O-Seco-RA-XXIV, a possible precursor of an antitumor peptide RA-XXIV, from Rubia cordifolia L.

Article abstract of DOI:10.1016/j.phytol.2012.02.014

O-Seco-RA-XXIV, a new cyclic peptide, cyclo-(d-alanyl-l-glutaminyl-N,O- dimethyl-l-tyrosyl-l-alanyl-N-methyl-l-tyrosyl-N-methyl-l-tyrosyl), was isolated from the roots of Rubia cordifolia L. along with RA-XXIV. Its structure and relative stereochemistry were determined by interpretation of the spectroscopic data and X-ray crystallography, and its absolute stereochemistry by the Marfey's amino acid analysis of its acid hydrolysate. Isolation of the two peptides from the same plant source may indicate that O-seco-RA-XXIV is a possible precursor of RA-XXIV and that the formation of the diphenyl ether linkage in the cycloisodityrosine moiety is to be formed after the formation of the cyclohexapeptide chain in this series of peptides.

Full text of DOI:10.1016/j.phytol.2012.02.014

Phytochemistry Letters 5 (2012) 335–339
Contents lists available at SciVerse ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
O-Seco-RA-XXIV, a possible precursor of an antitumor peptide RA-XXIV, from
Rubia cordifolia L.
Yukio Hitotsuyanagi, Jun-ichi Kusano, Ik-Hwi Kim, Tomoyo Hasuda, Haruhiko Fukaya, Koichi Takeya *
School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
A R T I C L E I N F O
A B S T R A C T
Article history:
O-Seco-RA-XXIV, a new cyclic peptide, cyclo-(D-alanyl-L-glutaminyl-N,O-dimethyl-L-tyrosyl-L-alanyl-N-
Received 3 December 2011
Received in revised form 21 February 2012
Accepted 28 February 2012
Available online 11 March 2012
methyl-L-tyrosyl-N-methyl-L-tyrosyl), was isolated from the roots of Rubia cordifolia L. along with RA-
XXIV. Its structure and relative stereochemistry were determined by interpretation of the spectroscopic
data and X-ray crystallography, and its absolute stereochemistry by the Marfey’s amino acid analysis of
its acid hydrolysate. Isolation of the two peptides from the same plant source may indicate that O-seco-
RA-XXIV is a possible precursor of RA-XXIV and that the formation of the diphenyl ether linkage in the
cycloisodityrosine moiety is to be formed after the formation of the cyclohexapeptide chain in this series
of peptides.
Keywords:
Antitumor
Bouvardin
O-Seco-RA-XXIV
RA-VII
ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
RA-XXIV
Rubia cordifolia
1
. Introduction
RA-VII (1), isolated from Rubia cordifolia L. and Rubia akane
2. Results and discussion
A methanol extract (3.6 kg) obtained from dried roots of R.
cordifolia (50 kg) was placed on an HP-20 column and eluted
Nakai (Rubiaceae), bouvardin (NSC 259968, 2), from Bouvardia
ternifolia (Cav.) Schltdl. (Rubiaceae), and deoxybouvardin (RA-V,
2
sequentially with H O/MeOH (1:0, 1:1, and 0:1), and acetone to
3
), from the above three plants are bicyclic hexapeptides
structurally characterized by the presence of a unique strained
4-membered cycloisodityrosine unit in the molecule (Fig. 1)
Jolad et al., 1977; Itokawa et al., 1983, 1997). These peptides have
give four fractions. The MeOH eluate (975 g) was subjected
sequentially to silica gel and charcoal column chromatography
with a series of solvent mixtures to give a fraction containing RAs
(176 g). The fraction was then placed on an aminopropyl-bonded
1
(
a promising antitumor activity, and peptide 1 underwent phase I
clinical trials as an anticancer drug in Japan (Majima et al., 1993;
Yoshida et al., 1994). The antitumor action of these peptides is
believed to be due to inhibition of protein synthesis through
interaction with eukaryotic ribosomes (Zalaca ı´ n et al., 1982;
Sirdeshpande and Toogood, 1995), and 1 has been shown also to
cause conformational changes in F-actin, which stabilizes the
actin filaments and induces G2 arrest (Fujiwara et al., 2004). In the
present study, we isolated a novel cyclic hexapeptide, O-seco-RA-
XXIV (4), along with known RA-series peptides from the roots of R.
