Structure and enantioselective synthesis of polyamine toxin MG30 from the venom of the spider Macrothele gigas

Article abstract of DOI:10.1016/j.tetlet.2004.05.076

A novel polyamine toxin, named MG30, was isolated from the venom of the spider, Macrothele gigas, and its structure was elucidated by two-dimensional NMR and mass analysis. In addition, the enantioselective synthesis of MG30 was achieved to assign its absolute stereochemistry.

Full text of DOI:10.1016/j.tetlet.2004.05.076

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 5371–5373
Structure and enantioselective synthesis of polyamine toxin
MG30 from the venom of the spider Macrothele gigas
Nahoko Yamaji,a,* Manabu Horikawa,a Gerardo Corzo,a Hideo Naoki,a Joachim Haupt,b
Terumi Nakajimaa and Takashi Iwashitaa
aSuntory Institute for Bioorganic Research, Instrumental analysis, 1-1-1 Wakayamadai,
Shimamoto-Cho, Mishima-Gun, Osaka 618-8503, Japan
bInstitute of Ecology, Technical University Berlin, 10623 Berlin, Germany
Received 13 April 2004; revised 12 May 2004; accepted 17 May 2004
Abstract—A novel polyamine toxin, named MG30, was isolated from the venom of the spider, Macrothele gigas, and its structure
was elucidated by two-dimensional NMR and mass analysis. In addition, the enantioselective synthesis of MG30 was achieved to
assign its absolute stereochemistry.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results
Spider venom contains various kinds of compounds such
as proteins, peptides and polyamine toxins. These toxins
are of interest as tools for studying neurophysiology and
pharmacology. Particularly, polyamine toxins have
shown specific activity as glutamate receptor blockers.1–3
M. gigas venom was obtained by electrical stimulation
of field-collected spiders from the Sonai area in Irio-
mote, Japan. The crude venom was frozen and stored at
)20 ꢁC until use.
The crude venom (70 lL) was resuspended in 0.1%
aqueous trifluoroacetic acid (TFA) containing 10%
acetonitrile, and the insoluble materials were removed
by centrifugation at 14,000g for 5 min. The supernatant
was purified by the established high-performance liquid
chromatography method (C18 / 10 · 250 mm, linear
gradient with 0–60% acetonitrile/H2O in 0.1% TFA in
60 min, flow rate 2.0 mL/min, UV detection at 220 nm).
The polyamine, named MG30, was a major component
of the venom to yield 150 lg.
In the previous report, we described six peptide toxins
(Magi 1–6), isolated from the venom of the Hexathe-
lidae spider Macrothele gigas collected at Iriomote-
Island in Japan.4 We have also isolated an unknown
polyamine compound, named MG30, as a major com-
ponent of the same spider venom. We report here the
structure determination of MG30 using two-dimen-
sional NMR, MS analysis and the enantioselective
synthesis of (R) and (S)-MG30 to assign the absolute
configuration of the native MG30.
1
The H NMR spectrum of MG30 showed peculiar sig-
nals to an indole ring at the low-field region (H-1, 3, 4, 5
16 ,1 7
21 ,2 2
1
O
and 6). H–1H COSY, TOCSY and HMBC analysis of
3
6
12
8
7
2
10
N
NH2
MG30 easily gave the structural information on an
indole-3-lactyl part, and the HMBC peaks from H-12
(dH 3.05 and 3.17) to C-11 (dC 178.8) revealed that an-
other unit connected to the indole-3-lactyl moiety
N
4
9
N
H
15
18
20
23
25
5
1
OH
N
H
MG30 (1)
1
through an amide linkage. H–1H COSY spectra led to
the connectivities from C-12 to C-15, C-18 to C-20 and
C-23 to C-25. This was also supported by TOCSY and
HMBC data. The methyl protons of H-16, 17, 21 and 22
(dH 2.98 and 3.17) had cross-peaks with dC 53.2 or 53.3
Keywords: Spider; Polyamine toxin; Macrothele gigas.
* Corresponding author. Tel.: +81-75-962-6142; fax: +81-75-962-2115;
e-mail: yamaji@sunbor.or.jp
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.05.076

