Minor constituents from the tubers of Gymnadenia conopsea

Article abstract of DOI:10.1080/10286020.2010.491476

Four new minor constituents including two cyclodipeptides (1 and 2) and two cyclopentene derivatives (3 and 4), together with four known cyclodipeptides, have been isolated from an ethanolic extract of the tubers of Gymnadenia conopsea. Their structures i

Full text of DOI:10.1080/10286020.2010.491476

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Minor constituents from the tubers of
Gymnadenia conopsea
Jia-Chen Zi a , Sheng Lin a , Cheng-Gen Zhu a , Yong-Chun Yang a &
Jian-Gong Shi a
a Institute of Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College (Key Laboratory of
Bioactive Substances and Resources Utilization of Chinese Herbal
Medicine, Ministry of Education) , Beijing, 100050, China
Published online: 15 Jun 2010.
To cite this article: Jia-Chen Zi , Sheng Lin , Cheng-Gen Zhu , Yong-Chun Yang & Jian-Gong Shi
(2010) Minor constituents from the tubers of Gymnadenia conopsea , Journal of Asian Natural
Products Research, 12:6, 477-484, DOI: 10.1080/10286020.2010.491476
To link to this article: http://dx.doi.org/10.1080/10286020.2010.491476
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Journal of Asian Natural Products Research
Vol. 12, No. 6, June 2010, 477–484
ORIGINAL ARTICLE
Minor constituents from the tubers of Gymnadenia conopsea
Jia-Chen Zi, Sheng Lin, Cheng-Gen Zhu, Yong-Chun Yang and Jian-Gong Shi*
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical
College (Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal
Medicine, Ministry of Education), Beijing 100050, China
(Received 27 February 2010; final version received 3 May 2010)
Four new minor constituents including two cyclodipeptides (1 and 2) and two
cyclopentene derivatives (3 and 4), together with four known cyclodipeptides, have
been isolated from an ethanolic extract of the tubers of Gymnadenia conopsea. Their
structures including absolute configurations were determined by spectroscopic data
interpretation combined with chemical methods. Among them, compound 1 contains
an abnormal S-(400-hydroxybenzyl)cysteine residue, 3 and 4 possess [(4-methylcyclo-
pentyl)methyl]benzene and (4-hydroxymethylcyclopentyl)benzene carbon skeletons,
respectively, both of which are first found from the natural source.
Keywords: Gymnadenia conopsea; Orchidaceae; cyclodipeptides; cyclopentene
derivatives
1. Introduction
was active in improving impaired memory
in mice caused by scopolamine and
cycloheximide, and from the extract, 20
glycosidic constituents including four new
4-diglycosyloxybenzyl alcohol derivatives
and six new bis- or mono-(4-glycosylox-
ybenzyl)-2-isobutyltartrate derivatives were
isolated [4]. Continuing our investigation of
less polar fractions from the same material,
we report herein the isolation and structural
elucidation of four new minor constituents
including two cyclodipeptides (1 and 2)
and two cyclopentene derivatives (3 and 4),
together with four known cyclodipeptides
identified as cyclo(L-Leu-L-Tyr) [5],
cyclo(L-Leu-L-Pro) [6], cyclo(L-Val-L-
Tyr), and cyclo(L-Ala-D-Phe) [7] by com-
paring with corresponding literature data.
Among them, compound 1 contains an
Gymnadenia conopsea R. Br. is a plant
belonging to the Orchidaceae family and
is distributed in Tibet, Xinjiang, Inner
Mongolia, Sichuan, Qinghai, and the
Gansu Provinces of China. The dried
tubers of this plant, other species of this
genus, and the genus Coeloglossum,
known as ‘Wangla’ (Chinese name), have
long been used as a traditional Tibetan
remedy to treat coughs, asthma, and
syndromes, and as a tonic in Chinese folk
medicine, with multiple indications such as
invigorating vital energy, promoting
the production of body fluids, having a
tranquilizing effect, and enhancing intelli-
gence [1–3]. We previously reported
that an ethanolic extract of the tubers of
G. conopsea collected in Sichuan Province
*Corresponding author. Email: shijg@imm.ac.cn
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2010 Taylor & Francis
DOI: 10.1080/10286020.2010.491476
http://www.informaworld.com

478
J.-C. Zi et al.
abnormal S-(400-hydroxybenzyl)cysteine
residue, 3 and 4 possess [(4-methylcyclo-
pentyl)methyl]benzene and (4-hydroxyme
thylcyclopentyl)benzene carbon skeletons,
respectively, both of which are first found
from the natural source (Figure 1).
