Cyclopentapeptides from Dianthus chinensis

Article abstract of DOI:10.1002/psc.2746

A new cyclopentapeptide dianthin I (1), together with two known ones pseudostellarin A (2) and heterophyllin J (3), was isolated from the aerial parts of Dianthus chinensis. The structure of 1 was elucidated as cyclo-(Gly¹- l-Phe²- l-Pro³- l-Ser⁴- l-Phe⁵) on the basis of extensive spectroscopic analyses and chemical methods.

Full text of DOI:10.1002/psc.2746

Special Issue Article
Received: 6 November 2014
Revised: 6 December 2014
Accepted: 24 December 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2746
Cyclopentapeptides from Dianthus
chinensis‡
Jing Han,a† Maobo Huang,a,b† Zhe Wang,a,b Yuqing Zheng,a Guangzhi Zeng,a
Wenjun Hea and Ninghua Tana*
A new cyclopentapeptide dianthin I (1), together with two known ones pseudostellarin A (2) and heterophyllin J (3), was isolated
from the aerial parts of Dianthus chinensis. The structure of 1 was elucidated as cyclo-(Gly1–L–Phe2–L–Pro3–L–Ser4–L–Phe5) on the
basis of extensive spectroscopic analyses and chemical methods.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: cyclopentapeptides; Caryophyllaceae; Dianthus chinensis; dianthin I
HPLC was performed on an Agilent 1100 with the Zorbax Eclipse-C18
(4.6 mm × 150 mm; 9.4 mm × 250 mm; 5 μm). Column chromatogra-
Introduction
Plant cyclopeptides, mainly formed with the peptide bonds of 2–37
protein or non-protein amino acids, have been divided into eight
types based on their structures and distributions in plants. Among
them, Caryophyllaceae-type cyclopeptides (CPs/type VI) were
the largest type with about 200 CPs, which are homomono-
cyclopeptides with mainly five to twelve α-amino acid residues,
isolated from over 40 higher plants [1].
phies were performed on silica gel (200–300 mesh, Qingdao
Yu-Ming-Yuan Chemical Co. Ltd., Qingdao, China), Sephadex LH-20
(Pharmacia Fine Chemical Co., Uppsala, Sweden), or Lichroprep
RP-18 (40–63 μM, Merck, Darmstadt, Germany). Fractions were
monitored by TLC (GF254, Qingdao Yu-Ming-Yuan Chemical Co.
Ltd., Qingdao, China), and spots were detected by spraying with
ninhydrin reagent for cyclopeptides [15].
The genus Dianthus plants (Caryophyllaceae) are widely used as
garden plants in Europe and Asia [2]. The aerial parts of D. chinensis
L., as well as D. superbus L., have been cited as a traditional Chinese
medicine (TCM) Dianthi Herba (Chinese name ‘Qu-Mai’) in Chinese
Pharmacopoeia (2010 Version), and are widely used for the treatment
of diuresis and strangury [3]. CPs are one of the chemical markers of
this family [1]. Some CPs isolated from D. susperbus have exhibited
cytotoxic and proliferative activities [4–8]. Eight saponins [9–11] and
one monosaccharide [12] have been reported from D. chinensis.
Herein we report a new cyclopentapeptide dianthin I (1) and two
known ones pseudosterllarin A (2) [13] and heterophyllin J (3) [14] iso-
lated from the aerial parts of D. chinensis (Figure 1). All compounds
were tested for cytotoxicity and antibacterial activities, but were
found to be inactive. The structural elucidation showed as follows.
Plant Material
TheaerialpartsofD.chinensiswerecollectedfromKunmingInstituteof
Botany, Yunnan Province, PRC, in May, 2009. The material was identi-
fied by Prof. Zhe-Kun Zhou at Kunming Institute of Botany. The
voucherspecimen(No.0270133)hasbeendepositedintheHerbarium
of Kunming Institute of Botany, Chinese Academy of Sciences.