cordifolia L., and elucidated its structure, which gave us a
significant clue for the possible biosynthetic route of RAs and
bouvardins.
silica gel column which was eluted sequentially with CHCl
(20:1, 10:1, and 0:1), and MeOH/AcOH (100:3) to give four
fractions. Repeated reversed-phase HPLC (ODS) of the CHCl
MeOH (10:1) eluate gave O-seco-RA-XXIV (4) (2.9 mg), and that of
the CHCl /MeOH (20:1) eluate gave deoxybouvardin (3), RA-I, RA-
3
/MeOH
3
/
3
XII, RA-XXIII, and RA-XXIV (5).
O-Seco-RA-XXIV (4) was obtained as colorless prisms. Its
molecular formula was determined to be C42
H
53
N
7
O
10 from the
+
+
[M+H] peak at m/z 816.3963 ([M+H] , calcd for C42H N O ,
54 7 10
1
l3
816.3932) in the HR-ESIMS. The H and C NMR spectra of 4 in
methanol-d (Table 1) resembled those of RA-series peptides, and
4
demonstrated the presence of four conformers in a ratio of
56:38:4:2 (Figs. S1 and S2, see Supplementary data). The structure
of 4 was determined by the analysis of the resonances caused by
the two major conformers-I and -II with the population of 56% and
3
8%, respectively. Those spectra showed characteristic signals of
two secondary methyl groups ascribable to two alanines (
.12/19.8 and 1.17/17.7 for conformer-I and 0.95/18.5
and 1.30/19.8 for conformer-II), three N-methyl groups (
H C
d /d
1
H C
d /d
*
Corresponding author. Tel.: +81 42 676 3007; fax: +81 42 677 1436.
E-mail address: takeyak@ps.toyaku.ac.jp (K. Takeya).
H C
d /d
1
874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2012.02.014

3
36
Y. Hitotsuyanagi et al. / Phytochemistry Letters 5 (2012) 335–339
1
1
Fig. 2. Key H– H COSY, HMBC, and ROESY correlations of O-seco-RA-XXIV (4).
Fig. 1. Structures of RA-series peptides and bouvardins.
2
3
.85/29.9, 2.87/30.0, and 2.96/32.2 for conformer-I and
0.4, 2.94/40.7, and 3.05/30.8 for conformer-II), and one O-methyl
3.74/55.7 for conformer-I and 3.76/55.6 for
H C
d /d 2.91/
group (
d
H
/d
C
H C
d /d
conformer-II), which are generally observed in the spectrum of RA-
series peptides. However, difference was noted in the aromatic
1
proton signals of isodityrosine. In the H NMR spectrum of 4,
characteristic signals due to inequivalent two
d and two e protons
of Tyr-5 and due to three protons of the tri-substituted phenyl ring
of Tyr-6, commonly observed in other RA-series peptides, were
missing, and instead, three para-substituted phenyl ring proton
signals ascribable to the three tyrosines (H
2
-
d
/H
.02/6.73, and 7.15/6.872 for conformer-I and
.74, and 7.10/6.868 for conformer-II) were observed. Analysis of
2
-
e
:
d
H
6.93/6.63,
7
6
d 6.93/6.67, 7.08/
H
1
1
the H– H COSY, HSQC, and HMBC spectra revealed that peptide 4
consisted of two alanines, two N-methyltyrosines, one N,O-
dimethyltyrosine, and one glutamine or one glutamic acid
(
4
Fig. 2). The NMR spectra in methanol-d were not able to decide
if the 6th amino acid was glutamine or glutamic acid, because the
signals due to the amide protons or the carboxylic acid proton
could not be observed due to the H/D exchange in this solvent. The
6
th amino acid was deduced to be glutamine from the molecular
formula of 4 containing seven nitrogens. The amino acid sequence
of 4 was determined to be as shown in Fig. 2 by the analysis of the
HMBC cross-peaks from the
a-proton or the N-methyl protons of
an amino acid to the carbonyl carbon of the adjacent i ꢀ 1 amino
acid. Its ROESY spectrum revealed that conformers-I and -II were
stereoisomers with respect to an N-methyl amide bond: in
conformer-I, the ROE correlation between Gln-2 H-
was observed, whereas in conformer-II, those between Gln-2 H-
Tyr-3 NMe and between Tyr-3 NMe/Tyr-3 H- were observed,
indicating that the amide configuration between the residues 2 and
was cis in conformer-I and trans in conformer-II. The correlations
between Ala-4 H- /Tyr-5 NMe, Ala-4 Me/Tyr-5 NMe, and Tyr-5 H-
/Tyr-6 H- were observed in both conformers-I and -II, implying
a/Tyr-3 H-a
a
/
a
3
Fig. 3. ORTEP representation of O-seco-RA-XXIV (4).