H-500). In addition, it displayed two AB
spin coupling systems assignable to two
isolated and deshielded methylenes at d
3.68 and 3.63 (each 1H, d, J ¼ 12.8 Hz,
H-700a and H-700b) and d 4.06 and 3.83
(each 1H, d, J ¼ 18.0 Hz, H-2a and H-2b),
as well as an ABX spin coupling system
attributed to a tri-substituted ethyl unit
(CH2CH) at d 2.97 (1H, dd, J ¼ 14.4,
4.4 Hz, H-30a), 2.76 (1H, dd, J ¼ 14.4,
3.6 Hz, H-30b), and 4.12 (1H, dd, J ¼ 4.4,
3.6 Hz, H-20). In addition to carbon
resonances corresponding to the above
units (see Section 3), the 13C NMR and
DEPT spectra of 1 showed resonances due
to two amide carbonyls at d 169.4 (C-10)
and 168.7 (C-1) which were consistent
with the strong IR absorption at
1677 cm21. Combined analysis of these
spectroscopic data suggested that 1 was a
cyclodipeptide containing glycine,
cysteine, and 400-hydroxybenzyl units,
which was further supported by the
amino acid analysis indicating the pre-
sence of glycine and cysteine in the acid
hydrolyzate of 1. The structure of 1 was
finalized by the 2D NMR spectroscopic
data analysis. The resonances of the
protonated carbons and their correspond-
ing protons in the NMR spectra were
assigned unambiguously by the HSQC
spectroscopic data analysis of 1. In the
HMBC spectrum, correlations of H2-2/C-1,
2. Results and discussion
The ethanolic extract of the dried tubers of
G. conopsea was suspended in water and
then partitioned with EtOAc. The aqueous
solution was chromatographed succes-
sively over macroporous resin, silica gel,
and reversed-phase medium-pressure
liquid chromatography and preparative
HPLC to yield 1–4.
Compound 1, a white amorphous
20
powder with ½aꢀD þ35.7 (c ¼ 0.56,
MeOH), showed the presence of hydroxyl
and/or amino (3340 and 3215 cm21),
amide carbonyl (1677 cm21), and aromatic
ring (1610, 1596, and 1515 cm21) func-
tional groups in its IR spectrum. The (þ)-
ESI-MS displayed a quasi-molecular ion
peak at m/z 289 [M þ Na]þ, and (þ)-HR-
ESI-MS at m/z 289.06232 [M þ Na]þ
indicated the molecular formula of 1
as C12H14N2O3S, with seven degrees of
unsaturation. The 1H NMR spectrum in
MeOH-d4 showed characteristic reson-
ances due to a 4-hydroxyphenyl unit at d
7.08 (2H, d, J ¼ 8.4 Hz, H-200 and H-600)
and 6.66 (2H, d, J ¼ 8.4 Hz, H-300 and
5
O
O
3"
5'
1
1
OH
OH
NH
2
NH
3
4
1"
7"
9'
S
HN
HN
O
5"
3'
1'
1'
3'
7'
O
O
1
2
O
O
1
OH
1'
1
7'
5
1'
5
3'
3'
3
OH
3
HO
6
OH
6
5'
OH
5'
4
3
Figure 1. The structures of compounds 1–4.

