Regiospecific Prenylation of Hydroxyxanthones by a Plant Flavonoid Prenyltransferase

Article abstract of DOI:10.1021/acs.jnatprod.6b00417

C-Prenylated xanthones are pharmacologically attractive specialized metabolites that are distributed in plants and microorganisms. The prenylation of xanthones often contributes to the structural diversity and biological activities of these compounds. However, efficient regiospecific prenylation of xanthones is still challenging. In this study, the regiospecific prenylation of a number of structurally different hydroxyxanthones (3-10) by MaIDT, a plant flavonoid prenyltransferase with substrate flexibility from Morus alba, is demonstrated. Among the enzymatic products, 2-dimethylallyl-1,3,7-trihydroxyxanthone (3a) effectively attenuated glutamate-induced injury in SK-N-SH neuroblastoma cells. These results suggest a potential approach for the synthesis of bioactive prenylated xanthones by a substrate-relaxed flavonoid prenyltransferase.

Full text of DOI:10.1021/acs.jnatprod.6b00417

Note
pubs.acs.org/jnp
Regiospecific Prenylation of Hydroxyxanthones by a Plant Flavonoid
Prenyltransferase
Ruishan Wang,† Ridao Chen,†,‡ Jianhua Li,† Xiao Liu,† Kebo Xie,† Dawei Chen,† Ying Peng,*,†
and Jungui Dai*,†,‡
‡
†State Key Laboratory of Bioactive Substance and Function of Natural Medicines and Key Laboratory of Biosynthesis of Natural
Products of National Health and Family Planning Commission, Institute of Materia Medica, Peking Union Medical College and
Chinese Academy of Medical Sciences, Beijing 100050, People’s Republic of China
S
* Supporting Information
ABSTRACT: C-Prenylated xanthones are pharmacologically
attractive specialized metabolites that are distributed in plants
and microorganisms. The prenylation of xanthones often
contributes to the structural diversity and biological activities
of these compounds. However, efficient regiospecific pre-
nylation of xanthones is still challenging. In this study, the
regiospecific prenylation of a number of structurally different
hydroxyxanthones (3−10) by MaIDT, a plant flavonoid prenyltransferase with substrate flexibility from Morus alba, is
demonstrated. Among the enzymatic products, 2-dimethylallyl-1,3,7-trihydroxyxanthone (3a) effectively attenuated glutamate-
induced injury in SK-N-SH neuroblastoma cells. These results suggest a potential approach for the synthesis of bioactive
prenylated xanthones by a substrate-relaxed flavonoid prenyltransferase.
anthones are naturally occurring compounds produced by
cell cultures.12 Two putative XPTs, XptA and XptB, have been
identified through gene deletion experiments in Aspergillus
nidulans.13 Additionally, XptB has been demonstrated to only
catalyze regiospecific O-prenylation of hydroxyxanthones in
vitro.14
In recent years, substantial progress has been achieved in
molecular biological and biochemical investigations on aromatic
prenyltransferases (PTs) from bacteria, fungi, and plants.15−18
Interestingly, a number of PTs exhibit obvious substrate
(aromatic acceptors and/or prenyl donors) tolerance, which
renders them powerful biocatalysts for the chemoenzymatic
prenylation of aromatic compounds.19−22 However, there are
rare aromatic PTs with substrate promiscuity toward various
xanthones and catalyzing prenylation. In the present work, the
efficient regiospecific prenylation of structurally different
xanthones by the promiscuous flavonoid PT MaIDT from the
medicinal plant Morus alba is described.23
1−4 Their common 9H-
Xplants, lichens, fungi, and bacteria.
xanthen-9-one scaffold has been described as a “privileged
structure” due to its potential to bind to a variety of targets;
many xanthones have been proven to possess diverse biological
and pharmacological activities.5,6 A host of xanthones with
medicinal value are functionalized with C-prenyl groups, most
commonly five-carbon dimethylallyl moieties.1−6 Decoration of
xanthones by prenyl moieties often enhances the biological
activities of these compounds.5,6 For example, α-mangostin, a
prenylated xanthone from the mangosteen fruit, possesses
multiple biological activities such as anticancer, antimycobacte-
rial, antioxidant, and anti-inflammatory.7−10 Owing to limited
resources in nature and the difficulty of regiospecificity in
organic synthesis approaches, an efficient approach for the
synthesis of structurally diverse prenylated xanthones for drug
discovery is of specific interest and importance.
