Total synthesis of the novel spider toxin NPTX-594 from Nephila madagascariencis

Article abstract of DOI:10.1246/bcsj.74.1743

A novel spider toxin, NPTX-594, comprised of four constituents: i.e., 2,4-dihydroxyphenylacetic acid (Dhpa), asparagine, 4,8-diaza-1,12-dodecanediamine (Dada), and lysine, was chemically synthesized. The synthetic compound was completely identical with the natural product as regards ¹H NMR and mass spectra. The structure of NPTX-594 was thus determined synthetically to be N¹²-(Dhpa-Asn)-N¹-Lys-Dada, as proposed by spectrometric elucidation.

Full text of DOI:10.1246/bcsj.74.1743

c. Jpn.
© 2001 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 74, 1743–1749 (2001) 1743
e Chemical
ciety of Ja-
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Total Synthesis of the Novel Spider Toxin NPTX-594 from Nephila
madagascariencis1
Synthesis of the Novel Spider Toxin NPTX-594
T. Wakamiya et al.
Tateaki Wakamiya,* Akinori Yamamoto, Keita Kawaguchi, Tomohiko Kinoshita,
Yoshihiro Yamaguchi, Yasuhiro Itagaki,†, # Hideo Naoki,† and Terumi Nakajima†
01
43
49
Faculty of Science and Technology, Kinki University, Higashi-osaka, Osaka 577-8502
†Suntory Institute for Bioorganic Research, Shimamoto-cho, Mishima-gun, Osaka 618-0024
SJA8
09-2673
092
(Received March 15, 2001)
01
01
A novel spider toxin, NPTX-594, comprised of four constituents: i.e., 2,4-dihydroxyphenylacetic acid (Dhpa), as-
paragine, 4,8-diaza-1,12-dodecanediamine (Dada), and lysine, was chemically synthesized. The synthetic compound
was completely identical with the natural product as regards 1H NMR and mass spectra. The structure of NPTX-594 was
thus determined synthetically to be N12-(Dhpa-Asn)-N1-Lys-Dada, as proposed by spectrometric elucidation.
.5
3
.3
Spider toxins are known to be potent and specific blockers
of glutamate receptor,2 and seem to be usable as unique tools
for elucidating the mechanism of glutamate neurotransmission
in the brain. A novel acylpolyamine toxin (NPTX-594) was
isolated from the venom of a Madagascar spider (Nephila
madagascariencis) and/or a Brazilian spider (Nephila clavi-
pes).3 On the basis of the mass spectrometric analysis of natu-
ral product, the plausible structure 1 was proposed to NPTX-
594; this compound is comprised of four constituents, i.e., 2,4-
dihydroxyphenylacetic acid (Dhpa), Asn, 4,8-diaza-1,12-dode-
canediamine (Dada), and Lys (Fig. 1).4 However, because of
shortage of natural sample, synthetic study was required to
confirm the structure of this novel spider toxin, and to eluci-
date the biological activity.
tive N-alkylation method. 3-(Troc-amino)propionaldehyde (4)
prepared from 3-aminopropionaldehyde diethyl acetal (2) was
connected with 2 in the presence of NaBH3CN. The secondary
amino group newly formed was protected with the Z group to
give 4-Z-7-(Troc-amino)-4-azaheptanal diethyl acetal (5),
which was then converted into the corresponding aldehyde 6
by acid hydrolysis (Scheme 1).
