A New Cyclic Peptide from Bulbs of Fritillaria hupehensis

Article abstract of DOI:10.1007/s10600-015-1448-8

A new cyclic peptide, cyclo(Leu-Val-Leu) (1), along with four known compounds (2-5), was isolated from the bulbs of Fritillaria hupehensis Hsiao and K.C. Hsia. The structure of compound 1 was elucidated by spectroscopic methods including 1D NMR and 2D NMR analyses as well as HR-ESI-MS techniques.

Full text of DOI:10.1007/s10600-015-1448-8

DOI 10.1007/s10600-015-1448-8
Chemistry of Natural Compounds, Vol. 51, No. 5, September, 2015
A NEW CYCLIC PEPTIDE FROM BULBS OF Fritillaria hupehensis
Ling Tong, Zhi Zhu, Hui-Fang Pi, Han-Li Ruan,
Ji-Zhou Wu, and Peng Zhang*
A new cyclic peptide, cyclo(Leu-Val-Leu) (1), along with four known compounds (2–5), was isolated from
the bulbs of Fritillaria hupehensis Hsiao and K.C. Hsia. The structure of compound 1 was elucidated by
spectroscopic methods including 1D NMR and 2D NMR analyses as well as HR-ESI-MS techniques.
Keywords: Fritillaria hupehensis, nonbasic constituents, cyclic peptide.
Fritillaria hupehensis Hsiao & K. C. Hsia is a liliaceous plant growing in the southwest district of Hubei Province,
China. Its bulbs have been recorded in Pharmacopoeia of the Peopleꢀs Republic of China as a principal Chinese traditional
medicine named “Hubeibeimu.” C-nor-D-homo steroidal alkaloids have been isolated from the bulbs of this plant, together
with a number of the nonbasic constituents, such as fritillebin C and fritillebin D [1]. In our continuing studies on the nonbasic
constituents, a new cyclic peptide named cyclo(Leu-Val-Leu) (1), together with four known compounds (2–5), was isolated
from this plant. In this article, we describe the isolation and structural elucidation of compound 1. The structures of compounds
2
–5 were respectively confirmed as 13ꢁ-hydroxy-7-oxoabiet-8(14)-en-19,6ꢁ-olide, thymidine, adenosine, and uridine.
OH
O
1
0
9
8
6
NH
4
5
H
H
7
2
1
1
2
HN
3
1
3
1
H
O
O
1
4
N
O
H
1
O
1
5
1
7
O
1
6
1
2
Compound 1 was obtained as a white amorphous powder. Its molecular formula was determined as C H N O
7 31 3 3
1
+
according to the HR-ESI-MS data (m/z 326.4290 [M + H] , calcd 326.4294), requiring four degrees of unsaturation. The
IR spectrum of 1 showed intense absorption bands for amide N–H at 3300 cm and amide C=O at 1650 cm . Analysis of the
H and C NMR data (Table 1) revealed the presence of three carbonyl signals (ꢂ 168.9, 168.8 and 167.5), three normal
–
1
–1
1
13
C
ꢃ-amino acid methine carbon resonances (ꢂ 31.3, 23.6 and 23.5), and three amide protons (ꢂ 8.11, 8.09, and 8.01), indicating
C
H
its peptide nature [2–5].
1
1
H– H COSY experiment (Fig. 1) constructed the three amino acid residues as a valine and two leucines, which were
in correspondence with that of amino acid analysis after complete acid hydrolysis (6N HCl, 110ꢄC, 24 h). These residues
accounted for three of the four degrees of unsaturation, indicating that compound 1 is a cyclopeptide. Since compound 1 gave
a negative response, but a positive response after its acid hydrolysis, in the ninhydrin test, it must be a cyclopeptide [6].
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Tongji School of Pharmaceutical
Sciences, Huazhong University of Science and Technology, 430030, Wuhan, P. R. China, fax: +86 2783692739, e-mail:
zpeng023@gmail.com. Published in Khimiya Prirodnykh Soedinenii, No. 5, September–October, 2015, pp. 792–793. Original
article submitted December 12, 2014.
0
0
009-3130/15/5105-0923 2015 Springer Science+Business Media New York
923

