Synthetic Investigations of Related Compounds

Article abstract of DOI:10.1021/acs.jnatprod.0c01269

During the course of our investigations of fairy chemicals (FCs), we found S-ICAr-H (8a), as a metabolite of imidazole-4-carboxamide (ICA) in rice and yeast (Saccharomyces cerevisiae). In order to determine its absolute configuration, an efficient synthetic method of 8a was developed. This synthetic strategy was applicable to the preparation of analogues of 8a that might be biologically very important, such as S-ICAr-M (9), S-AICAr-H (10), and S-AICAr-M (11).

Full text of DOI:10.1021/acs.jnatprod.0c01269

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Article
S‑Adenosylhomocysteine Analogue of a Fairy Chemical, Imidazole-
4-carboxamide, as its Metabolite in Rice and Yeast and Synthetic
Investigations of Related Compounds
Hitoshi Ouchi, Takuya Namiki, Kenji Iwamoto, Nobuo Matsuzaki, Makoto Inai, Mihaya Kotajima,
Jing Wu, Jae-Hoon Choi, Yoko Kimura, Hirofumi Hirai, Xiaonan Xie, Hirokazu Kawagishi,*
and Toshiyuki Kan*
Cite This: J. Nat. Prod. 2021, 84, 453−458
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ABSTRACT: During the course of our investigations of fairy
chemicals (FCs), we found S-ICAr-H (8a), as a metabolite of
imidazole-4-carboxamide (ICA) in rice and yeast (Saccharomyces
cerevisiae). In order to determine its absolute configuration, an
efficient synthetic method of 8a was developed. This synthetic
strategy was applicable to the preparation of analogues of 8a that
might be biologically very important, such as S-ICAr-M (9), S-
AICAr-H (10), and S-AICAr-M (11).
airy rings are zones of promoted or inhibited grass growth
their glycosides regulated the homeostasis of rice. In the course
of our continuing search for metabolite(s) of 2 from rice, we
found a new metabolite 8a. Herein, we describe the isolation of
the compound, determination of its absolute configuration by
chemical synthesis, and synthetic development of its related
compounds.
1
Fdue to an interaction between a fungus and a plant. This
phenomenon had been caused by unknown “fairies” before our
chemical discoveries. We disclosed that the fairies, the two
plant-growth regulators produced by a fairy-ring forming
fungus Lepista sordida, are 2-azahypoxanthine (1: AHX) and
imidazole-4-carboxamide (2: ICA).2 Furthermore, 2-aza-8-
oxohypoxanthine (3: AOH) was isolated as a metabolite of
AHX common in plants, and the compound also stimulated
plant growth.3 The three compounds were named fairy
chemicals (FCs) after the title of the article in Nature that
introduced our study.4 FCs exhibited growth regulatory
activity against all the plants tested, regardless of the families
they belong to, and provided tolerance to various stresses to
plants.5 In addition, yields of rice, wheat, and other crops were
increased by FCs treatment, suggesting that FCs can be
applied to agriculture.5,8 We have also proven the endogenous
existence of FCs in plants and mushrooms and discovered a
new route in the purine metabolic pathway through which FCs
are biosynthesized from 5-aminoimidazole-4-carboxamide (4:
AICA).3,5−7
RESULTS AND DISCUSSION
■
First of all, we investigated whether ICA (2) was converted to
further metabolites or not. Rice seedlings were treated with 2,
and their roots were extracted with EtOH and acetone,
successively. The solutions were combined and dried under
reduced pressure. The extracts were analyzed by high-
performance liquid chromatography (HPLC), and an un-
known peak having a UV absorption pattern similar to those of
2, 5, 6, and 7 was detected (Figure S1). Therefore, we tried to
isolate the unidentified metabolite, and the extracts were
fractionated by octadecylsilyl (ODS) gel flash column
chromatography, followed by HPLC, giving the target
metabolite 8a (Figure 2).
We hypothesized that FCs are a new family of plant
hormones, and the clarification of the metabolic pathways of
FCs is important to substantiate the hypothesis.7,9−11
A
Received: November 23, 2020
Published: January 22, 2021
riboside of ICA (ICAr, 5) and a glucoside of 5 (6) have been
isolated from ICA-treated rice, and endogenous existence of 5
and a ribotide of ICA (7) in rice has been proved (Figure 1).12
The metabolites showed no or weak activity against plant
growth, suggesting that the interconversion between FCs and
© 2021 American Chemical Society and
American Society of Pharmacognosy
https://dx.doi.org/10.1021/acs.jnatprod.0c01269
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Figure 1. Metabolic pathway of purine in plants, animals, and microorganisms.