In order to construct a C4-chain unit in the Dada residue, we
next required N-Boc-1,4-butanediamine (8). Although 8 can
be prepared from commercially available 1,4-butanediamine,
selective monoacylation of α,ω-diamines using general acylat-
ing reagents is often difficult and not reproducible.11c, 13 There-
fore, we employed an alternate method reported by Mattingly14
on the basis of the reduction of N-Boc-4-azidobutylamine (7)
prepared from 4-aminobutanol. Although Mattingly hydroge-
nated the azide compounds by use of 10% Pd–C as a catalyst
under pressure, we carried out the conversion of 7 into 8 with
PPh3 in THF and H2O. When the hydrogenation was carried
out using 10% Pd–C at atmospheric pressure, we observed a
very interesting result that a major product was not the primary
amine 8 but the secondary amine, i. e., (BocNHCH2CH2CH2-
CH2)2NH.15
The aldehyde derivative 6 and the amine component 8 were
connected in a manner similar to that mentioned in the prepa-
ration of 5, followed by benzyloxycarbonylation to obtain fully
protected Dada derivative 9 (Scheme 2). The other three acyl
components in the molecule of 1 were used as their active ester
derivatives for coupling with the amino groups. For this pur-
pose, Z-Lys(Z)-OSu (11)16 and Dhpa(Bzl)2-OSu (17)11c were
prepared according to known methods, and Boc-Asn-ONp (14)
was of commercial origin.
Results and Discussion
Many acylpolyamines, such as JSTX.5 NSTX.6 argiopine,7
NPTX,8, 9 and others10 have been found in the venom glands of
several kinds of spiders, such as Nephila clavata, Nephila
maculata, Argiope lobata, and Nephilengys borbonica. These
acylpolyamines are generally classified into six types based on
their structural features, as shown in Fig. 2.9c NPTX-594 is a
novel compound belonging to type B, i. e., the Lys residue in-
stead of the putreanine (8-amino-4-azaoctanoic acid) unit or
the Arg residue in known nephilatoxins binds to the 1-amino
group of Dada.
In the synthesis of acylpolyamine-type spider toxins, we
must find an effective strategy to introduce the acyl groups into
both the terminal amino groups of the polyamine residue.11,12
In the present study, the Lys-Dada bond was first formed, and
then the Dhpa-Asn part was introduced into the other amino
terminus of the Dada residue.
The connection of four components was carried out as
shown in Scheme 3. At first, the Troc group in the Dada deriv-
ative 9 was removed with Zn/AcOH, and the freed 1-amino
On the basis of the synthetic plan mentioned above, the
polyamine part was first prepared by application of the reduc-
group was coupled with 11. Next, the Boc group of the prod-
uct 12 was removed in the usual way, and 14 was coupled with
the 12-amino group of the Dada residue in the compound 13 to
# Department of Chemistry, Columbia University, 3000 Broad-
way, New York, NY10027, USA

1744 Bull. Chem. Soc. Jpn., 74, No. 9 (2001)
Synthesis of the Novel Spider Toxin NPTX-594
give 15. The Boc group of 15 was removed again, and 17 was
coupled with the α-amino group of the Asn residue to obtain
the fully protected NPTX-594 (18). Finally, all protecting
groups of 18 were removed by catalytic hydrogenation, and the
crude product was purified by preparative RPHPLC. The thus-
obtained synthetic compound 1 was completely identical with
natural product in respects of 1H NMR (Fig. 3) and FAB-MS/
MS17 spectra. As a result of the present work, we determined
Fig. 1. Proposed structure of NPTX-594.
Fig. 2. Classification of acylpolyamine-type spider toxins. In some cases, putreanine unit, –{COCH2CH2NH(CH2)4NH}nH, is
replaced by the arginine residue.
Scheme 1.

T. Wakamiya et al.
Bull. Chem. Soc. Jpn., 74, No. 9 (2001) 1745
Scheme 2.
Scheme 3.
the structure of NPTX-594 synthetically.
Mercury 300 (300 MHz, Varian Co., Ltd., USA), or Bruker
DMX-500 (500 MHz, Bruker Co., Ltd., Germany) spectrome-
ter. The chemical shifts are given in δ values from TMS used
as the internal standard. HRFAB-MS was carried out on a
JEOL JMS-700 TKM mass spectrometer (JEOL Co., Ltd., To-
kyo, Japan). Silica-gel column chromatography was carried
out with Merck silica gel 60 (Art. 7734, 70–230 mesh).
