Cyclic dipeptide of D-ornithine obtained from the dobsonfly, Protohermes grandis thunberg

Article abstract of DOI:10.1271/bbb.90090

A new compound was isolated from the water-soluble fraction of a methyl alcohol extract obtained from the larva of the dobsonfly (Protohermes grandis Thunberg). The novel compound had a 12-membered ring and was confirmed to be a cyclic dipeptide of D-orni

Full text of DOI:10.1271/bbb.90090

Biosci. Biotechnol. Biochem., 73 (7), 1669–1670, 2009
Note
Cyclic Dipeptide of D-Ornithine Obtained from the Dobsonfly,
Protohermes grandis Thunberg
y
Ryuichiro TANAKA and Masashi ODA
Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan
Received February 3, 2009; Accepted March 16, 2009; Online Publication, July 7, 2009
[doi:10.1271/bbb.90090]
A new compound was isolated from the water-soluble
+
195 [211–NH2]
fraction of a methyl alcohol extract obtained from the
larva of the dobsonfly (Protohermes grandis Thunberg).
The novel compound had a 12-membered ring and was
confirmed to be a cyclic dipeptide of D-ornithine by a
chiral analysis, using high-performance liquid chroma-
tography, 2D-nuclear magnetic resonance, and field
desorption mass spectrometry.
Key words: cyclic di-D-ornithinate; dobsonfly; larva;
Protohermes grandis
+
211 [227–NH2]
We have recently been searching for novel insect
ingredients to be used as crude drugs in traditional East
Asian medicine.1) Larvae of the dobsonfly, Protohermes
grandis Thunberg (Neuroptera, Corydalidae), an aquatic
227 [M–H]+
insect, have been prescribed as a crude drug in tradi-
tional Japanese medicine (called magotaro mushi in
m/z
Japanese). Previously characterized constituents of the
larvae included essential amino acids, fats, and some
steroids;2,3) however, other low-molecular-weight polar
constituents of the larvae have not been investigated.
Air-dried larvae of Protohermes grandis Thunberg
(from the Sai river in the Shiroishi district of Iwate
Prefecture, Japan) were purchased from Honzoukaku
(Nagoya, Japan). After a 40-d incubation period, the
larvae (24.5 g) were soaked in methanol (2 liters) at
room temperature and filtered. The filtrate gave a brown
extract (6.4 g). This extract was separated into two
portions, and CHCl3, MeOH, and H2O were added in a
final ratio of 1:2:1.4) Each solution was shaken, and the
upper (methanol-water) layer was isolated and concen-
trated in vacuo until dry, yielding the polar fraction
sample (3.1 g). This sample was subjected to open-
column chromatography (Diaion HP-20, Mitsubishi
Chemical, 40 mm I.D. ꢀ 175 mm; eluent, H2O !
50% MeOH/H2O ! MeOH ! EtOAcÞ. The eluted
H2O fraction (1820 mg) was separated into four frac-
tions by open-column chromatography (Sephadex LH-
20, Pharmacia Biotech., 45 mm I.D. ꢀ 450 mm; eluent,
50% MeOH/H2O). The first-eluted fraction (1030 mg)
of the previous separation contained trehalose, pyroglu-
tamate, and compound 1. Finally, the compounds were
refined by repeated high-performance liquid chromatog-
raphy (HPLC; Sugar-D, Nacalai Tesque, 4:6 mm I.D. ꢀ
250 mm; eluent, 85% CH3CN/H2O) which yielded 2.5,
3.0, and 1.0 mg, respectively.
Fig. 1. Part of the FD-MS Data (carbon emitter) Spectrum of
Compound 1.
25
1
rotation, ½ꢁꢂ
þ9:1ꢃ (c 0.1, H2O). The H-NMR and
D
13C-NMR spectra (in D2O) of compound 1 indicated the
presence of one methine group [Cb–H: ꢂC 64.0, ꢂH 4.13
(dd, J ¼ 7:6, 6.1 Hz)], three methylene groups [Cc–H2:
ꢂC 31.7, ꢂH 2.08 (1H, m), 2.34 (1H, m); Cd–H2: ꢂC 26.5,
ꢂH 2.02 (2H, m); Ce–H2: ꢂC 48.9, ꢂH 3.35 (1H, m), 3.44
(1H, m)], and one carbonyl (or carboxyl) carbon (Ca: ꢂC
177.0).
The 1H-1H COSY, HMQC (J constant ¼ 145 Hz),
and HMBC (long-range J constant ¼ 8 Hz) spectra of
compound 1 indicated connectivity of the O=Ca–CbH–
CcH2–CdH2–CeH2–NH–molecules. The 1H-NMR chemi-
cal shift of Cb–H suggested that a polar functional group
(–NH2) was linked to carbon Ca.
Compound 1 produced field desorption mass spectro-
metric (FD-MS) ion peaks at m=z 227 (8), 211 (30), and
195 (100) which were attributable to the ½M ꢁ Hꢂþ,
½227 ꢁ NH2ꢂþ, and ½211 ꢁ NH2ꢂþ ions for the molecu-
lar-related positive ions (hydride-fragment ions), respec-
tively (Fig. 1). The negative ion electrospray ionization
mass spectrum (ESI-MS) contained a ½M ꢁ Hꢂꢁ ion peak
at m=z 227 (5), and ½M ꢁ 2Hꢂ2ꢁ ion peak at m=z 113 (18).
The high-resolution (HR) FD-MS data indicated the
molecular ion composition, C10H19N4O2, for the ½M ꢁ
Hꢂþ ion (227.1503, calculated for C10H19N4O2: 227.1508).
Taken together, these results suggest that compound 1
had a 12-membered ring and was a cyclic dipeptide of
ornithine.
Compound 1 [ꢀmax (KBr) cmꢁ1: 1559 (HN–C=O),
1698 (C=O)] was a white powder with a positive optical
y
To whom correspondence should be addressed. Tel/Fax: +81-72-866-3139; E-mail: tanaka-r@pharm.setsunan.ac.jp