1
13
TABLE 1. H NMR (400 MHz) and C NMR (100 MHz) Data of Compound 1 (DMSO-d , ꢂ, ppm, J/Hz)
6
C atom
ꢂH
ꢂC
C atom
ꢂH
ꢂC
C atom
ꢂH
ꢂC
Val
1
2
3
4
5
N–H
Leu1
6
7
8
9
10
11
N–H
Leu2
12
13
14
15
16
17
N–H
167.4 (s)
59.2 (d)
31.3 (d)
16.8 (q)
18.3 (q)
168.8 (s)
52.3 (d)
41.3 (t)
23.6 (d)
22.9 (q)
21.9 (q)
168.9 (s)
52.4 (d)
41.7 (t)
23.5 (d)
3.64 (1H, m)
2.18 (1H, m)
0.84 (3H, d, J = 6.7)
0.94 (3H, d, J = 6.7)
8.01 (1H, br.s)
3.75 (1H, m)
1.55 (2H, m)
1.81 (1H, m)
3.75 (1H, m)
1.55 (2H, m)
1.81 (1H, m)
0.85 (3H, d, J = 3.9)
0.86 (3H, d, J = 3.9)
8.09 (1H, br.s)
0.86 (3H, d, J = 3.9) 22.8 (q)
0.87 (3H, d, J = 3.9) 22.1 (q)
8.11 (1H, br.s)
O
Leu1
NH
HN
Val
O
O
N
H
Leu2
1
COSY
HMBC
1
1
Fig. 1. Key H– H COSY and HMBC
correlations of compound 1.
1
1
Evidence of the sequence of these amino acid residues was provided by the H– H COSY and HMBC experiment.
HMBC correlations were observed between Val-NH (ꢂ 8.01)/Leu1-CO (ꢂ 168.8), Leu1-NH (ꢂ 8.09)/Leu2-CO (ꢂ 168.9),
H
C
H
C
and Leu2-NH (ꢂ 8.11)/Val-CO (ꢂ 167.4), indicating the peptide fragment Leu2-Val-Leu1.The absolute stereochemistry of
H
C
the amino acid residues in 1 was confirmed by the advanced Marfeyꢀs method [7]. Compound 1 was subjected to complete
hydrolysis with 6 N HCl at 110ꢄC for 24 h in a sealed tube. Each amino acid from the hydrolysate was purified through silica
gel TLC and then analyzed by comparing the R -values with standard amino acids on chiral-TLC [8]. Consequently, all amino
f
acids were established to be of the L-configuration. Therefore, the structure of compound 1 was assigned as cyclo(Leu-Val-Leu).
EXPERIMENTAL
General Procedures. NMR spectra were recorded on a Bruker AV-500 spectrometer with TMS as internal standard.
HR-ESI-MS spectra were measured with an Agilent 1100 LC/MSD TOF mass spectrometer.
Plant Material. The bulbs of F. hupehensis Hsiao & K. C. Hsia were obtained from Hubei Institute of Chinese
Materia Medica and identified by Peng De Tai, Li Chuan Institute of Chinese Materia Medica, China. A voucher speciman
(
No. 09-15-2010) was deposited at the Faculty of Pharmaceutical Sciences, Tongji Medical College of Huazhong University
of Science and Technology, Wuhan, China.
Extraction and Isolation. The powdered crude bulbs (7.3 kg) of F. hupehensis were extracted with MeOH under
reflux for three times. After removal of the solvent at reduced pressure, the MeOH extract (560 g) was suspended in water and
then partitioned with EtOAc and n-BuOH successively. The EtOAc extract (80 g) was subjected to silica gel column
chromatography (petroleum ether–EtOAc, 100:0–50:50) to obtain five fractions 1–5. Fraction 2 (8.5 g) was then
chromatographed on a silica gel column (petroleum ether–Me CO, 20:1–1:1) to afford four fractions (fractions 2.1–2.4).
2
Compound 2 (13.4 mg) was obtained from fraction 2.2 by recrystallization. The n-butanol extract (155 g) was chromatographed
on a silica gel column (CHCl –MeOH, 100:0–0:100) to give nine fractions 1–9. The combined fractions eluted with
3
CHCl –MeOH (fraction 7, 12.5 g) were separated on a silica gel H column (CHCl –MeOH, 50:1–1:2) to afford seven fractions
3
3
1
ꢀ–7ꢀ. Subfraction 3ꢀ (0.15 g) was purified repeatedly by semipreparative HPLC (20% MeOH–H O) to give compounds 3
2
(
15 mg), 4 (12 mg), and 5 (10 mg). Subfraction 5ꢀ (0.1 g) was further purified by Sephadex LH-20 column (CHCl –MeOH,
3
1
9
:1) to yield compound 1 (3 mg).
24

Compounds 2–5 were obtained from this plant for the first time. The structures of compounds were respectively
determined as 13ꢁ-hydroxy-7-oxoabiet-8(14)-en-19,6ꢁ-olide (2) [9], thymidine (3) [10], adenosine (4) [11], and uridine (5)
[
12] by comparison with the literature values.
ACKNOWLEDGMENT
This work was financially supported by the Fundamental Research Funds for the Central Universities (No. 2013QN009).
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