Figure 3. DQF-COSY and HMBC correlations of S-ICAr-H (8a).
cross-peaks (H-5′/C-1″; H-1″/C-5′) indicated that the
homocysteine was connected to the ribose at C-5′ via a
thioether bond. The complete assignment of all the proton and
carbon signals of NMR was accomplished as shown in Table 1.
Figure 2. Structures of S-ICAr-H (8a) and its derivatives.
Compound 8a was purified as a pale yellow, amorphous
material. The molecular formula was determined as
C13H20N4O6S by high-resolution electrospray ionisation mass
spectrometry (HRESIMS), indicating the presence of six
degrees of unsaturation in the molecule. Its structure was
elucidated by the interpretation of nuclear magnetic resonance
(NMR) spectra including distortionless enhancement by
polarization transfer (DEPT), double quantum filtered
correlation spectroscopy (DQF-COSY), heteronuclear multi-
ple quantum coherence (HMQC), and heteronuclear multiple
bond coherence (HMBC).
The DEPT experiment indicated the presence of three
methylenes, seven methines, and three nonprotonated carbons.
The 13C NMR data (δC 34.1, 72.8, 75.8, 84.8, 91.8, 121.4,
131.3, 136.9, 163.0) indicated that 8a had the same skeleton as
that of ICAr.12 The ribose part (C-1′−C-5′) was constructed
by the DQF-COSY correlations (H-1′/H-2′; H-2′/H-3′, 4′;
H-3′/H-4′; H-4′/H-5′) and the HMBC correlations (H-1′/C-
2′; H-2′/C-1′, C-4′; H-3′/C-1′, C-5′; H-4′/C-3′; H-5′/C-3′,
C-4′) (Figure 3). The linkage position between the sugar and
2 was determined from the cross-peaks (H-1′/C-2 and H-1′/
C-5) (Figure 3). This compound possessed four more carbons
than ICAr. The moiety constructed with the additional four
carbons, homocysteine (C-1″−C-4″), was suggested by the
characteristic 13C NMR chemical shifts at δC 28.1, 30.5, 53.1,
and 173.1, the DQF-COSY correlations (H-1″/H-2″; H-2″/H-
3″; H-3″/H-4″), and the HMBC correlations (H-1″/C-2″, C-
3″; H-2″/C-1″, C-3″, C-4″; H-3″/C-1″, C-2″, C-4″) (Figure
3). The molecular formula of the compound and its HMBC
1
Table 1. H and 13C NMR Data for 8a
8a (in D2O)
13C
1H
position
δC, type
δH, mult (J in Hz)
8.60, br.s
2
4
136.9, CH
131.3, C
5
6
121.4, CH
163.0, C
8.01, br.s
1′
2′
3′
4′
5′a
5′b
1″
2″a
2″b
3″
4″
91.8, CH
75.8, CH
72.8, CH
84.8, CH
34.1, CH2
5.78, d (4.6)
4.37, dd (4.6, 4.6)
4.16, dd (4.6, 5.2)
4.23, m
2.90, dd (5.2, 14.3)
2.82, dd (6.4, 14.3)
2.63, dd (7.5, 7.5)
2.11, m
28.1, CH2
30.5, CH2
2.04, m
53.1, CH
173.1, C
3.93, dd (6.3, 6.3)
Compound 8a, described as S-ICAr-H, was a new compound;
however, its absolute configuration could not be determined by
the spectroscopic data. Furthermore, very interestingly, we
found that yeast (Saccharomyces cerevisiae) also produced 8a by
treatment with 2 (Figure S6). Therefore, we tried to clarify the
absolute configuration of the compound by chemical synthesis.