Journal of Asian Natural Products Research
479
H2-30/C-10 and C-20, H-20/C-10, H-200 and
H-600/C-400 and C-700, H-300 and H-500/C-100,
and H2-700/C-200 and C-600 (Figure 2), in
combination with the shifts and coupling
patterns of these protons and carbons,
confirmed the presence of glycine, cysteine,
and 400-hydroxybenzyl residues in 1. In
addition, correlations of H2-30 with C-700 and
H2-700 with C-30 indicated unequivocally
that 400-hydroxybenzyl was located at the
sulfur atom of the cysteine unit to form an
abnormal amino acid residue, S-(400-hydro-
xybenzyl)cysteine. Meanwhile, correlations
ofC-1withH-20 andC-10 withH2-2revealed
a cyclodipeptide connection of the glycine
and S-(400-hydroxybenzyl)cysteine residues.
Bycomparingthe½aꢀ2D0 value of1withthose
of 3-[1-(4-methoxybenzylsulfanyl)-1-
methylethyl]piperazine-2,5-dione and
cyclo(Gly-L-Cys) [8,9], the absolute con-
figuration of the S-(400-hydroxybenzyl)cys-
teine residue in 1 was proposed to be L. This
was confirmed by Marfey’s analysis of the
acid hydrolyzate of 1 [10]. The acid
hydrolyzate of 1 was treated with Marfey’s
reagent, (1-fluoro-2,4-dinitrophenyl)-5-
L-alanine amide (FDAA), and the resulting
FDAA derivatives were analyzed by
reversed-phase HPLC. Peaks in the chro-
matogram were identified by comparing the
retention times with those of the FDAA
derivatives of authentic amino acids.
Marfey’s reagent derivative of the amino
acids liberated from 1 showed peaks
matching L-cysteine. Therefore, the struc-
ture of 1 was elucidated as cyclo[glycine-L-
S-(400-hydroxybenzyl)cysteine].
Compound 2 was obtained as a white
20
amorphous powder with ½aꢀD þ39.4
(c ¼ 0.22, MeOH). The (þ)-ESI-MS dis-
played a quasi-molecular ion peak at m/z
263 [M þ H]þ. The 1H NMR spectrum of
2 in DMSO-d6 showed diagnostic reson-
ances for a 4-hydroxyphenyl unit at d 6.94
(2H, d, J ¼ 8.5 Hz, H-20 and H-60) and
6.61 (2H, d, J ¼ 8.5 Hz, H-30 and H-50),
and an isopropyl unit at d 1.40 (1H, m,
H-3) and 0.59 (6H, d, J ¼ 7.0 Hz, H3-4 and
H3-5), in addition to resonances attribu-
table to two nitrogen-bearing methines at d
4.10 (1H, m, H-80) and 3.54 (1H, m, H-2)
and a deshielded methylene attached to
one of the methines at d 3.03 (1H, dd,
J ¼ 14.0, 4.0 Hz, H-70a) and 2.72 (1H, dd,
J ¼ 14.0, 4.5 Hz, H-70b). It also showed
exchangeable resonances assignable to a
phenolic hydroxyl group at d 9.22 (1H, s)
and two amide protons at d 8.01 and 7.84
(each 1H, s). These data are very similar to
those of the co-occurring cyclo(L-Val-L-
Tyr) [7]. However, the chemical shifts for
the two methyls of the valine residue of 2
were shifted significantly by Dd 20.09
and þ0.26 ppm, respectively, as compared
O
O
OH
NH
HN
S
OH
OH
O
O
3
1
O
OH
HO
OH
4
Figure 2. Important HMBC correlations ( ! ) of compounds 1, 3, and 4.

480
J.-C. Zi et al.
with those of cyclo(L-Val-L-Tyr). This
indicated that compound 2 is a stereo-
isomer of cyclo(L-Val-L-Tyr), which was
supported by the optical rotation data, and
confirmed by Marfey’s analysis of the acid
hydrolyzateof2usingthesameprocedureas
described above. Marfey’s reagent deriva-
tive of the amino acids liberated from 2
showed peaks matching L-valine and
D-tyrosine, respectively. Thus, the structure
of 2 was elucidated as cyclo(L-Val-D-Tyr).