Our previous work identified two flavonoid prenyltrans-
ferases (FPTs), MaIDT and CtIDT, from two moraceous
plants, M. alba and Cudrania tricuspidata, respectively.23 They
were able to regioselectively introduce a dimethylallyl
diphosphate into the ortho-position to the phenolic groups in
the common 2,4-dihydroxyacetophenone substructure shared
by three types of flavonoids, i.e., chalcones, isoflavones, and
flavones. Natural hydroxyxanthones and benzophenones
usually bear this substructure, emphasizing that MaIDT and
CtIDT might catalyze regiospecific prenylation of hydroxyx-
anthones.
Biosynthetically, xanthones result from a mixed origin
including both the polyketide (A-ring) and shikimate pathways
(C-ring) [Figure S1, Supporting Information (SI)].11 After the
formation of the C13 framework, which is catalyzed by
benzophenone synthase, the freely rotating intermediate
2,3′,4,6-tetrahydroxybenzophenone (1) undergoes a regiose-
lective cyclization to give rise to 1,3,7-trihydroxyxanthone (3)
and/or 1,3,5-trihydroxyxanthone (4), which are the precursors
of most prenylated xanthones. The introduction of prenyl
moieties into hydroxyxanthones may occur at the benzophe-
none and/or xanthone stages (Figure S1, SI). However, to date,
only one xanthone prenyltransferase (XPT), HcPT, which
catalyzes the regiospecific 8-prenylation of 1,3,6,7-tetrahydrox-
yxanthone, has been characterized from Hypericum calycinum
Received: May 10, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00417
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
To initiate the studies, the acceptance of the defined
precursor of prenylated xanthones, 2,3′,4,6-tetrahydroxybenzo-
phenone (1) and 2,4,6-trihydroxybenzophenone (2), as well as
1,3,7-trihydroxyxanthone (3) and 1,3,5-trihydroxyxanthone (4),
respectively, by MaIDT and CtIDT was tested. LC-MS analysis
of the incubation mixtures revealed that both MaIDT and
CtIDT accepted only 3 and 4 as substrates, and MaIDT
showed significantly higher activities toward these compounds
than CtIDT (Figures S2 and S3, SI). Consequently, MaIDT
was chosen for further exploration of its prenylation activities
toward a number of structurally different xanthones.
After incubation with 100 μg of recombinant yeast
microsomes containing MaIDT in a 100 μL assay at 30 °C
for 16 h, 3 and 4 were converted to 3a and 4a with 55% and
43% yield, respectively. Under these conditions, 90% of
isoliquiritigenin, a natural substrate of MaIDT, was converted
to 3′-dimethylallylisoliquiritigenin.23 Examining 3, for example,
by HPLC-UV/ESIMS analysis revealed that a product at tR 23.4
min with a 68 amu shift in its molecular ion peak was detected
in the incubation mixture with the yeast cell microsomes
transformed with MaIDT. In contrast, no PT activity was
observed when microsomes isolated from yeast cells trans-
formed with an empty vector or boiled recombinant protein
were used. These observations indicated the monoprenylation
of 3 (Figure 1). Product formation was strictly dependent on
the presence of MaIDT, DMAPP, Mg2+, and 3. A linear
dependence on the amount of microsomal protein for product
formation was found up to 200 μg per 100 μL assay and on a
reaction time up to 30 min at 30 °C.