RPHPLC was performed on Cosmosil 5C18-AR (4.6 × 150
mm, Nacalai Tesque, Kyoto, Japan) for analysis and YMC-
Pack ODS-AM (20 × 250 mm, YMC Co., Ltd., Kyoto, Japan)
for preparative purification. 3-Aminopropionaldehyde diethyl
acetal was purchased from Tokyo Chemical Industry Co., Ltd.,
Tokyo, Japan. Boc-Asn-ONp was purchased from Peptide In-
stitute, Inc., Osaka, Japan.
Although we are not able to compare the biological activity
of the synthetic compound with that of natural one due to
shortage of natural sample, NPTX-594 shows sufficient neuro-
toxicity, i. e., the ED50 value to paralyze crickets (Grillus bi-
maculatus) was of 0.38–0.30 µg/g insect.18 The syntheses of
several analogs of NPTX-594 are being currently undertaken
from the standpoint of the structure-activity relationship study
of type B nephilatoxins.
Experimental
The melting point is uncorrected and was measured by Ya-
naco MP-J3 (Yanaco Co., Ltd., Kyoto, Japan). Specific rota-
tions were measured by DIP-140 (Japan Spectroscopic Co.,
Ltd., Tokyo, Japan). 1H NMR spectra were recorded on JEOL
GX-270 (270 MHz, JEOL Co., Ltd., Tokyo, Japan), Varian
3-(2,2,2-Trichloroethoxycarbonylamino)propionalde-
hyde (4). To a solution of 3-aminopropionaldehyde diethyl

1746 Bull. Chem. Soc. Jpn., 74, No. 9 (2001)
Synthesis of the Novel Spider Toxin NPTX-594
Fig. 3. 500 MHz 1H NMR spectra of NPTX-594 in D2O: (a) synthetic compound and (b) natural product. Peaks asterisked are of
HDO.
acetal (2) (4.55 g, 30.9 mmol) in THF (100 mL) were added
TEA (3.44 g, 34.0 mmol) and 2,2,2-trichloroethoxycarbonyl
chloride (7.20 g, 34.0 mmol). The reaction mixture was stirred
for 10 min at 0 °C and additionally for 2 h at r. t. The precipi-
tated insoluble material was filtered off. The filtrate was con-
centrated in vacuo, and the residue was dissolved in AcOEt
(100 mL). The solution was washed with 10% citric acid (50
mL × 3), saturated aqueous NaHCO3 (50 mL × 3), and brine
(50 mL × 3). The organic layer was dried over anhydrous
MgSO4, and then the solvent was removed in vacuo to give 3-
(2,2,2-trichloroethoxycarbonylamino)propionaldehyde diethyl
acetal (3) as a colorless oil (9.96 g, quant.). To a solution of
the thus-obtained compound 3 in acetone (200 mL) was added
1 M HCl (10 mL). The reaction mixture was heated under re-
flux for 30 min. The solvent was removed in vacuo, and the
residue was dissolved in diethyl ether (200 mL). The solution
was washed with water (50 mL × 3), saturated aqueous
NaHCO3 (50 mL × 3), and brine (50 mL × 3). The organic
layer was dried over anhydrous MgSO4, and then the solvent
was removed in vacuo to give 4 as a colorless oil (7.23 g,
94.1%). The thus-obtained crude product was used for the
next reaction without further purification.