Extraction and Isolation
The air-dried and powdered aerial parts of D. chinensis (75.0 kg)
were extracted with 95% MeOH (4× 100L). After removal of the
solvent under reduced pressure, the MeOH extract (21.0kg) was
suspended in H2O and partitioned successively with EtOAc and
Materials and Methods
General Experimental Procedures
*
Correspondence to: Ninghua Tan, State Key Laboratory of Phytochemistry and
Plant Resources in West China, Kunming Institute of Botany, Chinese Academy
of Sciences, Kunming 650201, Yunnan, PR China. E-mail: nhtan@mail.kib.ac.cn
Melting points were obtained on an X-4 micromelting point appara-
tus. Optical rotations were measured with a Horiba SEPA-300 polar-
imeter. UV spectra were obtained using a Shimadzu UV-2401A
spectrophotometer. CD spectra were measured using a Chirascan
spectrophotometer. IR spectra were obtained on a Tenor 27 spectro-
photometer with KBr pellets. 1D and 2D NMR spectra were run on a
Bruker AV-600 or Bruker DRX-500 spectrometer with TMS as internal
standard. Mass spectra were recorded on a VG Autospec-3000
spectrometer or an API QSTAR Pulsar TOF spectrometer. LC-MS
spectra were run on a Waters Xevo TQ-S MS spectrometer or a
Waters Xevo TQD mass spectrometer. Analytical or semi-preparative
†
‡
These authors contributed equally.
Special issue of contributions presented at the 13th Chinese International Peptide
Symposium, Peptides: Treasure of Chemistry and Biology, June 30 - July 4, 2014 in
Datong, Shanxi, China
a State Key Laboratory of Phytochemistry and Plant Resources in West China,
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201,
Yunnan, PR China
b University of Chinese Academy of Sciences, Beijing 100049, PR China
J. Pept. Sci. 2015
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

HAN ET AL.
Figure 1. Structures of compounds 1–3.
n-BuOH, giving an EtOAc-soluble portion (6.4 kg) and an n-BuOH-
soluble portion (8.0kg). The EtOAc fraction (6.4 kg) was subjected
to silica gel column chromatography eluting with CHCl3/MeOH
(1:0, 95:5, 9:1, 8:2, 0:1) to afford a CP-containing fraction (1.2 kg,
CHCl3/MeOH, 95:5, 9:1, 8:2) and a CP-non-containing fraction
(2.5kg, CHCl3/MeOH, 1:0, 0:1). The CP-containing fraction was
loaded onto a silica gel column and eluted with gradient
CHCl3/CH3COOC2H5 (30:1–1:1). Seven fractions (I–VII) were
obtained, and fractions Ш contained CPs. Fraction Ш (100.3 g)
was subjected to silica gel column (CHCl3/Me2CO 10:1, 5:1),
and sub-fraction Ш-3 (30.0 g) was subjected to Lichroprep RP-18
(MeOH/H2O, 70:30–100:0), then Sephadex LH-20 (CHCl3/MeOH,
1:1), and further purified by HPLC (Eclipse XDB-C18, 90 μM,
9.4mm× 250 mm, 2.0mL/min, UV detection at 205, 215, 230, 254,
and 280 nm) eluting with 60–70% CH3CN which contained
0.4‰ TFA to get 1 (21.1mg). Sub-fraction Ш-4 (13.7 g) was sub-
jected to CC on silica gel (CHCl3/MeOH, 14:1) to get 2 (22.6mg)
and 3 (20.0 mg).
Table 1. 1H NMR and 13C NMR Data of 1 in pyridine-d5 (δ in ppm, J in Hz)
1H NMR
13C NMR
Assignment
Gly1
δH (int. mult, J (Hz))
δC
α
3.69 (1H, d, 16.1)
4.84 (1H, dd, 16.1, 9.5)
8.17 (1H, d, 9.5)
43.9 (CH2)
171.1 (C)
NH
C¼O
L-Phe2
α
5.40 (1H, dd,13.6, 8.8)
3.45 (1H, dd, 13.6, 7.5)
3.94 (1H, dd, 13.6, 7.5)
56.1 (CH)
βa
βb
γ
40.3 (CH2)
138.7 (C)
δ
ε
ζ
7.57 (2H, overlap)
7.42 (2H, t, 7.5)
7.25 (1H, overlap)
8.68 (1H, d, 8.8)
130.5 (CH)
129.2 (CH)
127.2 (CH)
NH
C¼O
Dianthin I (1)
171.6 (C)
L-Pro3
C28H33N5O6, colorless powder, mp. 157–160 °C; [α]21.4 D ꢀ153.0
(c 0.10, MeOH); UV (MeOH) λmax (log ε) 204 (1.85) nm; CD (MeOH)
205 (Δϵ ꢀ34.6), 214 (Δϵ ꢀ37.2); IR (KBr) νmax 3423, 2927, 1644,
α
4.46 (1H, d, 7.6)
1.75 (1H, overlap)
2.05 (1H, m)
62.0 (CH)
32.5 (CH2)
βa
βb
γa
γb
δa
δb
C¼O
1
1531, 1452, 1204, 1134, 749, 702 cmꢀ1; H NMR (C5D5N, 500 MHz)
and 13C NMR (C5D5N, 125 MHz) spectroscopic data see Table 1;
positive ESI-MS m/z 558 [M+ Na]+; positive HREI-MS, m/z 535.2419
[M]+ (calcd for C28H33N5O6, 535.2431).