a
a
a
that in both, the configuration between the residues 4 and 5 was
trans and that between the residues 5 and 6 was cis.
opposite configuration as in other RA-series peptides. This crystal
structure of 4 was unique in that it had two type-VI
b-turn
The relative stereochemistry of 4 was determined by its X-ray
crystallography to be as shown in Fig. 3: the amino acid residues 2–
structures, one centered at Gln-2 and Tyr-3 and the other at Tyr-5
and Tyr-6, which were stabilized by the hydrogen bonds between
6
had the same relative configuration, whereas the residue 1 the
D-Ala-1 C 55 O and Ala-4 NH and between Ala-4 C 55 O and D-Ala-1 NH,

Y. Hitotsuyanagi et al. / Phytochemistry Letters 5 (2012) 335–339
337
Table 1
a
NMR data of O-seco-RA-XXIV (4).
Residue
Conformer-I
Conformer-II
d
H
d
C
d
H
d
C
D
-Ala-1
a
b
4.24 (q, 6.9)
50.2
19.8
4.29 (q, 6.9)
49.62
19.8
1.12 (d, 6.9, 3H)
1.30 (d, 6.9, 3H)
C5O
174.8
174.6
Gln-2
Tyr-3
a
b
g
d
4.31 (m)
49.56
27.8
4.53 (dd, 8.4, 6.0)
1.99–1.89 (m, 2H)
2.27 (t, 6.9, 2H)
50.9
26.9
b
1.77 , 0.77 (m)
b
1.88 (m), 1.77
32.5
31.8
177.1
174.0
177.2
173.8
C5O
a
5.28 (dd, 10.2, 5.0)
3.03 (dd, 14.4, 5.0)
2.98 (dd, 14.4, 10.2)
63.3
34.6
3.94 (m)
68.9
b
b
g
d
e
a
3.27 (dd, 13.7, 4.5)
3.16 (dd, 13.7, 11.6)
34.13
b
130.3
131.55
131.46
c
c
7.15 (d-like, 8.5, 2H)
6.872 (d-like, 8.5, 2H)
131.8
7.10 (d-like, 8.6, 2H)
6.868 (d-like, 8.6, 2H)
c
c
115.3
115.1
z
160.3
169.5
29.9
160.0
171.4
40.7
C5O
NMe
OMe
2.85 (s, 3H)
3.74 (s, 3H)
2.94 (s, 3H)
3.76 (s, 3H)
55.7
55.6
Ala-4
Tyr-5
a
b
C5O
4.72 (q, 6.8)
48.4
17.7
4.70 (q, 6.7)
47.8
18.5
1.17 (d, 6.8, 3H)
0.95 (d, 6.7, 3H)
173.2
173.6
a
5.08 (t, 7.8)
57.2
35.1
5.59 (dd, 9.8, 5.8)
53.0
b
b
b
b
g
d
e
a
2.94
2.85
34.09
b
2.51 (dd, 13.5, 8.3)
2.21 (dd, 14.3, 5.8)
128.4
128.4
c
c
7.02 (d-like, 8.5, 2H)
6.73 (d-like, 8.5, 2H)
131.2
6.93 (d-like, 8.5, 2H)
6.67 (d-like, 8.5, 2H)
131.0
c
c
116.7
116.32
157.3
172.1
30.4
z
C5O
157.7
174.2
32.2
NMe
2.96 (s, 3H)
2.91 (s, 3H)
Tyr-6
a
4.77 (t, 7.9)
63.7
35.7
5.02 (dd, 9.8, 5.3)
3.02 (dd, 13.6, 5.3)
2.73 (dd, 13.6, 9.8)
63.2
36.9
b
b
g
d
e
a
2.81 (dd, 13.1, 8.4)
1.86 (dd, 13.1, 7.1)
b
128.75
128.78
c
c
6.93 (d-like, 8.5, 2H)
6.63 (d-like, 8.5, 2H)
131.6
7.08 (d-like, 8.5, 2H)
6.74 (d-like, 8.5, 2H)
131.3
c
c
116.29
157.4
170.0
30.0
116.7
z
C5O
157.7