Compound 3 was obtained as a
H-30 and H-50), and a tri-substituted double
bond at d 6.82 (1H, br s, H-5). In addition,
it showed two singlets ascribed to a
methylene at d 3.07 (2H, H2-70) and an
olefinic tertiary methyl at d 1.94 (3H, H3-6),
respectively, as well as two broadened
exchangeable singlets due to two hydroxyl
groups at d 4.90 and 2.69 (each 1H, br s). In
addition to the carbon signals corresponding
to the above units (Table 1), the 13C NMR
and DEPT spectra indicated the presence of
an oxygen-bearing quaternary carbon at d
75.6 (C-2) and two conjugated carbonyls at
d 201.5 (C-1) and 203.1 (C-3) in 3. Based on
the above spectroscopic data, a structure of
cyclopentenedione derivative with substitu-
ents of 40-hydroxybenzyl, hydroxyl, and
methyl groups was proposed for 3. The
structure of 3 was constructed finally by
HSQC and HMBC data analysis. Further-
more, the resonances of the protonated
carbons and their bonded protons in the
NMR spectra (Table 1) were assigned
unambiguously by the HSQC experiment.
In the HMBC spectrum, two- and three-
bond correlations of H-5 with C-1, C-2, C-3,
20
yellowish gum with ½aꢀD < 0 (c ¼ 0.12,
CHCl3). The IR spectrum showed absorp-
tion bands for hydroxyl (3392 cm21),
carbonyl (1750 and 1700 cm21), and
aromatic ring (1612 and 1515 cm21
)
groups. The (þ)-ESI-MS spectrum of 3
exhibited a quasi-molecular ion peak at
m/z 255 [M þ Na]þ, and (þ)-HR-ESI-MS
at m/z 255.0679 [M þ Na]þ indicated a
molecular formula C13H12O4 for 3. The 1H
NMR spectrum of 3 in CDCl3 displayed
resonances attributed to a 40-hydroxyphe-
nyl group at d 6.83 (2H, d, J ¼ 8.0 Hz, H-
20 and H-60) and 6.68 (2H, d, J ¼ 8.0 Hz,
Table 1. 1H and 13C NMR spectroscopic data (d) of compounds 3 and 4a.
3
4
Position
dH
dC
dH
dC
1
2
3
4
5
201.5
75.6
203.1
161.5
144.4
202.4
148.8
139.7
37.7
3.32 m
a: 2.46 dd (J ¼ 18.0, 6.0)
6.82 s
1.94 s
36.5
b: 2.25 d (J ¼ 18.0)
6
11.5
a: 3.64 dd (J ¼ 10.5, 3.0)
b: 3.19 dd (J ¼ 10.5, 6.0)
63.8
10
125.2
131.0
115.6
155.8
115.6
131.0
41.3
125.1
129.9
115.6
158.1
129.9
115.6
20
6.83 d (J ¼ 8.0)
6.68 d (J ¼ 8.0)
7.68 d (J ¼ 8.5)
6.81 d (J ¼ 8.5)
30
40
50
6.68 d (J ¼ 8.0)
6.83 d (J ¼ 8.0)
3.07 s
4.90 br s
2.69 br s
6.81 d (J ¼ 8.5)
7.68 d (J ¼ 8.5)
60
70
40-OH
2-OH
9.60 br s
4.65 br s
Notes: a Data were measured at 500 MHz for 1H NMR and at 125 MHz for 13C NMR, in CDCl3 for 3 and in
DMSO-d6 þ D2O for 4. Proton coupling constants (J) in Hz are given in parentheses. The assignments were based
on HSQC and HMBC experiments.

Journal of Asian Natural Products Research
481
C-4, and C-6 and of H3-6 with C-3, C-4, and
C-5 (Figure 2), in combination with the
chemicalshiftsoftheseprotonsandcarbons,
revealed that 3 had a parent structure moiety
of 2-hydroxy-4-methylcyclopent-4-ene-
1,3-dione. In addition, HMBC correlations
ofH-30 and/orH-50 withC-10, C-20, C-40, and
C-60; H-20 and/or H-60 with C-10, C-30, C-40,
C-50, andC-70;andH2-70 withC-10, C-20, and
C-60, together with chemical shifts of these
protons and carbons, proved the presence
of the 40-hydroxybenzyl group in 3. Mean-
while, HMBC correlations of H2-70 with
C-1, C-2, and C-3 demonstrated unequi-
vocallythatthe40-hydroxybenzylgroupwas
located at C-2 of the parent structure.