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N. Yamaji et al. / Tetrahedron Letters 45 (2004) 5371–5373
Table 1. 13C NMR and 1H NMR spectral data of MG30 in D2O
O
Position
dC
dH (mult., J in Hz)
N
N
NH2
N
H
1
127.6
111.9
121.8
122.1
124.6
114.5
138.8
130.3
32.2
7.30 (s)
OH
N
H
2
3
7.69 (d, 7.8)
7.19 (t, 7.4)
7.27 (t, 7.7)
7.52 (d, 7.8)
38 9
13 0 16 0 18 8
25 9
4
5
Figure 1. Fragmentation of MG30 by ESI-Q-TOF MS/MS.
6
7
8
9
3.24, 3.29 (dd, 15.0, 5.5)
4.53 (t, 5.5)
In order to determine the absolute configuration of the
secondary hydroxy group of MG30, both enantiomeri-
cally pure (R) and (S)-MG30 were synthesized in short
steps as shown in Scheme 1. The preparation of the (S)-
indole-3-lactic acid, (S)-3, was carried out by coupling
of indole and methyl (2S)-glycidate with tin tetra-
chloride in carbon tetrachloride, followed by hydroly-
sis.5 The monoprotected polyamine 10 having two
quaternary ammonium salts was synthesized step-by-
step. Protection of the amine 4 with di-tert-butyl dicar-
bonate (Boc2O) and subsequent treatment of excess
amounts of 1,3-dibromopropane gave the monoammo-
nium salt 6.6 Coupling of 6 and 8, which was prepared
by protection of the amine 7 with pyrocarbonic acid
dibenzyl ester (Cbz2O), was performed by treatment
with sodium bicarbonate in ethanol at 50 ꢁC to give the
bisammonium salt 9. Subsequent removal of the Boc
group with trifluoroacetic acid (TFA) furnished the
mono-primary amine 10. Coupling of 10 with (S)-3 was
accomplished by treatment with 1-ethyl-3-(3-dimeth-
ylaminopropyl)carbodiimide hydrochloride (EDCI) and
1-hydroxybenzotriazole (HOBt) in dimethylformamide
(DMF) at room temperature to give the protected
MG30, (S)-11. Deprotection of (S)-11 with thioanisole
and trimethylsilyl trifluoromethanesulfonate in TFA
10
11
12
13
14
15
16,17
18
19
20
21,22
23
24
25
74.4
178.8
40.5
27.9
3.05, 3.17 (m)
1.23 (m)
1.37 (m)
3.15 (m)
2.98 (s)
21.7
67.1
53.2 or 53.3
62.9
3.22 (m)
2.25 (m)
3.37 (m)
3.17 (s)
19.3
63.6
53.2 or 53.3
65.0
24.8
3.46 (m)
2.12 (m)
2.97 (m)
39.5
methyl carbons on HSQC. These methyl carbons were
assumed to attach to the positively charged nitrogen
atoms from the chemical shift values. The methyl
protons H-16 and H-17 (dH 2.98) showed HMBC
correlations with C-15 and C-18 (dC 67.1 and 62.9), and
H-21 and H-22 (dH 3.17) showed HMBC correlations
with C-20 and C-23 (dC 63.6 and 65.0). These data
showed that MG30 has two dimethyldialkyl ammonium
salts in the main chain backbone (Table 1).7
Mass spectral data for MG30 are presented in Figure 1.8
The molecular weight of 223.68 [M]2þ was obtained by
ESI-Q-Tof MS, and MS/MS fragmentation patterns
clearly showed that MG30 was composed of a main
chain with two dimethyldialkyl ammonium salts acyl-
ated by an indole-3-lactyl group. Combining NMR and
MS data, the structure of MG30 was elucidated as
shown in 1.
gave the desired (S)-1 (½aꢀ +3.5 (c 0.43, H2O)). (R)-1
D
(½aꢀ )3.0 (c 0.58, H2O)) was obtained by using (R)-3
D
instead of (S)-3.
The CD spectra of the two synthetic enantiomers and
the native polyamine are shown in Figure 2.9 From these
spectra, the absolute stereostructure of the native poly-
amine is (S)-1.
1
N
H
CO H
2
CO Me
SnCl ,CCl
2
4
4
b
N
Br
N
Br
R HN
1
BocHN
OH
2. NaOH aq, THF
31%
O
N
H
4 : R =H
5 : R =Boc
1
a
6
(S)-2
(S)-3
1
O
d
f
N
X
NHCbz
N
N
X
N
NHR
4
NHR
N
2
R HN
3
N
6
(S)-3
H
OH
CF CO
3
CF CO
3 2
2
N
H
7 : R =H
2
c
9 : R =Boc
10 : R =H
X=Br
X=CF CO
2
3
e
8 : R =Cbz
11: R =Cbz
(S)-1: R =H
2
4
g
3
3
4
(R)-2
(R)-1
Scheme 1. Reagents and conditions: (a) Boc2O, THF, rt (99%); (b) dibromopropane, rt (74%); (c) Cbz2O, THF, 60 ꢁC (54%); (d) NaHCO3, EtOH,
50 ꢁC; (e) TFA, CH2Cl2, rt (2 steps 58%); (f) EDCI, HOBt, DMF, rt; (g) thioanisole, TMSOTf, TFA, 0 ꢁC (2 steps 31%).