To determine the prenylation properties, the prenylated
product (3a) was subsequently prepared from a scaled-up
enzymatic reaction and subjected to NMR spectroscopic
1
analysis (Figures S4 and S5, SI). In the H NMR spectrum,
the presence of the prenyl moiety was suggested by the signals
for two methyls (δH 1.65 and 1.79, s, 3H each), a methylene
(δH 3.37, d, J = 7.2 Hz, 2H), and a methine (δH 5.29, t, J = 7.2
Hz, 1H). The location of the prenyl moiety at C-2 was inferred
by the fact that the H-2 signal (δH 6.20) in 3 had disappeared
and that the resonance of the active 1-OH proton shifted to
lower field (from δH 12.70 to δH 13.27), which indicated the
introduction of a prenyl substituent at the ortho-position.24 This
deduction is supported by a deshielded chemical shift of C-2
from δC 97.8 in 3 to δC 111.3 in 3a (Figure S5, SI). The NMR
data for 3a were in good agreement with those for 2-
dimethylallyl-1,3,7-trihydroxyxanthone.25 Therefore, it was
proven that MaIDT promoted the specific 2-prenylation of
1,3,7-trihydroxyxanthone (3). Likewise, 4a was isolated from
the preparative-scale incubation and determined to be 2-
dimethylallyl-1,3,5-trihydroxyxanthone by MS and NMR
spectroscopic data analyses (Figures S6 and S7, SI). Notably,
3 and 4 turned out to be specifically prenylated at C-2, although
their C-ring substitution patterns were different.
Encouraged by the aforementioned results, the activities of
MaIDT utilizing nine potential substrates including various
types of hydroxyxanthones with different substituents (Figure
2) were tested. As shown in Figure 2A, all of the
hydroxyxanthones with 1,3-dihydroxy substitution were accep-
ted by MaIDT, specifically 1,3-dihydroxyxanthone (5, 51.1%
yield), 1,3,6-trihydroxyxanthone (6, 19.1% yield), 1,3,8-
trihydroxyxanthone (7, 28.5% yield), 1,3,5,6-tetrahydroxyxan-
thone (8, 19.3% yield), 1,3,6,8-tetrahydroxyxanthone (9, 30.1%
total yield), and 1,3,6,7-tetrahydroxyxanthone (10, 3.9% yield).
In contrast, the absence of these phenolic groups inhibited
Figure 1. HPLC-DAD/ESIMS analysis of the enzymatic reaction
mixture from microsomal fractions containing 3, DMAPP, and Mg2+.
(A) Reaction catalyzed by recombinant MaIDT. (B) Enzymatic
reaction gives 55% yield with recombinant MaIDT. (C) Enzymatic
reaction with no yield using microsomes from yeast containing the
empty vector. (D) Selected ion chromatogram for a prenylated
product in positive mode. (E) MS spectra for 3a.
enzymatic product formation (11, 12, and 13). The data
showed that the presence of C-1 and C-3 phenolic groups was
crucial for the prenylation of hydroxyxanthones by MaIDT.
The enzymatic products (5a, 6a, 7a, 9a, and 9b) were then
prepared from large-scale reactions and subjected to MS and
NMR data analyses (Figures S8−S13, SI). However, the
prenylation of 1,3,5,6-tetrahydroxyxanthone (8) and 1,3,6,7-
tetrahydroxyxanthone (10) were confirmed using only HPLC-
UV/ESIMS analysis (Figures S14 and S15, SI) due to the low
yields. The results clearly showed that one dimethylallyl moiety
was regiospecifically introduced at C-2 of the A-ring, although
the positions of the substituents on the C-ring were different.
Interestingly, 1,3,6,8-tetrahydroxyxanthone (9) was dipreny-
lated at C-2 and C-7 due to its symmetric structure, whereas
1,3,6-trihydroxyxanthone (6) and 1,3,8-trihydroxyxanthone (7)
could be monoprenylated only as a result of the absence of an
8/6-OH group. This observation confirmed the importance of
B
DOI: 10.1021/acs.jnatprod.6b00417
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
All of the enzymatic products and their corresponding
substrates were evaluated for their neuroprotective effects on L-
glutamic acid-induced cellular damage in SK-N-SH human
neuroblastoma cells using the MTT method; resveratrol was
used as a positive control. The results (Table S1, SI) indicate
that the neuroprotective effects of 3a, 9a, and 9b are
significantly higher than those of their corresponding substrates
3 and 9 (p < 0.05) at a concentration of 10 μM. In particular,
when the cells were preincubated with 10 μM 2-dimethylallyl-
1,3,7-trihydroxyxanthone (3a), the cell survival rate was
82.87%, which was comparable with that for the positive
control resveratrol (79.94%).