170.7
30.8
NMe
2.87 (s, 3H)
3.05 (s, 3H)
a
1
13
3
In CD OD, 600 MHz for H and 150 MHz for C; J-values given in Hz in parentheses. Population of conformers-I and -II in this solution was 56% and 38%, respectively.
b
c
Multiplicity not determined due to signal overlapping.
Two carbons.
respectively (Chou and Fasman, 1977; Zimmerman and Scheraga,
977). Its absolute configuration was determined by amino
acid analysis. When subjected to the hydrolysis – Marfey’s
in the cycloisodityrosine moiety takes place. A number of RA-series
and related antitumor bicyclic peptides have been isolated from
rubiaceous plants, R. cordifolia, R. akane (Itokawa et al., 1997), R.
yunnanensis (Fan et al., 2010), and B. ternifolia (Jolad et al., 1977).
However, they had given no clue to those problems. Isolation of O-
seco-RA-XXIV (4) with RA-XXIV (5) (Lee et al., 2008) from R.
cordifolia in the present study may suggest that the 18-membered
cyclohexapeptide ring is formed first to produce 4, which, by
subsequent phenolic oxidative coupling reaction, produces bicyclic
RA-XXIV (5) in the plant body. The other RA-series peptides and
bouvardins having a cycloisodityrosine unit may also be considered
to be biosynthesized analogously from their O-seco-analogues.
Peptide 4 showed a cytotoxic effect on human promyelocytic
1
a
derivatization with N -(2,4-dinitro-5-fluorophenyl)-
mide ( -FDAA), and then to HPLC, 4 gave three peaks correspond-
ing to -DAA derivatives of - and -alanines, and -glutamic acid
Marfey, 1984). The residues 1, 2, and 4 of 4 were thus identified as
L-alanina-
L
L
D
L
L
(
D
-alanine,
L
-glutamine, and
L
-alanine, respectively, and the
-alanyl- -glutami-
-alanyl-N-methyl- -tyrosyl-N-
-tyrosyl), as shown in Fig. 1.
The RA-series peptides are usually composed of one
residue 1), one -alanine (residue 4), one -amino acid which
includes -alanine, -glutamic acid, -glutamine, -serine, and
threonine (residue 2), and three modified N-methyl- -tyrosines
residues 3, 5, and 6). One of those N-methyl- -tyrosines (residue 3)
structure of 4 was determined to be cyclo-(
nyl-N,O-dimethyl- -tyrosyl-
methyl-
D
L
L
L
L
L
D-alanine
(
L
L
L
L
L
L
L-
leukemia HL-60 cells (IC50 58
mg/ml), which was much weaker than
L
that of RA-XXIV (5) (0.50
m
g/ml) or of RA-VII (1) (0.0030 g/ml).