N. Yamaji et al. / Tetrahedron Letters 45 (2004) 5371–5373
5373
between legs 2 and 3. After injection, their paralyses
occurred depending on the concentration of the poly-
amines, and most insects recovered within a few hours.
ED50s for paralysis were calculated using the program
Prism version 3.0C (Graphpad Software, Inc). The
results indicated that the respective enantiomers, (R)
and (S)-1, had similar potency.
5
4
(S)-1
3
native MG30
2
1
0
-1
-2
-3
-4
-5
Acknowledgements
(R)-1
This work was supported in part by a grant from the
Research for the Future Program from the Japanese
Society for the Promotion of Science (JSPS).
200
220
wavelength (nm)
240
260
Figure 2. CD spectrum of native MG30, (R)-1 and (S)-1 in H2O.
References and notes
1. McCormick, K. D.; Meinwald, J. J. Chem. Ecol. 1993, 19,
2411–2451.
2. Strømgaard, K.; Andersen, K.; Krogsgaard-Larsen, P.;
Jaroszewski, J. W. Mini Res. Med. Chem. 2001, 1, 317–
338.
100
synthetic-(R)-1
synthetic-(R)-1
3. Skinner, W. S.; Dennis, P. A.; Lui, A.; Carney, R. L.;
Gary, B. Toxicon 1990, 28, 541–546.
50
4. Corzo, G.; Gilles, N.; Satake, H.; Villegas, E.; Dai, L.;
Nakajima, T.; Haupt, J. FEBS Lett. 2003, 547, 43–50.
5. Entzeroth, M.; Kucnzik, T.; Jaenicke, L. Liebig Ann.
Chem. 1983, 226–230.
6. Spencer, T. A.; Onofrey, T. J.; Cann, R. O.; Russel, J. S.;
Lee, L. E.; Blanchard, D. E.; Castro, A.; Gu, P.; Jiang, G.;
Shechter, I. J. Org. Chem. 1999, 64, 807–818.
7. NMR spectra were recorded by a DMX-750 spectrometer
(Bruker Biospin, Germany) in D2O using a microcell
NMR tube (Shigemi, Japan).
0
10-2
10-1
100
µg/g insect
101
102
103
Figure 3. Dose–response curves resulting from injection of synthetic
(R)-1 and (S)-1 into crickets.
8. Mass spectra were recorded by a Q-Tof mass spectrometer
(Micromass, UK).
9. CD spectra were obtained on a JASCO-725 spectropolar-
imeter (JASCO, Japan). Data were collected at 0.1 nm
with a scan rate of 100 nm/min and a time constant of 0.5 s
at room temperature using a 0.5 cm path-length cell.
10. Escoubas, P.; Stankiewicz, M.; Takaoka, T.; Pelhate, M.;
Romi-Lebrun, R.; Wu, F. Q.; Nakajima, T. FEBS Lett.
2000, 483, 175–180.
The preliminary toxicity assay was performed with the
synthetic polyamines using crickets (Gryllus bimacula-
tus) (Fig. 3).10 The polyamines were dissolved in distilled
water, and controls were injected with water only.
Injections were made using a micro-syringe into crickets