4-Benzyloxycarbonyl-7-(2,2,2-trichloroethoxycarbonyl-
amino)-4-azaheptanal Diethyl Acetal (5). To a solution of
compound 4 (4.23 g, 17.0 mmol) in MeOH (200 mL) were
added 2 (3.00 g, 20.4 mmol) and AcOH (2.93 mL, 51.0 mmol),
and the reaction mixture was stirred for 1 h at r. t. To the solu-
tion was added dropwise NaBH3CN (2.14 g, 34.0 mmol) in
MeOH (20 mL) at r. t. over a 30-minute period, and the reac-
tion mixture was additionally stirred overnight. The solvent
was removed in vacuo, and the residue was dissolved in AcOEt
(200 mL). The solution was washed with 10% citric acid (50
mL × 3), saturated aqueous NaHCO3 (50 mL × 3), and brine
(50 mL × 3). The organic layer was dried over anhydrous
MgSO4, and then the solvent was removed in vacuo. To a solu-
tion of the residue in THF (100 mL) was added TEA (1.89 g,
18.7 mmol) under cooling at 0 °C. To this solution was added
dropwise ZCl (3.19 g, 18.7 mmol) in THF (40 mL) at 0 °C
over a 15-minute period, and the reaction mixture was addi-
tionally stirred for 3 h at r. t. The solvent was removed in vac-
uo, and the residue was dissolved in AcOEt (100 mL). The so-
lution was washed with 10% citric acid (30 mL × 3), saturated
aqueous NaHCO3 (30 mL × 3), and brine (30 mL × 3). The
organic layer was dried over anhydrous MgSO4, and then the
solvent was removed in vacuo. The crude product was purified
by silica-gel column chromatography (180 g, 2.0 × 70 cm,
benzene:AcOEt = 5:1). The fractions containing the desired
product were combined, and concentrated in vacuo to give 5 as
1
a colorless oil (4.62 g, 52.9%). H NMR (300 MHz, CDCl3) δ
1.23 (6H, t, CH3 × 2), 1.88 (4H, m, CH2 × 2), 3.18–3.75
(12H, m, CON–CH2 × 3 and OCH2 × 2), 4.50 (1H, t, O–CH–
O), 4.73 (2H, s, CH2/Troc), 5.17 (2H, dd, PhCH2) and 7.35
(5H, s, Ph); HRFAB-MS: found m/z 513.1309 (M+H)+ (calcd
for C21H31Cl3N2O6 + H: 513.1326).
4-Benzyloxycarbonyl-7-(2,2,2-trichloroethoxycarbonyl-
amino)-4-azaheptanal (6). To a solution of compound 5
(3.35 g, 6.52 mmol) in acetone (100 mL) was added 1 M HCl
(6 mL), and the reaction mixture was heated under reflux for
30 min. The solvent was removed in vacuo, and the residue
was dissolved in diethyl ether (100 mL). The solution was
washed with water (20 mL × 3), saturated aqueous NaHCO3
(20 mL × 3), and brine (20 mL × 3). The organic layer was
dried over anhydrous MgSO4, and then the solvent was con-
centrated in vacuo to give 6 as a colorless oil (2.87 g, quant.).
The thus-obtained crude product was used for the next reaction
without further purification.

T. Wakamiya et al.
Bull. Chem. Soc. Jpn., 74, No. 9 (2001) 1747
N-t-Butoxycarbonyl-1,4-butanediamine (8). To a solu-
tion of N-Boc-4-azidobutylamine (7)14 (22.6 g, 106 mmol) in
THF (50 mL) were added PPh3 (30.4 g, 116 mmol) and water
(5 mL) under cooling at 0 °C. The mixture was stirred for 2 h
at 0 °C and for 21 h at r. t. The solvent was removed in vacuo,
and the residue was treated with 10% citric acid (100 mL) and
AcOEt (30 mL). The aqueous layer separated was basified
with 2 M NaOH, and the alkaline solution was extracted with
CHCl3 (50 mL × 3). The extract was dried over anhydrous
MgSO4, and the solvent was then removed in vacuo to give 8
were added TEA (0.184 g, 1.82 mmol) and Z-Lys(Z)-OSu
(11)16 (0.930 g, 1.82 mmol). The reaction mixture was stirred
for 24 h at r. t. The solvent was removed in vacuo, and the res-
idue was dissolved in AcOEt (100 mL). The solution was
washed with 10% citric acid (30 mL × 3), saturated aqueous
NaHCO3 (30 mL × 3), and brine (30 mL × 3). The organic
layer was dried over anhydrous MgSO4, and then the solvent
was removed in vacuo. The crude product was purified by sili-
ca-gel column chromatography (40g, 3.0
× 40 cm,
CHCl3:MeOH = 100:1). The fractions containing the desired
product were combined and concentrated in vacuo to give 12
1
as a colorless oil (18.3 g, 92.1%). H NMR (270 MHz, CDCl3)
1
δ 1.44 (9H, s, (CH3)3C), 1.51–1.71 (4H, m, CH2 × 2), 2.76
(2H, t, NH2CH2) and 3.14 (2H, m, BocNHCH2); HRFAB-MS:
found m/z 189.1604 (M + H)+ (calcd for C9H20N2O2 + H:
189.1594).