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We deduced that the ribose in 8a was D, because all the
known naturally occurring ribosides have D-ribose. In order to
determine the absolute configuration of the homocysteine part
in 8a, we decided to condense 5 and each of the optical
isomers of homocysteine derivative 14. However, both of the
enantiomers of homocysteine are very expensive for the
synthetic starting material and we needed a more practical way
to synthesize the target compound.13 Then, we came up with
an idea to use both enantiomers of methionine that are much
less expensive (L-form: $ 21/25 g and D-form: $ 84/25 g; TCI)
for synthesis of the corresponding homocysteine.
After the selective removal of the methyl group of L-
methionine (12) by Birch condition, without purification of
the produced homocysteine, oxidative dimerization with
hydrogen peroxide proceeded smoothly to provide the stable
homocystine.13 In order to accomplish the efficient C−S bond
formation of 8a,14,15a the protection of the carboxy and amino
groups by methyl esterification and incorporation of a Boc
group provided 13. The cleavage of the disulfide bond of 13
was performed by treatment with Zn in the presence of acetic
acid to give 14, which was subjected to the coupling without
purification. On the contrary, ICAr part 16 was synthesized
from protected ICAr (15), which was readily synthesized from
inosine by mesylation and iodine displacement.15b Upon the
treatment of 14 and 16 with triethylamine in DMF, the desired
condensation proceeded smoothly to afford 17. Subsequently,
the stepwise deprotection of 17 was carried out by the removal
of the TBS ether and Boc groups and the hydrolysis of the
methyl ester to give 8a. Furthermore, employing the same
sequence for the synthesis of 8a, the synthesis of its
diastereomer 8b was also accomplished using D-methionine.
A remarkable difference of the retention times of 8a and 8b
was observed in HPLC analysis by using a chiral-phase
column, CROWNPAK CR (+) (Figure S8). As a result, the
isolated compound was determined to be 8a.
derivatives should be expected to be significant compounds
for the biological investigations.
We thought that it was highly possible that the analogue of
SAH, 8a, was generated from the analogue of SAM, S-ICAr-M
(9), in plants, and therefore, we synthesized the analogue.
Upon the treatment of 8a with methyl iodide and AgOAc in
HCO2H and CH3CO2H, the methylation reaction proceeded
smoothly to give 9. Because the alkyl sulfide derivative
possessed a labile nature, the purification of 9 by general
methods was not possible. Therefore, the purification of the
compound was performed according to the reported
procedure19 for the preparation of similar S-dimethyladenosyl
methionine; briefly, 0.1% TFA in H2O was added to the
reaction mixture and resulting precipitates were removed by
filtration. After washing the filtrate with Et2O and removing the
organic solvent under reduced pressure at room temperature to
avoid degradation, the purification of 9 was performed by
HPLC of the resulting aqueous solution (Scheme 1).
Scheme 1. Synthesis of S-ICAr-H (8a) and S-ICAr-M (9)
The plant growth regulatory activities of 8a and 8b were
examined using rice. Both diastereomers inhibited the shoot
growth only at a high concentration (0.1 mM) like 2, while
they did not show any activity against the root unlike 2 (Figure
S9).
The structure of 8a is similar to that of S-adenosylhomo-
cysteine (SAH). SAH is derived from S-adenosylmethionine
(SAM) in many living organisms (Figure 4). SAM is a key
Next, we turned our attention to S-AICAr-H (10), as AICAr
(18: acadesine) is known as a metabolite in the purine
metabolic pathway like FCs21 and its aglycon, AICA (4), is
converted to ICA (2) in plants, and 10 has been found in the
plasma and urine of homocystinurics patients.20 However, the
detailed spectroscopic data of 10 as well as its chemical
synthesis have not been reported. We expected that the
employment of a similar procedure for the preparation of 8a
would readily provide the desired 10 and the methionine
analogue of 10, S-AICAr-M (11). After protection of all the
hydroxy groups of 18 as TBS ethers and selective hydrolysis by
acidic conditions, the resultant primary alcohol was converted
to the corresponding iodide derivative 19 by treatment with
PPh3 and I2. Upon the treatment of 14 and 19 with
triethylamine in DMF, the desired coupling reaction proceeded
smoothly to give 20. After the deprotection of the TBS ethers
of 20, the deprotection of the Boc group and the hydrolysis of
the methyl ester provided the desired S-AICAr-H (10).