Therefore, the structure of 3 was elucid-
ated as 2-hydroxy-2-(40-hydroxybenzyl)-4-
methylcyclopent-4-ene-1,3-dione. Because
of the optical inactive nature and no Cotton
effect in the CD spectrum of 3, it is
considered to be a racemate.
DEPT spectra of 4 displayed a pair of
olefinic quaternary carbons at d 148.8
(C-2) and 139.7 (C-3) and a conjugated
carbonyl carbon at d 202.4 (C-1). The
above spectroscopic data indicated that 4
was a cyclopentenone derivative substi-
tuted by hydroxyl, hydroxymethyl, and 40-
hydroxyphenyl groups. The structure of 4
was constructed further by HSQC and
HMBC data analysis. In particular, in the
HMBC spectrum of 4, two- and three-bond
correlations of H-4 with C-1, C-2, C-3,
C-5, and C-6; H2-5 with C-1, C-2, C-3, C-
4, and C-6; and H2-6 with C-3, C-4, and C-
5 (Figure 2), together with the chemical
shifts of these protons and carbons,
demonstrated a parent structure of 2-
hydroxyl-4-hydroxymethylcyclopent-2-
enone for 4. Meanwhile, HMBC corre-
lations of H-20 and H-60 with C-3 and C-40
and H-30 and H-50 with C-10 and C-40, in
combination with their chemical shifts,
revealed the presence of the 40-hydroxy-
phenyl located at C-3 of the parent
structure. Therefore, the structure of 4
was elucidated as 2-hydroxy-4-hydroxy-
methyl-3-(40-hydroxyphenyl)cyclopent-2-
enone. Because of optical inactivity and no
Cotton effect observed in the CD spectrum
of 4, it is also considered to be a racemate.
Compound 4, a yellowish amorphous
20
powder, ½aꢀD < 0 (c ¼ 0.17, MeOH),
showed IR absorption bands for hydroxyl
(3365 cm21),
conjugated
carbonyl
(1677 cm21), and aromatic ring (1605
and 1515 cm21) groups. The (2)-ESI-
MS of 4 exhibited a quasi-molecular ion
peak at m/z 219 [M 2 H]2, and (2)-HR-
ESI-MS at m/z 219.0687 [M 2 H]2
indicated the molecular formula of 4 as
3. Experimental
1
C12H12O4. The H NMR spectrum of 4 in
3.1 General experimental procedures
DMSO-d6 showed resonances assignable
to a methine at d 3.32 (1H, m, H-4), which
was coupled with two methylenes at d 2.46
(1H, dd, J ¼ 18.0, 6.0 Hz, H-5a) and 2.25
(1H, d, J ¼ 18.0 Hz, H-5b) and at d 3.64
(1H, dd, J ¼ 10.5, 3.0 Hz, H-6a) and 3.19
(1H, dd, J ¼ 10.5, 6.0 Hz, H-6b). In
Optical rotations were measured on a PE
model 343 polarimeter. UV spectra were
measured on a Cary 300 spectrometer. IR
spectra were recorded on a Nicolet 5700
FT-IR microscope spectrometer (FT-IR
microscope transmission). 1D and 2D
NMR spectra were obtained at 400 or
1
addition, the H NMR spectrum showed
1
resonances attributed to a 40-hydroxyphe-
nyl unit at d 7.68 (2H, d, J ¼ 8.5 Hz, H-20
and H-60) and 6.81 (2H, d, J ¼ 8.5 Hz, H-
30 and H-50), along with resonances for
three exchangeable hydroxyl protons at d
9.60 (2H, br s) and 4.65 (1H, br s). In
addition to the carbon signals correspond-
ing to the above units, the 13C NMR and
500 MHz for H, and 100 or 125 MHz for
13C, respectively, on INOVA 400 and
500 MHz spectrometers in MeOH-d4,
CDCl3, DMSO-d6, or DMSO-d6 þ D2O
with solvent peaks as references. ESI-MS
data were measured with a Q-Trap
LC/MS/MS (Turbo Ionspray source) spec-
trometer. HR-ESI-MS data were measured

482
J.-C. Zi et al.