1.50 (1H, m)
22.1 (CH2)
49.0 (CH2)
175.9 (C)
1.75 (1H, overlap)
3.41 (1H, overlap)
3.61(1H, dd, 10.8, 7.6)
Pseudostellarin A (2)
L-Ser4
C25H35N5O6, colorless powder. mp. 151–152 °C; [α]21.4 D ꢀ201.0
(c 0.80, MeOH); UV (MeOH) λmax (log ε) 202 (1.91), 278 (0.88) nm;
CD (MeOH) 200 (Δϵ ꢀ50.4), 206 (Δϵ ꢀ49.1); IR (KBr) νmax 3428,
α
β
5.09 (1H, dd, 13.0, 6.5)
4.25 (2H, t, 6.5)
58.0 (CH)
61.9 (CH2)
2959, 2930, 1664, 1517, 1452, 1203, 1139, 835, 802, 721 cmꢀ1
;
NH
C¼O
8.66 (1H, d, 6.5)
1H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spec-
troscopic data see Table 2; positive ESI-MS m/z 524 [M + Na]+,
1025 [2 M + Na]+; positive HREI-MS, m/z 501.2582 [M]+ (calcd for
C25H35N5O6, 501.2587).
174.1 (C)
L-Phe5
α
4.30 (1H, overlap)
3.76 (1H, d, 11.2)
58.4 (CH)
βa
βb
γ
35.6 (CH2)
3.89 (1H, dd, 11.2, 3.7)
Heterophyllin J (3)
140.4 (C)
δ
ε
ζ
NH
7.45 (2H, d, 7.5)
7.30 (2H, t, 7.5)
7.25 (1H, overlap)
10.86 (1H, d, 6.9)
130.4 (CH)
129.0 (CH)
126.9 (CH)
C24H33N5O6, colorless powder. mp. 147–151 °C; [α]21.6 D ꢀ328.4
(c 0.10, MeOH); UV (MeOH) λmax (log ε) 202 (2.11), 222 (1.83), 278
(1.00) nm; CD (MeOH) 205 (Δϵ ꢀ77.3), 215 (Δϵ ꢀ63.9); IR (KBr) νmax
1
3387, 2967, 1659, 1516, 1450, 1202, 1137, 834, 721 cmꢀ1; H NMR
C¼O
171.7 (C)
(C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spectroscopic
data see Table 2; positive ESI-MS m/z 488 [M + H]+; positive
HREI-MS, m/z 487.2438 [M]+ (calcd for C25H35N5O6, 487.2431).
1H NMR were recorded at 500 MHz, and 13C NMR were recorded at
125 MHz.
Configuration Analysis of 1–3 (Advanced Marfey’s
Method [16])
hydrolyzate was divided into two portions (0.5mg each), then
dried. Each hydrolyzate was dissolved in 100 μL of acetone, 1 M
NaHCO3 (20 μL), and 1% 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide
(L- or D-FDLA, Marfey’s reagent, Sigma Aldrich, 100 μL) were added.