Additionally, the treatment of 10, similar to that for the
Figure 4. Structures of SAM and SAH.
compound for living organisms, because the compound serves
as the methyl group donor for various biological materials such
as DNA, peptide, choline, creatinine, and many other small
molecules, with SAH as the byproduct. SAM is also a
biosynthetic precursor of polyamines, spermidine and
spermine, and a gaseous plant hormone, ethylene.16,17 In
addition, SAH is a strong inhibitor of several methyltrans-
ferases.18 Therefore, new analogues of SAH and SAM
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[1H NMR (500 MHz), 13C NMR (125 MHz)] spectra were
determined on a JEOL ECZ-500R instrument. Chemical shifts for 1H
NMR were reported in ppm relative to the singlet at 7.26 ppm for
deuteriochloroform, the broad singlet at 3.30 ppm for deuteriome-
thanol, or the singlet at 4.65 ppm for deuterium oxide. Chemical shifts
for 13C NMR were reported in parts per million relative to the
centerline of a triplet at 77.0 ppm for deuteriochloroform or the
centerline of a septet at 49.0 ppm for deuteriomethanol. High-
resolution mass spectra (HRMS) were obtained on a Bruker
Daltonics micrOTOF (ESI). Analytical thin layer chromatography
(TLC) was performed on Merck precoated analytical plates, 0.25 mm
thick, silica gel 60 F254. Preparative TLC separations were made on 7
cm × 20 cm plates prepared with a 0.25 mm layer of Merck silica gel
60 F254. Compounds were eluted from the adsorbent with 10%
MeOH in CHCl3. Silica gel column chromatography separations were
performed on Chromatorex PSQ 60B or PSQ 100B purchased from
Fuji Silysia Chemical Ltd. HPLC analyses and separations were
performed with a SHIMADZU HPLC Prominence system. Reagents
and solvents were of commercial grade and were used as supplied with
the following exceptions: (1) CH2Cl2, dimethylformamide, tetrahy-
drofuran, and toluene were dried over molecular sieves 4A; (2)
MeOH was dried over molecular sieves 3A. All reactions sensitive to
oxygen and/or moisture were conducted under an argon atmosphere.
Detection of ICA (2) and its Metabolite 8a by HPLC. All
plants were divided into two parts: the shoot and root. The shoot and
root were extracted with EtOH. After removing the solvent of each
extracts under reduced pressure, they were dissolved in 5% MeOH
and subjected to HPLC analysis. The extracts were analyzed by
reversed-phase HPLC using a Develosil C30-UG-5 column (ϕ 4.6
mm × 250 mm, 5 μm) with a gradient manner (5% MeOH (B) in
(A) for 30 min, 5%−100% B in A for 45 min, and 100% B for 30 min)
at a flow rate of 0.5 mL/min at a multiple wavelength using a
photodiode array detector12 (Figure S1, Supporting Information).
Purification of 8a from ICA-Treated Rice. Rice seeds were
germinated in nursery boxes, incubated for a week, and then treated
with ICA (0.5 mM) in a growth chamber under a 16 h photoperiod at
28 °C for 2 weeks. The treated seedlings were divided into two parts,
shoot and root. The root (1.9 kg) was extracted with EtOH and then
acetone. After the solution was combined and concentrated under
reduced pressure, the concentrate was extracted with CH2Cl2 to
remove lipophilic substances and dried under reduced pressure. The
residue (14.6 g) was fractionated by ODS gel flash column
chromatography (Cosmosil 140 C18-OPN, Nacalai Tesque, Japan;
5%, 10%, 25%, 50% MeOH, MeOH) to obtain five fractions. Fraction
2 (263.5 mg) was further separated by HPLC with a Develosil C30-
UG-5 column (ϕ 20 mm × 250 mm, 5 μm; flow rate 5 mL/min;
injection volume 1 mL; 5% MeOH; UV detection 210 nm) to obtain
21 fractions (fraction 2-1 to 2-21). Fraction 2-10 was separated by
HPLC with Cosmosil 5PYE (ϕ 10 mm × 250 mm, 5 μm; flow rate 2
mL/min; injection volume 0.5 mL; 2% MeOH; UV detection 210
nm) to afford 8a (2.0 mg). 8a: pale yellow amorphous. [α]D25 −31 (c
0.31, H2O). IR (film): 3342, 664 cm−1. 1H and 13C NMR data: Table
1. ESIMS m/z 359 [M − H]−. HRESIMS m/z 383.1022 [M + Na]+
(calcd for C13H20N4NaO6S, 383.1001).
conversion of 8a to 9, afforded 11 without providing any N-
alkyl derivatives (Scheme 2).