as a colorless oil (1.30 g, 81.8%). H NMR (300 MHz, CDCl3)
δ 1.44 (9H, s, (CH3)3C), 1.19–1.90 (6H, m, β-, γ-, and δ-CH2/
Lys; 8H, m, CH2/Dada × 4), 2.88–3.49 (14H, m, CON–CH2 ×
6 and ε-CH2/Lys), 4.13 (1H, m, α-CH/Lys), 5.07 (8H, dd,
PhCH2 × 4), 7.32 (20H, s, Ph × 4); HRFAB-MS: found m/z
967.5144 (M+H)+ (calcd for C53H70N6O11 + H: 967.5151);
[α]D19 −2.7° (c 1.0, MeOH).
4,8-Bis(benzyloxycarbonyl)-N12-t-butoxycarbonyl-N1-
(2,2,2-trichloroethoxycarbonyl)-4,8-diaza-1,12-dodecane-
diamine (9). To a solution of compound 6 (1.50 g, 3.41
mmol) in MeOH (100 mL) were added 8 (0.770 g, 4.09 mmol)
in MeOH (10 mL) and AcOH (0.584 mL, 10.2 mmol). The re-
action mixture was stirred for 1 h at r. t., and then NaBH3CN
(0.430 g, 6.82 mmol) in MeOH (10 mL) was added dropwise
at r. t. over a 30-minute period; the reaction mixture was next
stirred overnight. The solvent was removed in vacuo, and the
residue was dissolved in AcOEt (100 mL). The solution was
washed with 10% citric acid (30 mL × 3), saturated aqueous
NaHCO3 (30 mL × 3), and brine (30 mL × 3). The organic
layer was dried over anhydrous MgSO4, and then the solvent
was removed in vacuo. To a solution of the oily residue in
THF (100 mL) was added TEA (0.387 g, 3.75 mmol) at 0 °C.
To the solution was added dropwise ZCl (0.640 g, 3.75 mmol)
in THF (10 mL) at 0 °C over a 15-minute period, and the reac-
tion mixture was additionally stirred for 3 h at r. t. The solvent
was removed in vacuo, and the residue was dissolved in AcOEt
(100 mL). The solution was washed with 10% citric acid (30
mL × 3), saturated aqueous NaHCO3 (30 mL × 3), and brine
(30 mL × 3). The organic layer was dried over anhydrous
MgSO4, and then the solvent removed in vacuo. The crude
product was purified by silica-gel column chromatography
(180 g, 2.0 × 70 cm, benzene:AcOEt = 6:1). The fractions
containing the desired product were combined and evaporated
in vacuo to give 9 as a colorless oil (1.22 g, 48.0%). 1H NMR
(300 MHz, CDCl3) δ 1.44 (9H, s, (CH)3C), 1.29–1.88 (8H, m,
CH2/Dada × 4), 2.80–3.40 (12H, m, CON–CH2 × 6), 4.72
(2H, s, CH2/Troc), 5.11 (4H, dd, PhCH2 × 2), 7.37 (10H, s, Ph
× 2 ); HRFAB-MS: found m/z 745.2586 (M+H)+ (calcd for
C34H47Cl3N4O8 + H: 745.2538).
4,8-Bis(benzyloxycarbonyl)-N1-[Nα,Nε-bis(benzyloxycar-
bonyl)lysyl]-N12-[Nα-(t-butoxycarbonyl)asparaginyl]-4,8-
diaza-1,12-dodecanediamine (15). Compound 12 (1.30 g,
1.35 mmol) was dissolved in CH2Cl2 (24 mL) and TFA (6 mL).