In summary, the regiospecific 2-prenylation of a variety of
structurally diverse hydroxyxanthones catalyzed by a plant FPT,
MaIDT, was demonstrated. Seven prenylated products were
efficiently obtained by incubation with MaIDT. One of the
enzymatic products, 2-dimethylallyl-1,3,7-trihydroxyxanthone
(3a), exhibited a strong neuroprotective effect. Thus, MaIDT
should be explored as an efficient biocatalyst in combinatorial
biosyntheses to create structurally various prenylated hydrox-
yxanthones for drug discovery due to its high efficiency and
catalytic promiscuity.
EXPERIMENTAL SECTION
■
1
General Experimental Procedures. H and 13C NMR spectra
were recorded on Varian NMR System 600 spectrometers (Varian
Inc., Palo Alto, CA, USA). The HPLC-UV/ESIMS analyses and
isolation of the enzymatic products were performed as previously
described.23
Figure 2. Recombinant MaIDT substrate specificity. (A) Relative
enzymatic activities with isoliquiritigenin and various xanthones and
benzophenones acting as the prenyl acceptors and DMAPP acting as
the prenyl donor. (B) The reactions catalyzed by recombinant MaIDT
and the chemical structures of the substrates used for the assay.
Chemicals. The prenyl donor dimethylallyl diphosphate
(DMAPP) was chemically synthesized as described previously.28
Benzophenones 1 and 2 were synthesized according to the
literature.29,30 Hydroxyxanthones 3−9 and 12 were prepared
according to the literature.31,32 Other tested substrates were purchased
from J&K Chemical Ltd. (Beijing, People’s Republic of China) and
BioBio-Pha (Kunming, Yunnan, People’s Republic of China).
Prenyltransferase Activity. The expression of MaIDT and the
preparation of the microsomal fraction from recombinant yeast
YPH499 were described previously.23 A basic reaction mixture (100
μL) containing 100 mM Tris-HCl (pH 9.0), 10 mM MgCl2, 200 μM
prenyl acceptor, 400 μM DMAPP, and 100 μg of recombinant yeast
microsome protein was used to quantitatively determine the PT
activity. The reaction mixtures were incubated at 30 °C for 16 h, and
the reactions were terminated by the addition of 200 μL of MeOH.
The protein was removed by centrifugation at 14000g for 20 min. The
enzymatic products were analyzed by HPLC-UV/ESIMS under
previously described conditions.23 Three parallel assays were routinely
carried out to quantitatively measure the enzyme activity. The assays
for determining the prenyl acceptors’ kinetic parameters (100 μL)
contained 400 μM DMAPP, 40 μg of recombinant yeast microsome
protein, and prenyl acceptors at final concentrations of 5, 10, 20, 40,
80, 160, and 400 μM. The reactions were incubated for 20 min. The
resulting initial velocities were then fitted to the Lineweaver−Burk
equation using OriginPro 8 (Originlab, Co., USA) to extract the Km
parameters.
C-1 and C-3 (or C-6 and C-8) phenolic groups for
hydroxyxanthone prenylation by MaIDT.
The C-prenylation catalyzed by MaIDT is essentially a
Friedel−Crafts electrophilic aromatic alkylation, via the reactive
carbocation generated from DMAPP at the ortho-position of
electron-donating hydroxy groups, leading to regiospecific C−
C bond formation.15,19−23 For example, the low electron
density at C-2 of 1-hydroxyxanthone (12) in comparison with
1,3-tetrahydroxyxanthone (5) might prevent prenylation.
It may also be concluded from Figure 2A that MaIDT has an
apparently higher conversion rate for its natural substrate
isoliquiritigenin (14) than the tested hydroxyxanthones, which
is supported by the MaIDT kinetic study. The apparent MaIDT
Km value for 14 was calculated as 25.9 μM, which is lower than
those determined for the tested hydroxyxanthones (42.8, 70.2,
51.4, 143.6, and 104.6 μM for 3, 4, 5, 6, and 7, respectively).