Scheme 2. Synthesis of S-AICAr-H (10) and S-AICAr-M
(11)
As mentioned above, SAM is a methyl group donor in living
organisms. The methylation is catalyzed by various methyl
transferases. The ability of the synthesized SAM analogues, S-
ICAr-M (9) and S-AICAr-M (11), as substrates of one of the
enzymes, caffeic acid 3-O-methyltransferase, was compared
with that of SAM. The transferase of rice was heterologously
expressed in Escherichia coli, and caffeic acid was used as the
acceptor. In the presence of SAM, caffeic acid was completely
methylated by the enzyme to form ferulic acid. Compounds 9
and 11 also served as methyl donors; however, their
conversion rates of caffeic acid to ferulic acid was much
lower than that of SAM: 9, 60% and 11, 49% compared to
SAM, 100% (Figure S10).
In summary, we discovered a unique metabolite of ICA, S-
ICAr-H (8a), in rice and yeast and developed an efficient
synthetic method for 8a. The absolute configuration of 8a was
determined by comparison with synthetic epimers 8a and 8b.
Furthermore, our synthetic strategy was applicable to the
preparation of S-ICAr-M (9), S-AICAr-H (10), and S-AICAr-
M (11). Our new synthesized compounds possess potential as
chemical biology tools for the investigation of extremely
important biomolecules, SAM and SAH. Some of the
synthesized compounds might exist in nature in addition to
8a, and we are now trying to confirm their endogenous
existence.
EXPERIMENTAL SECTION
■
Yeast (Saccharomyces cerevisiae). Saccharomyces cerevisiae
strains (W303) were precultured in YPAD medium [1% yeast extract,
2% peptone, 2% glucose, and 150 μM adenine] for 12 h (25 °C, 100
rpm). Then, 10 μL of the culture was diluted in 70 mL of YPAD
medium. The yeast strains were cultured until the OD600 reached 0.5,
and the cells obtained by centrifugation (10 000g, 5 min) were
washed with distilled water. The cells were diluted with SC medium
[0.5% ammonium sulfate, 2% glucose, 0.5% casamino acids, 0.17%
yeast nitrogen, 0.0006% tryptophan, 0.01% uracil, and 150 μM
adenine/ICA] so that the OD600 at the start of culture was 0.2 and
were cultured for 5 days. The cells collected by centrifugation
(10 000g, 5 min) were extracted and fractionated according to
previous report.5
General Experimental Procedures for Isolation. The specific
rotation values were measured by a Jasco DIP-1000 polarimeter, and
infrared (IR) spectra were recorded on a Jasco FT/IR-4100
spectrometer. 1H NMR spectra were recorded on a JEOL ECZ-
500R spectrometer at 500 MHz, while 13C NMR spectra were
recorded on the same instrument at 125 MHz. The HRESIMS spectra
were measured on a JMS-T100LC mass spectrometer. The HPLC
separations were performed with a Jasco Gulliver system using
reverse-phase HPLC columns (Develosil C30-UG-5, Nomura
chemical and Cosmosil 5PYE waters Nacalai Tesque). An ODS gel
(Cosmosil 140 C18-OPN, Nacalai Tesque) was used for flash column
chromatography. All solvents used throughout the experiments were
obtained from Kanto Chemical Co.
General Experimental Procedures for Synthesis. Optical
rotations were measured on a JASCO P-2200 polarimeter. Infrared
(IR) spectra were recorded on a SHIMADZU IRPrestige-21. NMR
Identification and Quantification of 8a. The identification and
quantification of S-ICAr-H (8a) from the ICA-treated samples was
completed using a triple linear ion trap instrument (LIT)
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(QTRAP5500; AB Sciex) with an electrospray ionization (ESI)
source and coupled to a UHPLC system (Nexera X2; Shimadzu) in
MRM (Multiple Reaction Monitoring) analysis mood. MS/MS
spectra were recorded in product ion scan mode using LIT. The
MRM analysis was performed at 25 V collision energy, and the
transitions of m/z 361 > 134 and 361 > 88 were monitored for S-
ICAr-H in the ESI positive ion mode. The data acquisition and
analysis were performed with the Multi Quant software (ver. 3.0.1).