using an AccuToFCS JMS-T100CS spec-
trometer. Column chromatography was
performed with silica gel (200–300 mesh;
Qingdao Marine Chemical Inc., Qingdao,
China) and Sephadex LH-20 (Pharmacia
Biotech AB, Uppsala, Sweden). HPLC
separation was performed on an instru-
ment consisting of a Waters 600 controller,
a Waters 600 pump, and a Waters 2487
dual l absorbance detector, with an
Alltima (250 mm £ 10 mm i.d.) prepara-
tive column packed with C18 (5 mm). TLC
was carried out with precoated silica gel
GF254 glass plates. Spots were visualized
under UV light or by spraying with 7%
H2SO4 in 95% EtOH followed by heating.
Amino acids were analyzed using a
Hitachi model L-8500 amino acid analyzer
equipped with a column (number 2622:
4.6 £ 40 mm; Hitachi, Tokyo, Japan).
Marfey’s analysis was performed on an
Agilent HP1100 HPLC system, with an
Alltima (250 £ 4.6 mm i.d.) column
packed with C18 (5 mm).
of the column with H2O, 15% EtOH, 40%
EtOH, and 95% EtOH (5000 ml each)
yielded four fractions after removing the
solvents. Theactivefraction(100.2g)eluted
by 40% EtOH was suspended in H2O
(500ml) and then partitioned with n-BuOH
(5 £ 400 ml) to give H2O and n-BuOH
fractions. The n-BuOH fraction (50 g) was
separated by medium-pressure liquid chro-
matography over reversed-phase silica gel
eluting with a gradient of increasing MeOH
(0–90%) in H2O to give five fractions
(A–E) on the basis of TLC analysis.
Fraction E (2.05 g) was separated by
normal-phase silica gel column chromatog-
raphy, eluting with a gradient of increasing
MeOH (0–100%) in CHCl3, afforded nine
subfractions (E1 –E9). Subfraction E7
(153mg) was subjected to column chroma-
tography over Sephadex LH-20, using
petroleum ether–CHCl3–MeOH (5:5:1) as
the eluting solvent, to afford 1 (21 mg) and 2
(2.1mg). Separation of fraction E1 (272mg)
on normal-phase silica gel column chroma-
tography, eluting with petroleum ether–
EtOAc (3:1), afforded 3 (37 mg). Subfrac-
tion E4 (353mg) was chromatographed over
silica gel column chromatography using
CHCl3–MeOH (20:1) as the eluting solvent
and purified further by Sephadex LH-20,
eluting with CHCl3 –MeOH (1:1), to
afford 4 (46 mg).
3.2 Plant materials
The tubers of G. conopsea were collected
at Ganzi, Sichuan Province, China in
September 2005. The plant identification
was verified by Prof. Lin Ma (Institute of
Materia Medica). A voucher specimen
(No. 05916) has been deposited at the
Herbarium of the Department of Medicinal
Plants, Institute of Materia Medica.