Therefore, the MaIDT substrate relaxation led to its robust
prenylation capacity toward various hydroxyxanthones. The
unique regiospecificity of MaIDT has not been demonstrated
previously for other identified PTs. Furthermore, because
neofunctionalization can be achieved through the exploitation
of ancestral enzyme promiscuity in specific metabolism,26,27 the
acceptance of hydroxyxanthones by MaIDT reveals character-
istics of its promiscuous ancestor. Additionally, not only
moraceous FPTs may have evolved from this promiscuous
progenitor but also other unidentified moraceous XPTs. The
putative homology shared by moraceous FPTs with undis-
covered XPTs might be valuable for the identification of XPTs
in plants.
Preparation and Identification of the Reaction Products. To
prepare the enzymatic products for structural elucidation, reaction
mixtures containing 100 mM Tris-HCl (pH 9.0), 10 mM MgCl2, 1
mM DMAPP, 500 μM prenyl acceptors, and 30 mg of recombinant
yeast microsome protein in a total volume of 10−15 mL were
incubated at 30 °C for 16 h. The reaction mixtures were subsequently
extracted with EtOAc (5 × 20 mL). After evaporating the solvent, the
residues were dissolved in MeOH and purified by RP semipreparative
HPLC under the previously described conditions.23 The isolated
1
products were subjected to MS and H and 13C NMR spectroscopic
analyses.
C
DOI: 10.1021/acs.jnatprod.6b00417
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
2-Dimethylallyl-1,3,7-trihydroxyxanthone (3a): 1H NMR (600
MHz, acetone-d6) δ 13.27 (1H, s, 1-OH), 7.58 (1H, d, J = 3.0 Hz, H-
8), 7.42 (1H, d, J = 9.0 Hz, H-5), 7.34 (1H, dd, J = 9.0, 3.0 Hz, H-6),
6.50 (1H, s, H-4), 5.29 (1H, t, J = 7.2 Hz, H-2′), 3.37 (2H, d, J = 7.2
Hz, H-1′), 1.79 (3H, s, H-4′), 1.65 (3H, s, H-5′); 13C NMR (150
MHz, acetone-d6) δ 181.3 (C-9), 164.0 (C-3), 161.5 (C-1), 155.7 (C-
4a), 154.8 (C-7), 150.8 (C-10a), 131.7 (C-3′), 125.0 (C-2′), 123.3 (C-
6), 122.0 (C-8a), 119.8 (C-5), 111.3 (C-2), 109.4 (C-8), 103.5 (C-9a),
94.0 (C-4), 26.0 (C-5′), 22.0 (C-1′), 18.0 (C-4′); ESIMS, m/z 312.9
[M + H]+.25
software. The differences were considered statistically significant at p <
0.05.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00417.
NMR spectra of prenylated xanthones (3a−7a, 9a, and
9b), HPLC-DAD/ESIMS analysis of enzymatic reaction
mixtures of 3, 4, 8, and 10, a proposed prenylxanthone
biosynthetic pathway in plants, and neuroprotective
effects of 3−7 and 9 and their prenylated products
(PDF)
2-Dimethylallyl-1,3,5-trihydroxyxanthone (4a): 1H NMR (600
MHz, acetone-d6) δ 13.26 (1H, s, 1-OH), 7.68 (1H, d, J = 7.8 Hz, H-