m
(
L
is often O-methylated, and the other two (residues 5 and 6) are
3. Experimental
connected to form a cycloisodityrosine with an ether linkage
between the hydroxyl oxygen at the
z
position of the residue 5 and
3.1. General experimental procedures
the carbon atom at the position of the residue 6. How those RA-
e
series peptides are biosynthesized in plants? Especially, it is of great
Melting point was determined on a Yanaco MP-3 apparatus and
is uncorrected. Optical rotation was measured on a JASCO P-1030
interest to know at what stage the diphenyl ether linkage formation

3
38
Y. Hitotsuyanagi et al. / Phytochemistry Letters 5 (2012) 335–339
digital polarimeter, UV spectrum on a JASCO V-530 spectropho-
tometer, IR spectrum on a JASCO FT/IR-620 spectrophotometer,
NMR spectra on a Bruker AVANCE 600 spectrometer, and mass
Center as supplementary publication number CCDC 852755.
Copies of the data can be obtained, free of charge, on application
to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44 0 1223
336033 or deposit@ccdc.cam.ac.uk
1
spectrum on a Micromass LCT spectrometer. In the H NMR
spectrum, the chemical shifts (
d
) are given in parts per million
OD at 3.31 ppm, and in
the C NMR spectrum, the chemical shifts are given in parts per
million relative to the resonance at 49.0 ppm for CD OD. Single-
relative to the resonance of residual CHD
2
3.5. Amino acid analysis
13
3
Compound 4 (1.1 mg) was suspended in 6 M HCl (1 ml) and
heated in a sealed glass vial at 110 8C for 16 h. The solution was
crystal X-ray analysis was carried out on a Bruker AXS APEX II
ULTRA CCD area detector diffractometer with a rotating anode
˚
evaporated to dryness under a N
2
stream. The residue was
(100 l) and the solution
treated with N -(2,4-dinitro-5-fluorophenyl)- -alaninamide (1%
w/v acetone solution, 200 l). The mixture was heated (40 8C,
90 min), cooled, and quenched with 2 M HCl (25 l). Standard
samples of - and -glutamic acids and - and -alanines were also
converted to their -DAA derivatives by the same procedure. The
solutions obtained by derivatization were analyzed by HPLC under
the following conditions: column, GL Sciences ODS-3 (5 m,
4.6 mm ꢁ 250 mm); mobile phase, MeOH/MeCN/0.02 M phos-
phate buffer (pH 2.5) (20:20:60); detection, UV = 340 nm; flow
rate, 1.0 ml/min. The solution from 4 gave three peaks with the
retention times of t 15.82 min, t 19.70 min, and t 31.98 min,
which corresponded to those derived from authentic -glutamic
acid (t 15.80 min), -alanine (t 19.67 min), and -alanine (t
31.90 min), respectively. The derivative from authentic -glutamic
source (Mo K
a
radiation,
l
= 0.71073 A). Preparative HPLC was
redissolved in 0.5 M aqueous NaHCO
3
m
a
performed by using Shimadzu LC-6AD pump unit equipped with a
L
Wakosil-II 5C18 HG Prep column (5
Shimadzu SPD-10A UV detector.
m
m, 20 mm ꢁ 250 mm) and a
m
m
D
L
D
L
3.2. Plant material
L
The roots of R. cordifolia L. were commercially obtained in Tokyo
m
in March 2006. The material was identified by Prof. Koichi Takeya,
and a voucher specimen (Tko-0601g01) has been deposited at the
Herbarium of Tokyo University of Pharmacy and Life Sciences.
l
R
R
R
3.3. Extraction and isolation
L
R
L
R
D
R
The dried roots (50 kg) of R. cordifolia were extracted with MeOH
D
(
3 ꢁ 180 l). After removal of MeOH under reduced pressure, the
residue (3.6 kg) was placed on a column of HP-20 (18 kg) and
sequentially eluted with H O (72 l), H O/MeOH (1:1, 78 l), MeOH
78 l), and acetone (63 l) to give four fractions. After removal of the
R
acid gave the retention time of t 20.66 min.