Compounds 1–3 (1mg each) were separately dissolved in 6 N HCl
(1mL) in a sealed glass tube and incubated at 115 °C for 24 h. The
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CYCLOPENTAPEPTIDES FROM DIANTHUS CHINENSIS
Table 2. 1H-NMR and 13C-NMR data for 2 and 3 in pyridine-d5 (δ in ppm, J in Hz)
2
3
Assignment
Gly1
δH (int. mult, J (Hz)
δC
Assignment
δH (int. mult, J (Hz)
δC
Gly1
α
4.06 (1H, brs)
42.6 (CH2)
α
3.99 (1H, dd, 12.3, 3.9)
4.43 (1H, dd, 12.3, 3.9)
8.84 (1H, brs)
42.9 (CH2)
4.30 (1H, d, 13.4)
9.44 (1H, overlap)
NH
NH
C¼O
170.0 (C)
C¼O
169.6 (C)
L-Pro2
L-Tyr3
L-Leu4
L-Pro2
α
β
4.52 (1H, brs)
62.7 (CH)
α
β
4.59 (1H, overlap)
2.04 (1H, m)
63.1 (CH)
30.4 (CH2)
1.26 (1H, m)
30.3 (CH2)
1.90 (1H, overlap)
1.60 (1H, overlap)
1.93 (1H, overlap)
3.35 (1H, m)
2.12 (1H, m)
γ
25.3 (CH2)
48.2 (CH2)
172.7 (C)
γ
1.70 (1H, m)
25.6 (CH2)
47.7 (CH2)
173.1 (C)
1.98 (1H, m)
δ
δ
3.34 (1H, dd, 13.6, 5.9)
3.93 (1H, m)
4.23 (1H, brs)
C¼O
C¼O
L-Val3
α
β
γ
δ
ε
ζ
5.32 (1H, dd, 17.5, 9.8)
3.48 (2H, m)
57.4 (CH)
37.8 (CH2)
129.1 (C)
131.5 (CH)
116.6 (CH)
158.1 (C)
α
β
γ
4.59 (1H, overlap)
2.40 (1H, m)
61.6 (CH)
31.4 (CH)
19.6 (CH3)
20.4 (CH3)
0.95 (3H, d, 5.6)
1.03 (3H, d, 5.6)
8.03 (1H, d, 7.0)
7.43 (2H, d, 8.0)
7.14 (2H, t, 8.0)
NH
C¼O
173.0 (C)
NH
C¼O
8.41 (1H, brs)
L-Tyr4
173.2 (C)
α
β
4.84 (1H, overlap)
59.5 (CH)
37.2 (CH2)
3.47 (1H, dd, 11.3, 7.5)
3.59 (1H, dd, 11.3, 7.5)
α
β
4.65 (1H, brs)
56.2 (CH)
1.88 (1H, overlap)
2.26 (1H, m)
40.4 (CH2)
γ
δ
ε
ζ
129.0 (C)
131.4 (CH)
116.8 (CH)
158.2 (C)
7.34 (2H, d, 7.0)
7.14 (2H, d, 7.0)
γ
δ
1.70 (1H, overlap)
0.83 (3H, d, 6.5)
0.85 (3H, d, 6.5)
9.47 (1H, overlap)
25.6 (CH)
22.3 (CH3)
23.5 (CH3)
NH
C¼O
9.49 (1H, overlap)
NH
172.6 (C)
C¼O
173.6 (C)
L-Ala5
α
β
NH
C¼O
4.84 (1H, overlap)
1.61 (3H, d, 5.9)
9.51 (1H, overlap)
50.7 (CH)
17.4 (CH3)
L-Ala5
α
β
NH
4.95 (1H, d, 7.0)
1.59 (3H, d, 7.0)
9.21 (1H, brs)
51.1 (CH)
18.1 (CH3)
174.6 (C)
C¼O
175.5 (C)
1H NMR were recorded at 500 MHz, and 13C NMR were recorded at 125 MHz.
The mixture was incubated at 45 °C for 2 h. The reaction was
quenched by adding 2 N HCl (10 μL) after being cooled, and the
dried mixture was dissolved in 50% aqueous CH3CN (600μL). Ten
microliters of each solution of FDLA derivatives was analyzed by
LC-MS.
20%–80%, 40 min) at a flow rate of 1 mL/min. A Waters Xevo TQD
mass spectrometer was used for detection in ESI (negative) mode.
The capillary voltage was kept at 3.5KV and the ion source at
450°C. Nitrogen gas was used as a sheath gas at 750 L/h. A mass
range of m/z 200–1400 was scanned in 0.3 s.
The analysis of the L- and D,L-FDLA (mixture of D- and L-FDLA) de-
rivatives of 1 was performed using an Agilent Eclipse XDB-C18 col-
umn (4.6× 150mm, 5 μm) maintained at 40 °C. Acetonitrile–1‰
HCOOH/H2O was used as the mobile phase under a linear gradient
eluent (acetonitrile, 10%–70%, 50 min) at a flow rate of 1 mL/min. A
Waters Xevo TQ-S mass spectrometer was used for detection in ESI
(negative) mode. The capillary voltage was kept at 2.5KV and the
ion source at 350 °C. Nitrogen gas was used as a sheath gas at
400 L/h. A mass range of m/z 100–1000 was scanned in 0.2 s. The
LC-MS analysis of 2–3 was performed using a Waters SunFireTMC18
5 μm (4.6×150 mm, 5 μm) maintained at 35 °C. Acetonitrile–1‰
HCOOH/H2O was used under a linear gradient eluent (acetonitrile,
Results and Discussion
Air-dried and powdered aerial parts of D. chinensis were extracted
with 95% MeOH. The CP-containing fraction was fractionated on
a silica gel column eluted with increasingly polar mixtures of
CHCl3/MeOH, and used the thin layer chromatography (TLC) detec-
tion method from the crude extracts. Further purification was
achieved by chromatographies on Sephadex LH-20 (CHCl3/MeOH),
RP-18 (MeOH/H2O), and HPLC (CH3CN/H2O containing 0.4‰ TFA or
1‰ HCOOH), yielding 3 cyclopeptides (1–3).