The solution was stirred for 30 min at r. t. and then concentrat-
ed in vacuo. The residue was dissolved in AcOEt (100 mL),
and the solution was washed with saturated aqueous NaHCO3
(30 mL × 3) and brine (30 mL × 3). The organic layer was
dried over anhydrous MgSO4, and then the solvent was re-
moved in vacuo to give 4,8-bis(benzyloxycarbonyl)-N1-[Nα,Nε-
bis (benzyloxycarbonyl) lysyl ] - 4,8 - diaza -1,12-dodecanedi-
amine (13) as a colorless oil (1.16 g, 99.1%). To a solution of
the thus-obtained compound 13 in DMF (30 mL) were added
Nα-(t-butoxycarbonyl)asparagine p-nitrophenyl ester (14)
(0.522 g, 1.47 mmol) and TEA (0.148 g, 1.47 mmol), and the
reaction mixture was stirred for 3 h at r. t. The solvent was re-
moved in vacuo, and the residue was dissolved in AcOEt (100
mL). The solution was washed with 10% citric acid (20 mL ×
3), saturated aqueous NaHCO3 (20 mL × 3), and brine (20 mL
× 3). The organic layer was dried over anhydrous MgSO4,
and then the solvent was concentrated in vacuo. The crude
product was purified by silica-gel column chromatography (50
g, 3.0 × 20 cm, CHCl3:MeOH = 20:1). The fractions con-
taining the desired product were combined and concentrated in
vacuo to give 15 as a colorless oil (1.32 g, 91.0%). 1H NMR
(300 MHz, CDCl3) δ 1.44 (9H, s, (CH3)3C), 1.22–1.90 (6H, m,
β-, γ-, and δ-CH2/Lys; 8H, m, CH2/Dada × 4), 2.46–2.91 (2H,
m, β-CH2/Asn), 2.92–3.40 (14H, m, CO–N–CH2 × 6 and ε-
CH2/Lys), 4.10 (1H, m, α-CH/Lys), 4.40 (1H, m, α-CH/Asn),
5.07 (8H, dd, PhCH2 × 4), 7.32 (20H, s, Ph × 4); HRFAB-
MS: found m/z 1081.5625 (M+H)+ (calcd for C57H76N8O13
+H: 1081.5610); [α]D19 −3.3° (c 1.0, MeOH).
4,8-Bis(benzyloxycarbonyl)-N1-[Nα,Nε-bis(benzyloxycar-
bonyl)lysyl]-N12-t-butoxycarbonyl-4,8-diaza-1,12-dodec-
anediamine (12). To a solution of compound 9 (1.23 g,1.65
mmol) in AcOH (36 mL) and water (4 mL) was added zinc
dust (3.24 g, 49.5 mmol). The suspension was stirred for 4 h at
r. t., and then insoluble materials were filtered off. The filtrate
was concentrated in vacuo, and the residue was dissolved in di-
ethyl ether (50 mL). The solution was extracted with water (10
mL × 5), and the extract was lyophilized to obtain 4,8-
bis(benzyloxycarbonyl)-N12-t-butoxycarbonyl-4,8-diaza-1,12-
dodecanediamine acetate (10) as a colorless powdery sub-
stance. To a solution of the thus-obtained 10 in DMF (20 mL)
4,8-Bis(benzyloxycarbonyl)-N1-[Nα,Nε-bis(benzyloxycar-
bonyl)lysyl]-N12-{Nα-[2,4-bis(benzyloxy)phenylacetyl]as-
paraginyl}-4,8-diaza-1,12-dodecanediamine (18). A solu-
tion of compound 15 (1.32 g, 1.22 mmol) in 20% TFA/CH2Cl2
(20 mL) was stirred for 30 min at r. t. The solvent was re-
moved in vacuo, and the residue was dissolved in AcOEt (100
mL). The solution was washed with saturated aqueous
NaHCO3 (30 mL × 3) and brine (30 mL × 3). The organic
layer was dried over anhydrous MgSO4, and then the solvent

1748 Bull. Chem. Soc. Jpn., 74, No. 9 (2001)
Synthesis of the Novel Spider Toxin NPTX-594
was concentrated in vacuo to give 4,8-bis(benzyloxycarbonyl)-
N1-[Nα,Nε-bis(benzyloxycarbonyl)lysyl]-N12-asparaginyl-4,8-
diaza-1,12-dodecanediamine (16) as a colorless oil (1.11 g,
92.5%).