1670
R. TANAKA and M. ODA
solv.
O
H2N
R
NH
7
1a + L-Orn
1
R
HN
NH2
O
inj.
Fig. 3. Structure of Compound 1.
1a + D-Orn
structure are a group of compounds which have strong
characteristic bioactivity. Since compound 1 had a
lactam structure with a 12-membered ring, its physio-
logical significance is of interest.
inj.
inj.
1a
Instruments. The optical rotation of compound 1 was
measured at 25 ꢃC with a polarimeter (P-1020; Jasco,
Tokyo, Japan). Mass spectra (MS) and HR-MS were
recorded by a JMX-HX 110 spectrometer (Jeol). The
NMR spectra were recorded by JNM-GSX 400 and
JNM-ECA 600 spectrometers (Jeol). HPLC was per-
formed by using 510 pump (Waters, Milford, MA,
USA), U-620 column oven (Sugai, Tokyo, Japan), UV-
visible detector (Jasco), 504R RI detector (GL Sciences,
Tokyo, Japan), and C-R-8A integrator (Shimadzu,
Kyoto, Japan). The Chiralpak CR(þ) HPLC column
was obtained from Daicel (Hiroshima, Japan), the
Sumichiral OA-6100 column was obtained from Sumika
Chemical Analysis Service (Osaka, Japan), and the
Sugar-D column was purchased from Nacalai Tesque
(Kyoto, Japan).
0
2.0
4.0
(min)
Fig. 2. HPLC Profiles of the Acid Hydrolysis Product of Compound
1.
Column, Chiralpak CR(þ), 4:6 mm I.D. ꢀ 150 mm; solvent, H2O,
pH adjusted to 1.5 with HClO4; flow rate, 0.4 ml/min; detection,
210 nm
The product (1a, FD-MS, m=z 133 (40) ½M þ Hꢂþ and
132 (3) [M]þ; neg. ion-FAB MS, glycerol matrix, m=z
149 (40) ½M þ H2O ꢁ Hꢂꢁ) of the acid hydrolysate (3 N
HCl, 100 ꢃC, 4 h) of compound 1 (0.4 mg) yielded a
single peak (Rt 3.54 min) after an HPLC analysis
(Chiralpak CR(þ), 4:6 mm I.D. ꢀ 150 mm; solvent,
H2O, pH adjusted with HClO4 at 1.5; flow rate,
0.4 ml/min; detection, 210 nm; Rt values: D-Orn, 3.54
min; L-Orn, 3.84 min). The co-HPLC chromatography
suggested that the product from acid hydrolysis was
Chemicals. D- and L-ornithine (special grade; Wako,
Osaka, Japan) were obtained commercially.
Acknowledgments
a
D-isomer (Fig. 2). Furthermore, the chiral HPLC
analysis of the hydrolysate using a Sumichiral OA-
6100 column (4:6 mm I.D. ꢀ 250 mm, solvent of 1.5 mM
copper (II) sulfate in H2O, flow rate of 1.0 ml/min,
detection at 254 nm, Rt values: D-Orn, 8.28 min; L-Orn,
16.76 min) also indicated the presence of D-ornithine.
Two-dimensional nuclear magnetic resonance (2D-
NMR), FD-MS, and chemical data indicated that the
structure of compound 1 was (3R,9R)-2,8-dioxo-1,7-
diazacyclododecane-3,9-diamine (a cyclic dipeptide of
D-ornithine) (Fig. 3).
The author thanks Dr. Masatoshi Nishi and Dr. Shouji
Yamaguchi of the Central Analytical Laboratory of
Setsunan University for measuring the 600-MHz NMR
and MS data.
References
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Osaka, pp. 345–346 (2000).
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compound from a natural material. One of the anti-
biotics, ꢃ-lactams and macrolides that have a lactone
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