J. Pept. Sci. 2015
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HAN ET AL.
Science Foundation of Yunnan Province (No. 2012GA003). The au-
thors are grateful to the members of the analytical group from
the State Key Laboratory of Phytochemistry and Plant Resources
in West China, Kunming Institute of Botany, Chinese Academy of
Sciences, for measuring optical rotations, and for the IR, UV, CD,
NMR, and mass spectra.
Figure 2. Key 2D NMR correlations of 1.
References
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Compound 1 was obtained as colorless powder, [α]21.4 D ꢀ153.0
(c 0.10, MeOH), which had the molecular formula C28H33N5O6 by
HREI-MS (m/z 535.2419 [M]+), indicating 15 degrees of
unsaturation. The IR spectrum indicated the presence of amino
(3423 cmꢀ1), amide (1644 cmꢀ1), and phenol ether (1204 and
1134 cmꢀ1) groups. The 13C NMR spectrum revealed that there
were five carbonyls (δC 171.1, 171.6, 171.7, 174.1, 175.9), twelve
phenyl carbons (126.9, 129.0, 129.0, 130.4, 130.4, 140.4; 127.2,
129.2, 129.2, 130.5, 130.5, 138.7), four α-amino methines (δC 56.1,
58.0, 58.4, 62.0), and seven methylenes (δC 22.1, 32.5, 35.6, 40.3,
43.9, 49.0, 61.9). The 1H NMR spectrum presented typical four NH
(δH 8.17, 8.66, 8.68, 10.86), two mono-substitued phenyl groups
(7.45, 7.45, 7.30, 7.30, 7.25; 7.57, 7.57, 7.42, 7.42, 7.25), and six α-
H (δH 3.69, 4.84; 4.30, 4.46, 5.09, 5.40). These data suggested that
1 was a cyclic pentapeptide, which contained one proline, two
phenylalanines, one serine, and one glycine. Correlations of Phe2
NH to Gly1 C¼O, Ser4 NH to Pro3 C¼O, and Phe5 NH to Ser4
C¼O in the HMBC spectrum indicated the connections of Gly1–
Phe2 and Pro3–Ser4–Phe5. Interactions between Gly1 NH and
Phe5 αH, and Phe2 αH and Pro3 αH observed in the ROESY spec-
trum indicated the linkages of Phe5–Gly1 and Phe2–Pro3 (Figure 2).
Thus, the planar structure of 1 was determined as Cyclo-(Gly1–
Phe2–Pro3–Ser4–Phe5). Amino acid analysis of the hydrolysates
of 1 showed L-Ser (11.96/398, Rt/ESI¯-MS), L-Pro (16.34/408,
Rt/ESI¯-MS), and L-Phe (24.85/458) according to the advanced
Marfey’s method. Therefore 1 was elucidated as cyclo-(Gly1-L-
Phe2-L-Pro3-L-Ser4-L-Phe5) and named as dianchin I.
10 Li HY, Koike K Ohmoto T. Triterpenoid saponins from Dianthus chinensis.
Phytochemistry 1994; 35: 751–756.
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amides from Pseudostellaria heterophylla (Caryophyllaceae). Helv. Chim.
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of cyclopeptides in plants. Chin. Sci. Bull. 2000; 45: 1825–1831.
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using LC/MS for determination of the absolute configuration of
Conclusions
A new cyclopentapeptide dianthin I (1) was isolated from the
aerial parts of D. chinensis L. Its structure was identified by exten-
sive NMR and MS analysis, and the absolute configuration was
determined by the advanced Marfey’s method. This finding
expands the cyclopeptide diversity in D. chinensis.
constituent amino acids in
a peptide: combination of Marfey’s
method with mass spectrometry and its practical application. Anal.
Chem. 1997; 69: 5146–5151.
Acknowledgements
Supporting Information
This work was supported by the National Natural Science Founda-
tion of China (31470428, 30725048), the National New Drug Innova-
tion Major Project of China (2011ZX09307-002-02), and the Natural
Additional supporting information may be found in the online ver-
sion of this article at the publisher’s web site.
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Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2015