2
2
3.6. Cytotoxic assay
(
solvent, the residue of the MeOH fraction (975 g) was subjected to
silica gel (5 kg) column chromatography eluting sequentially with
Cytotoxic assay was performed by using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)
assay method. The human HL-60 promyelocytic leukemia cells
were precultured in RPMI 1640 medium (Nissui Co. Ltd., Japan)
supplemented with 10% heat-inactivated fetal bovine serum
CHCl
The CHCl
400 g) column chromatography eluting sequentially with MeOH/
CHCl (1:0, 3:1, 1:1, and 1:10, 18 l each) to give four fractions. The
3
3
(14 l), EtOAc (28 l), CHCl /MeOH (3:2, 36 l), and MeOH (8 l).
3
/MeOH (3:2) fraction (400 g) was subjected to charcoal
(
3
(FBS) and kanamycin (100 mg/l) in a humidified atmosphere of
4
residue of the first fraction, i.e., the MeOH eluate (176 g), was
subjected to aminopropyl-bonded silica gel (750 g) column
93% air and 7% CO
100
2
at 37 8C. The cell suspension (3 ꢁ 10 cell/ml,
3
m
l) was added to each well (3 ꢁ 10 cells/well) of a 96-
chromatography eluting sequentially with CHCl
3
0:1, and 0:1) and MeOH/AcOH (100:3) (3 l each). The CHCl /
3
/MeOH (20:1,
microwell plate (flat bottomed, polystyrene treated) and
1
incubated for 24 h. Test compound solutions in dimethyl
MeOH (20:1) eluate (18.6 g) was crystallized from MeOH/isopropyl
ether to give crystals of crude RAs (1.2 g, mainly RA-XXIII and RA-
XXIV) and mother liquor (ML). After removal of the solvent, ML
sulfoxide (DMSO) (100, 30, 10, 3, 1, 0.3, 0.1
mg/ml) were
prepared, and 10 l of the test solution or DMSO (control) was
m
added to each well. The plate was kept in an incubator for 48 h.
(
17.2 g) was subjected to HPLC with MeOH/H
to give six fractions, FA1–FA6. Fraction FA2 (3.45 g) was subjected to
HPLC with MeOH/H O (30:70) to give RA-I (451 mg), RA-XII
264 mg), and RA-XXIV (864 mg), and fraction FA3 (4.59 g) with
2
O (60:40, then 100:0)
After termination of the cell culture by adding 5% MTT in PBS
(20
m
l) to each well, the plate was kept in the incubator for 4 h.
l of 10% SDS-0.01 M HCl to each well, the
2
After addition of 100
m
(
plate was read on a microplate reader (MPR A4i, Tosoh Co., Japan)
at 550/700 nm. A dose-response curve was plotted for each
compound, and the concentrations giving 50% inhibition of the
cell growth (IC50) were recorded.
MeCN/H
2
O (32:68) to give RA-XXIII (501 mg) and 3 (2165 mg).
/MeOH (10:1) eluate (19.9 g) of the aminopropyl-
The CHCl
3
bonded silica gel column chromatography was treated with MeOH,
and insoluble material was removed by filtration. The filtrate was
subjected to HPLC with MeOH/H
eight fractions, FB1–FB8. Repeated HPLC of fraction FB2 (3.28 g)
with MeOH/H O (45:55) and then MeCN/H O (24:76) afforded 4
2
O (50:50, then 100:0) to give
Appendix A. Supplementary data
2
2
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytol.2012.02.014.
(2.9 mg).
3
.3.1. O-Seco-RA-XXIV (4)
25
References
Colorless prisms; [
log ) 225 (4.40), 278 (3.54), 284 (3.48) nm; IR (film)
921, 2850, 1659, 1651, 1624, 1614, 1513, 1243, 1029, 804,
a
]
D
ꢀ62 (c 0.21, MeOH); UV (MeOH)
l
max
max 3282,
(
2
7
e
n
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1
1
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H
54
N
7
O
10 [M+H] 816.3932, found 816.3963.
1
3.4. X-ray crystallographic study of 4
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2
arrest
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