3.3.1 Cyclo[glycine-L-S-(400-hydroxy-
benzyl)cysteine] (1)
20
A white amorphous powder; ½aꢀD þ35.7
3.3 Extraction and isolation
(c ¼ 0.56, MeOH); UV (MeOH) lmax
The air-dried tubers of G. conopsea
(16.2 kg) were powdered and extracted
with 20.0liters of 75% EtOH at room
temperature for 4 £ 42h. The EtOH extract
was evaporated under reduced pressure at
,408C to yield a residue (618.2g). The
residue was suspended in H2O (2000 ml)
and then partitioned with EtOAc
(5 £ 1200 ml). The aqueous phase was
applied to an HP-20 macroporous adsorbent
resin (1500 g) column. Successive elution
(log 1): 229 (3.59), 279 (0.69) nm; IR
n
max: 3340, 3215, 3074, 2915, 1677, 1610,
1595, 1515, 1470, 1332, 1262, 1237, 1071,
816 cm21 1H NMR (400 MHz, MeOH-
;
d4): d 7.08 (2H, d, J ¼ 8.4 Hz, H-200 and H-
600), 6.66 (2H, d, J ¼ 8.4 Hz, H-300 and
H-500), 4.12 (1H, dd, J ¼ 4.4 and 3.6 Hz,
H-20), 4.06 (1H, d, J ¼ 18.0 Hz, H-2a),
3.83 (1H, d, J ¼ 18.0 Hz, H-2b), 3.68 (1H,
d, J ¼ 12.8 Hz, H-700a) and 3.63 (1H, d,
J ¼ 12.8 Hz, H-700b), 2.97 (1H, dd,

Journal of Asian Natural Products Research
483
J ¼ 14.4 and 4.4 Hz, H-30a), 2.76 (1H, dd,
J ¼ 14.4 and 3.6 Hz, H-30b); 13C NMR
(100 MHz, MeOH-d4): d 169.4 (s, C-10),
168.7 (s, C-1), 157.7 (s, C-400), 131.3 (d, C-
200), 131.3 (d, C-600), 129.9 (s, C-100), 116.3
(d, C-300), 116.3 (d, C-500), 56.5 (d, C-20),
45.7 (d, C-2), 37.6 (t, C-700), 37.0 (t, C-30);
(þ)-ESI-MS: m/z 267 [M þ H]þ and 289
[M þ Na]þ; (þ)-HR-ESI-MS: m/z
289.06232 [M þ Na]þ (calcd for C12H14
NaN2O3S, 289.06228).
1244, 1176, 1129, 1048, 1025, 1000, 827,
765 cm21; 1H NMR (500 MHz, DMSO-d6)
and 13C NMR (125 MHz, DMSO-d6
þ D2O) data: see Table 1; positive-mode
ESI-MS: m/z 243 [M þ Na]þ; (2)-ESI-
MS: m/z 219 [M 2 H]2; (2)-HR-ESI-MS:
m/z 219.0687 [M 2 H]2 (calcd for
C12H11O4, 219.0657).
3.3.5 Acid hydrolysis of 1 and 2, and
Marfey’s analyses of the hydrolyzates
Compound 1 (1.6 mg) or compound 2
(0.9 mg) was dissolved in 6 mol/l HCl
(0.5 ml), and heated at 1108C in a sealed
vial for 13 h. The cooled reaction
mixture was evaporated to dryness under
reduced pressure, and HCl was removed
from the residue of the acid hydrolyzate by
repeated evaporation from frozen water
(1.5 ml). The amino acid mixture was
dissolved in water (40 ml), and then treated
with 1% FDAA in acetone (100 ml) and
6% triethylamine (Et3N) in 50 ml of water
at 408C for 1 h. After cooling to room
temperature, the derivative and the stan-
dard amino acid samples were derivatized
in the same manner as the acid hydrolyzate
above (1% FDAA and 6% Et3N), were
filtered, and the filtrates were analyzed
by HPLC (Alltima RP-C18, 5 mm,
250 £ 4.6 mm, flow rate: 1 ml/min,
UV detection at 340 nm, mobile phase:
3.3.2 Cyclo(L-Val-D-Tyr) (2)
20
A white amorphous powder; ½aꢀD þ39.4
1
(c ¼ 0.22, MeOH); H NMR (500 MHz,
DMSO-d6): d 9.22 (1H, s, HO-40), 8.01
(1H, s, NH), 7.84 (1H, s, NH), 6.94 (2H, d,
J ¼ 8.5 Hz, H-20 and H-60), 6.61 (2H, d,
J ¼ 8.5 Hz, H-30 and H-50), 4.10 (1H, m,
H-80), 3.54 (1H, m, H-2), 3.03 (1H,
dd, J ¼ 14.0, 4.0 Hz, H-70a), 2.72
(1H, dd, J ¼ 14.0, 4.5 Hz, H-70b), 1.40
(1H, m, H-3), 0.59 (6H, d, J ¼ 7.0 Hz,
H3-4 and H3-5); (þ)-ESI-MS: m/z 263
[M þ H]þ.