The quantification of S-ICAr-H was conducted using synthetic
standards. The exudate samples were dissolved in 50% aqueous
MeOH and filtered through a spin column (PVDF 0.45 μm,
Millipore). An aliquot of the filtered 50% aqueous MeOH sample
solution was diluted with a volume of either pure 50% MeOH or 50%
MeOH containing known amounts of S-ICAr-H standard. The
increase in peak area on the chromatogram corresponded to the
amounts of S-ICAr-H standards added, enabling the amount of 8a in
the samples to be deduced (Figure S6).
Bioassay. Rice seeds (Oryza sativa L. cv. Nipponbare) were
sterilized in EtOH for 5 min and then 1% sodium hypochlorite for 30
min on a plastic container (18.5 × 14.5 × 4.5 cm3). The seeds were
washed completely in sterile H2O and germinated for 2 days at 30 °C
with intensive light. The germinated seeds (n = 4) were planted onto
test tube (ϕ 5.5 cm × 10 cm) containing samples (1, 10, and 100
μM) and incubated for a week at 30 °C with intensive light. The
lengths of the root and shoot were measured to an accuracy of 0.01
mm with an absolute digimatic caliper (Mitutoyo Co.) (Figure S9).
Expression of Recombinant OsCOMT1. RNA was isolated from
root of rice (Oryza sativa L. cv. Nipponbare) using an RNeasy Plant
Mini Kit (QIAGEN), according to the manufacturer’s protocol. The
cDNA was synthesized with the PrimeScript RT Reagent Kit
(TaKaRa). The full-length cDNA from rice that corresponds to
caffeic acid 3-O-methyltransferase (COMT1) was amplified with the
following primers: 5′-GAG CTC GAA TTC GGA CTA CTT GGT
GAA CTC GAT GGC-3′ (forward) and 5′-GCG GCA GCC ATA
TAA TGG GTT CTA CAG CCG CC-3′ (reverse). The cDNA
sequence ligated into the pET-28a (+) vector linearized by BamHI
and Pvu II to yield pET-28a (+)-COMT1 plasmid. For the expression
of the COMT1 protein, Escherichia coli BL21-Codon Plus (DE3) was
transformed with the pET-28a (+)-COMT1 plasmid. Recombinant E.
coli was cultured in lysogeny broth (LB) broth containing 20 mg/L
kanamycin sulfate at 37 °C overnight. Four milliliters of the overnight
culture was added to 500 mL of fresh LB containing 20 mg/L
kanamycin sulfate, and it continued to incubate for 3−4 h and was
shaken at frequency of 150 rpm at 37 °C. Upon the culture reaching
an OD600 of 0.5, isopropyl-β-D-thiogalactopyranoside (IPTG) was
added to the culture at a final concentration of 1 mM to induce
protein expression and the culture was incubated for 3 h at 37 °C.
The cells were harvested by centrifugation, resuspended in lysis buffer
(20 mM Tris-HCl, 100 mM NaCl, 30 mM imidazole, pH 7.4), and
sonicated on ice. After centrifugation, the supernatant was loaded
onto a Histrap HP column (1 mL, GE Health Sciences) equilibrated
with buffer A (20 mM Tris-HCl, 100 mM NaCl, 30 mM imidazole,
pH 7.4) and the His-tagged protein was eluted with buffer B (20 mM
Tris-HCl, 100 mM NaCl, 500 mM imidazole, pH 7.4). The purified
enzyme concentration was determined using a Pierce 660 nm Protein
Assay Reagent (Thermo Fisher Scientific).
the negative FTMS mode at a resolution of 30 000 at m/z 400 with
the following source parameters: sheath gas flow, 50; auxiliary gas flow
rate, 10; tube lens, −63 V; capillary voltage, −16 V; ion spray voltage,
3 kV (Figure S10).
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c01269.