8), 7.33 (1H, d, J = 7.8 Hz, H-6), 7.27 (1H, dd, J = 7.8, 7.8 Hz, H-7),
6.57 (1H, s, H-4), 5.29 (1H, t, J = 7.2 Hz, H-2′), 3.37 (2H, d, J = 7.2
Hz, H-1′), 1.79 (3H, s, H-4′), 1.65 (3H, s, H-5′); 13C NMR (150
MHz, acetone-d6) δ 181.6 (C-9), 164.2 (C-3), 161.6 (C-1), 156.4 (C-
4a), 146.9 (C-5), 146.0 (C-10a), 131.7 (C-3′), 124.7 (C-2′), 123.2 (C-
7), 122.3 (C-8a), 121.7 (C-6), 116.4 (C-2), 111.6 (C-8), 103.6 (C-9a),
94.3 (C-4), 25.9 (C-5′), 22.0 (C-1′), 17.9 (C-4′); ESIMS m/z 312.9
[M + H]+.25
AUTHOR INFORMATION
■
Corresponding Authors
2-Dimethylallyl-1,3-dihydroxyxanthone (5a): 1H NMR (600
MHz, acetone-d6) δ 13.21 (1H, s, 1-OH), 8.22 (1H, d, J = 8.4 Hz,
H-8), 7.83 (1H, t, J = 8.4 Hz, H-6), 7.52 (1H, d, J = 8.4 Hz, H-5), 7.46
(1H, t, J = 7.8, H-7), 6.55 (1H, s, H-4), 5.29 (1H, t, J = 7.2 Hz, H-2′),
3.37 (2H, d, J = 7.2 Hz, H-1′), 1.79 (3H, s, H-4′), 1.65 (3H, s, H-5′);
13C NMR (150 MHz, acetone-d6) δ 181.4 (C-9), 164.4 (C-3), 161.6
(C-1), 156.9 (C-4a), 156.8 (C-10a), 136.1 (C-6), 131.8 (C-3′), 126.4
(C-8), 125.0 (C-7), 121.4 (C-2′), 118.5 (C-8a), 113.4 (C-5), 111.6
(C-2), 103.8 (C-9a), 94.4 (C-4), 26.0 (C-5′), 22.0 (C-1′), 18.0 (C-4′);
ESIMS m/z 312.9 [M + H]+.33
*Tel: +86 10 6316 5173. Fax: +86 10 6301 7757. E-mail:
ypeng@imm.ac.cn (Y. Peng).
*Tel: +86 10 6316 5195. Fax: +86 10 6301 7757. E-mail:
jgdai@imm.ac.cn (J. Dai).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The study was supported by National Natural Science
Foundation of China Grant 81273405.
2-Dimethylallyl-1,3,6-trihydroxyxanthone (6a): 1H NMR (600
MHz, acetone-d6) δ 13.40 (1H, s, 1-OH), 8.07 (1H, d, J = 9.0 Hz, H-
8), 6.96 (1H, dd, J = 9.0, 2.4 Hz, H-7), 6.86 (1H, d, J = 2.4 Hz, H-5),
6.48 (1H, s, H-4), 5.28 (1H, t, J = 7.2 Hz, H-2′), 3.36 (2H, d, J = 7.2
Hz, H-1′), 1.78 (3H, s, H-4′), 1.65 (3H, s, H-5′); ESIMS m/z 312.9
[M + H]+.34
REFERENCES
■
(1) El-Seedi, H. R.; El-Ghorab, D. M.; El-Barbary, M. A.; Zayed, M.
F.; Goransson, U.; Larsson, S.; Verpoorte, R. Curr. Med. Chem. 2009,
2-Dimethylallyl-1,3,8-trihydroxyxanthone (7a): 1H NMR (600
MHz, acetone-d6) δ 12.18 (1H, s, 8-OH), 11.94 (1H, s, 1-OH), 7.68
(1H, t, J = 8.4 Hz, H-6), 6.95 (1H, d, J = 8.4 Hz, H-5), 6.77 (1H, d, J =
8.4 Hz, H-7), 6.54 (1H, s, H-4), 5.27 (1H, t, J = 7.2 Hz, H-2′), 3.37
(2H, d, J = 7.2 Hz, H-1′), 1.79 (3H, s, H-4′), 1.66 (3H, s, H-5′);
ESIMS m/z 312.9 [M + H]+.33
16, 2581−2626.
(2) Vieira, L. M. M.; Kijjoa, A. Curr. Med. Chem. 2005, 12, 2413−
2446.
(3) Peres, V.; Nagem, T. J.; de Oliveira, F. F. Phytochemistry 2000, 55,
683−710.
(4) Masters, K. S.; Brase, S. Chem. Rev. 2012, 112, 3717−3776.