3.3.3 2-Hydroxy-2-(40-hydroxybenzyl)-
4-methylcyclopent-4-ene-1,3-dione (3)
20
A yellowish gum; ½aꢀD < 0 (c ¼ 0.12,
CHCl3); UV (MeOH) lmax (log 1): 227
(4.37), 270 (0.75) nm; IR nmax: 3392,
2920, 1750, 1700, 1670, 1612, 1515, 1441,
1377, 1313, 1236, 1174, 1114, 1070,
35%
CH3CN/H2O þ 1%
HOAc).
The FDAA derivatives of amino
acids liberated from 1 showed a peak at
9.21 min, matching the retention time of
FDAA L-cysteine (9.17 min; 12.14 min for
D –); those liberated from 2 showed peaks at
14.81 and 12.79 min, matching the retention
times of FDAA L-valine (14.86 and
26.83 min for D –) and D-tyrosine (12.85
and 10.24 min for L –).
1028, 930, 896, 836, 790, 754 cm21; H
1
NMR (500 MHz, CDCl3) and 13C NMR
(125 MHz, CDCl3) data: see Table 1; (þ)-
ESI-MS: m/z 255 [MþNa]þ; (þ)-HR-
ESI-MS: m/z 255.0679 [MþNa]þ (calcd
for C13H12NaO4, 255.0633).
3.3.4 2-Hydroxy-4-hydroxymethyl-3-
(40-hydroxyphenyl)cyclopent-2-enone (4)
20
Acknowledgements
A yellowish amorphous powder; ½aꢀD < 0
Financial support from the National Natural
Sciences Foundation of China (NNSFC;
Grant Nos 30825044 and 20932007),
and the Program for Changjiang Scholars
(c ¼ 0.17, MeOH); UV (MeOH) lmax
(log1): 227 (0.66), 325 (1.52) nm; IR nmax:
3365, 1677, 1605, 1515, 1444, 1386, 1275,

484
J.-C. Zi et al.
and Innovative Research Team in
University (PCSIRT, Grant No. IRT0514) is
acknowledged.
[4] J.C. Zi, S. Li, M.T. Liu, M.L. Gan, S. Lin,
W.X. Song, Y.L. Zhang, X.N. Fan, Y.C.
Yang, J.J. Zhang, J.G. Shi, and D.L. Di,
J. Nat. Prod. 71, 799 (2008).
[5] W.Q. Yin, Y.C. Lin, and S.N. Zhou,
Acta Scientiarum Naturalium Universita-
tis Sunyatseni: Nat. Sci. 41, 56 (2002).
[6] D. Hendea, S. Laschat, A. Baro, and W.
Frey, Helv. Chim. Acta 89, 1894 (2006).
[7] T. Stark and T. Hofmann, J. Agric. Food
Chem. 53, 7222 (2005).
[8] L.S. Richter and T.R. Gadek, Tetrahe-
dron: Asymm. 7, 427 (1996).
[9] F.G. Hopkins and L.J. Harris, J. Biol.
Chem. 84, 269 (1929).
References
[1] Beijing Institute of Botany, The Chinese
Academy of Sciences, Iconographia
Cormophytorum Sinicorum, Tomus V
(Science Press, Beijing, 1976), Vol. 5,
p. 631.
[2] Jiangsu New Medical College, Dictionary
of Traditional Chinese Medicine (Shang-
hai Science and Technology Publishing
House, Shanghai, 1977), Vol. 1,
pp. 436–437.
[10] M.Y. Summers, F. Kong, X. Feng,
M.M. Siegel, J.E. Janso, E.I. Graziani,
and G.T. Carter, J. Nat. Prod. 70, 391
(2007).
[3] K.Y. Lang, X.Q. Chen, and G.H. Zhu, in
Chinese Flora (Zhongguo Zhiwu Zhi)
(Science Press, Beijing, 1999), Vol. 17,
pp. 389–390.