Figures of HPLC profiles, H and 13C NMR spectra,
1
HMQC, HMBC, and COSY spectra, MRM chromato-
grams and product ion spectra, growth regulatory of
ICA, and LC−MS/MS chromatograms and discussions
of synthetic methods (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Hirokazu Kawagishi − Graduate School of Integrated Science
and Technology, Research Institute of Green Science and
Technology, and Graduate School of Integrated Science and
Technology, Shizuoka University, Shizuoka 422-8529,
Japan; orcid.org/0000-0001-5782-4981;
Email: kawagishi.hirokazu@shizuoka.ac.jp
Toshiyuki Kan − School of Pharmaceutical Sciences,
University of Shizuoka, Shizuoka 422-8526, Japan;
orcid.org/0000-0002-9709-6365; Email: kant@u-
shizuoka-ken.ac.jp
Authors
Hitoshi Ouchi − School of Pharmaceutical Sciences, University
of Shizuoka, Shizuoka 422-8526, Japan
Takuya Namiki − School of Pharmaceutical Sciences,
University of Shizuoka, Shizuoka 422-8526, Japan
Kenji Iwamoto − School of Pharmaceutical Sciences,
University of Shizuoka, Shizuoka 422-8526, Japan
Nobuo Matsuzaki − Graduate School of Integrated Science
and Technology, Shizuoka University, Shizuoka 422-8529,
Japan
Makoto Inai − School of Pharmaceutical Sciences, University
of Shizuoka, Shizuoka 422-8526, Japan; orcid.org/0000-
0001-7074-0520
Mihaya Kotajima − Graduate School of Integrated Science and
Technology, Shizuoka University, Shizuoka 422-8529, Japan
Jing Wu − Research Institute of Green Science and Technology,
Shizuoka University, Shizuoka 422-8529, Japan
Jae-Hoon Choi − Graduate School of Integrated Science and
Technology, Research Institute of Green Science and
Technology, and Graduate School of Integrated Science and
Technology, Shizuoka University, Shizuoka 422-8529, Japan
Yoko Kimura − Graduate School of Integrated Science and
Technology, Shizuoka University, Shizuoka 422-8529, Japan
Hirofumi Hirai − Graduate School of Integrated Science and
Technology, Research Institute of Green Science and
Technology, and Graduate School of Integrated Science and
Technology, Shizuoka University, Shizuoka 422-8529, Japan
Xiaonan Xie − Center for Bioscience Research and Education,
Utsunomiya University, Utsunomiya, Tochigi 321-8505,
Japan
Enzyme Assay. Enzymatic reactions with purified recombinant
OsCOMT1 were carried out according to Koshiba et al.22 S-adenosyl-
L-methionine (SAM) and the analogues, S-ICAr-M and S-AICAr-M,
were used as a methyl donor. The typical reaction mixture (100 μL)
contained 50 mM Tris-HCl (pH 7.4), 200 μM caffeic acid (CA), 200
μM methyl donors, and 1 μM enzyme solution. After incubation for 2
h at 30 °C, the reaction was terminated by adding 50 μL of MeCN.
Following centrifugation to remove protein, the reaction products
were analyzed by a Shimadzu UPLC system (Shimadzu) coupled to
an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific)
equipped with an electrospray ionization probe. An ACQUITY UPLC
BEH C18 column (ϕ 2.1 mm × 50 mm, 1.7 μm, Waters) was used in
the analysis (injection volume, 5 μL; solvent, 30% MeCN with 0.05%
formic acid; flow rate; 0.2 mL/min). MS analysis was performed in
Complete contact information is available at:
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Notes
The authors declare no competing financial interest.
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J. Nat. Prod. 2021, 84, 453−458

Journal of Natural Products
pubs.acs.org/jnp
Article
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ACKNOWLEDGMENTS
■
This work was financially supported by Grant-in-Aid for
Scientific Research (JP17H03973, JP19K22330, and
JP19K05460) from JSPS, a Grant-in-Aid for Specially
Promoted Research “Science of fairy chemicals and their
application development” (JP20H05620) from JSPS, a Grant-
in Aid for Scientific Research on Innovative Areas “Middle
Molecular Strategy” (JP16H01160) from MEXT, a Grant-in
Aid for Scientific Research on Innovative Areas “Frontier
Research on Chemical Communications” (JP17H06402) from
MEXT, Specific Research Grant from Takeda Science
Foundation, and Platform Project for Supporting Drug
Discovery and Life Science Research “Basis for Supporting
Innovative Drug Discovery and Life Science Research
(BINDS)” (JP19am0101099) from AMED.
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