̈
2-Dimethylallyl-1,3,6,8-tetrahydroxyxanthone (9a): 1H NMR
(600 MHz, acetone-d6) δ 12.27 (1H, s, 1-OH), 12.06 (1H, s, 8-
OH), 6.49 (1H, s, H-4), 6.38 (1H, s, H-5), 6.24 (1H, s, H-7), 5.27
(1H, t, J = 7.2 Hz, H-2′), 3.35 (2H, d, J = 7.2 Hz, H-1′), 1.78 (3H, s,
H-4′), 1.65 (3H, s, H-5′); ESIMS m/z 328.9 [M + H]+.35
2,7-Dimethylallyl-1,3,6,8-tetrahydroxyxanthone (9b): 1H NMR
(600 MHz, acetone-d6) δ 12.35 (2H, s, 1-OH, 8-OH), 6.46 (2H, s, H-
4, 5), 5.27 (2H, t, J = 7.2 Hz, H-2′, 2″), 3.35 (2H, d, J = 7.2 Hz, H-1′,
1″), 1.78 (6H, s, H-4′, 4″), 1.65 (6H, s, H-5′, 5″); ESIMS m/z 396.8
[M + H]+.
(5) Lesch, B.; Brase, S. Angew. Chem., Int. Ed. 2004, 43, 115−118.
̈
(6) Pinto, M. M. M.; Sousa, M. E.; Nascimento, M. S. J. Curr. Med.
Chem. 2005, 12, 2517−2538.
(7) Matsumoto, K.; Akao, Y.; Kobayashi, E.; Ohguchi, K.; Ito, T.;
Tanaka, T.; Iinuma, M.; Nozawa, Y. J. Nat. Prod. 2003, 66, 1124−
1127.
(8) Suksamrarn, S.; Suwannapoch, N.; Phakhodee, W.;
Thanuhiranlert, J.; Ratananukul, P.; Chimnoi, N.; Suksamrarn, A.
Chem. Pharm. Bull. 2003, 51, 857−859.
(9) Jung, H. A.; Su, B. N.; Keller, W. J.; Mehta, R. G.; Kinghorn, A. D.
J. Agric. Food Chem. 2006, 54, 2077−2082.
In Vitro Biassay.36 Human neuroblastoma SK-N-SH cells were
grown in Dulbecco’s modified Eagle’s medium containing 10% fetal
bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin.
The cell cultures were incubated at 37 °C in a humid 5% CO2/95% air
environment. SK-N-SH cells were cultured in 96-well microplates at a
density of 1 × 105 cells/well. The compounds were prepared in
DMSO as 100 mM stock solutions. Glutamate was freshly prepared
prior to each experiment. The cells were preincubated with the
compounds for 4 h before 30 mM glutamate was added to the cells.
After 4 h of co-incubation, MTT solution (5 mg/mL) was added for
another 4 h at 37 °C. The MTT formazan crystals were solubilized
with DMSO and spectrophotometrically measured at 570 nm. The cell
viability was expressed as a percentage of the control group. All of the
data presented in the study were obtained from at least three
(10) Chen, L. G.; Yang, L. L.; Wang, C. C. Food Chem. Toxicol. 2008,
46, 688−693.
(11) El-Seedi, H. R.; El-Barbary, M. A.; El-Ghorab, D. M. H.; Bohlin,
L.; Borg-Karlson, A. K.; Goransson, U.; Verpoorte, R. Curr. Med.
Chem. 2010, 17, 854−901.
(12) Fiesel, T.; Gaid, M.; Muller, A.; Bartels, J.; El-Awaad, I.; Beuerle,
̈
T.; Ernst, L.; Behrends, S.; Beerhues, L. Molecules 2015, 20, 15616−
15630.
(13) Sanchez, J. F.; Entwistle, R.; Hung, J. H.; Yaegashi, J.; Jain, S.;
Chiang, Y. M.; Wang, C. C. C.; Oakley, B. R. J. Am. Chem. Soc. 2011,
133, 4010−4017.
(14) Pockrandt, D.; Ludwig, L.; Fan, A.; Konig, G. M.; Li, S.-M.
̈
ChemBioChem 2012, 13, 2764−2771.
independent experiments and expressed as the mean
SEM.
(15) Heide, L. Curr. Opin. Chem. Biol. 2009, 13, 171−179.
(16) Yazaki, K.; Sasaki, K.; Tsurumaru, Y. Phytochemistry 2009, 70,
1739−1745.
Significant differences between groups were compared using one-
way ANOVA followed by an LSD post hoc test using SPSS ver. 10.0
D
DOI: 10.1021/acs.jnatprod.6b00417
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
(17) Winkelblech, J.; Fan, A.; Li, S.-M. Appl. Microbiol. Biotechnol.
2015, 99, 7379−7397.
(18) Li, W. Trends Biochem. Sci. 2016, 41, 356−370.
(19) Fan, A.; Winkelblech, J.; Li, S.-M. Appl. Microbiol. Biotechnol.
2015, 99, 7399−7415.
(20) Kuzuyama, T.; Noel, J. P.; Richard, S. B. Nature 2005, 435,
983−987.
(21) Chen, R.; Liu, X.; Zou, J.; Yin, Y.; Ou, B.; Li, J.; Wang, R.; Xie,
D.; Zhang, P.; Dai, J. Adv. Synth. Catal. 2013, 355, 1817−1828.
(22) Li, J.; Chen, R.; Wang, R.; Liu, X.; Xie, D.; Zou, J.; Dai, J.
ChemBioChem 2014, 15, 1673−1681.
(23) Wang, R.; Chen, R.; Li, J.; Liu, X.; Xie, K.; Chen, D.; Yin, Y.;
Tao, X.; Xie, D.; Zou, J.; Yang, L.; Dai, J. J. Biol. Chem. 2014, 289,
35815−35825.
(24) Helboe, P.; Arends, P. Acta Chem. Scand. 1973, 27, 2237−2238.
(25) Cortez, D. A. G.; Young, M. C. M.; Marston, A.; Wolfender, J.
L.; Hostettmann, K. Phytochemistry 1998, 47, 1367−1374.
(26) Weng, J. K.; Philippe, R. N.; Noel, J. P. Science 2012, 336, 1667−
1670.
(27) Weng, J. K. New Phytol. 2014, 201, 1141−1149.
(28) Davisson, V. J.; Woodside, A. B.; Poulter, C. D. Methods
Enzymol. 1985, 110, 130−144.
(29) Rancon, S.; Chaboud, A.; Darbour, N.; Comte, G.; Bayet, C.;
Simon, P. N.; Raynaud, J.; Pietro, A. D.; Cabalion, P.; Barron, D.
Phytochemistry 2001, 57, 553−557.
(30) Lin, C. M.; Huang, S. T.; Lee, F. W.; Kuo, H. S.; Lin, M. H.
Bioorg. Med. Chem. 2006, 14, 4402−4409.
(31) Liu, Y.; Zou, L.; Ma, L.; Chen, W. H.; Wang, B.; Xu, Z. L. Bioorg.
Med. Chem. 2006, 14, 5683−5690.
(32) Genoux-Bastide, E.; Lorendeau, D.; Nicolle, E.; Yahiaoui, S.;
Magnard, S.; Di Pietro, A.; Baubichon-Cortay, H.; Boumendjel, A.
ChemMedChem 2011, 6, 1478−1484.
(33) Zhang, X.; Li, X.; Ye, S.; Zhang, Y.; Tao, L.; Gao, Y.; Gong, D.;
Xi, M.; Meng, H.; Zhang, M.; Gao, W.; X, X.; Guo, Q.; You, Q. Med.
Chem. 2012, 8, 1012−1025.
(34) Lim, C. K.; Tho, L.-Y.; Lim, Y. M.; Shah, S. A. A.; Weber, J.-F. F.
Lett. Org. Chem. 2012, 9, 549−555.
(35) Lim, C. K.; Tho, L.-Y.; Lim, C. H.; Lim, Y. M.; Shah, S. A. A.;
Weber, J.-F. F. Lett. Drug Des. Discovery 2011, 8, 523−528.
(36) Yang, L.; Wang, Z.; Lei, H.; Chen, R.; Wang, X.; Peng, Y.; Dai, J.
Tetrahedron 2014, 70, 8244−8251.
E
DOI: 10.1021/acs.jnatprod.6b00417
J. Nat. Prod. XXXX, XXX, XXX−XXX