This work was partly supported by a glant from the Re-
search for the Future Program from the Japan Society for the
Promotion of Science (JSPS), and a SUNBOR GRANT from
Suntory Institute for Bioorganic Research.
To a solution of a part of compound 16 (0.214 g, 0.219
mmol) in DMF (10 mL) were added 2,4-bis(benzyloxy)phen-
ylacetic acid succinimidyl ester11c (17) (0.107 g, 0.241 mmol)
and TEA (24.4 mg, 0.241 mmol), and the reaction mixture was
stirred for 3 h at r. t. The solvent was removed in vacuo, and
the residue was dissolved in AcOEt (50 mL). The solution was
washed with 10% citric acid (10 mL × 3), saturated aqueous
NaHCO3 (10 mL × 3), and brine (10 mL × 3). The organic
layer was dried over anhydrous MgSO4, and then the solvent
was removed in vacuo. The crude product was purified by sili-
ca-gel column chromatography (10 g, 1.0 × 20 cm, CHCl3:
MeOH = 100:1). The fractions containing desired product
were combined and concentrated in vacuo. The residue was
triturated with hexane to obtain 18 as colorless crystals, and
the thus-obtained crude product was recrystallized from dieth-
References
1
A part of this work was presented at the 25th European
Peptide Symposium: T. Wakamiya, A. Yamamoto, K. Kawaguchi,
Y. Yamaguchi, T. Fujita, M. Hisada, Y. Kan, Y. Itagaki, T.
Nakajima, and M. Andriantsiferana, “Peptides 1998,” ed by S.
Bajusz and F. Hudecz, Akadémiai Kiadó, Budapest (1999), p. 462.
2
(1982).
3
N. Kawai, A. Niwa, and T. Abe, Brain Res., 247, 169
NPTX-594 indicates a nephilatoxin whose molecular
weight is 594. Details about the isolation and structure determina-
tion will be reported soon in Natural Toxins.
4
Abbreviations according to IUPAC-IUB commission, Eur.
J. Biochem., 138, 9 (1984), are used. Asn: L-asparagine; Boc: t-
butoxycarbonyl; Boc2O: di-t-butyl dicarbonate; Bzl: benzyl;
DCHA: dicyclohexylamine; DMF: N,N-dimethylformamide; DM-
SO: dimethyl sulfoxide; EDC: 1-(3-dimethylaminopropyl)-3-eth-
ylcarbodiimide; HRFAB-MS: high resolution fast atom bombard-
ment mass spectrometry; HOBt: 1-hydroxybenzotriazole; HOSu:
N-hydroxysuccinimide; Lys: L-lysine; Ms: methanesulfonyl; Np:
p-nitrophenyl (or 4-nitrophenyl); RPHPLC: reversed-phase high-
performance liquid chromatography; TEA: triethylamine; TFA:
trifluoroacetic acid; THF: tetrahydrofuran; Troc: 2,2,2-trichloro-
ethoxycarbonyl; TrocCl: 2,2,2-trichloroethoxycarbonyl chloride;
Z: benzyloxycarbonyl; ZCl: benzyloxycarbonyl chloride.
1
yl ether and hexane (0.181 g, 63.3%). Mp 130–134 °C; H
NMR (500 MHz, DMSO-d6) δ 1.20–1.72 (6H, m, β-, γ-, and δ-
CH2/Lys; 8H, m, CH2/Dada × 4), 2.39 and 2.44 (each 1H, dd,
β-CH2/Asn), 2.90–3.21 (14H, m, CO–N–CH2/Dada × 6 and ε-
CH2/Lys), 3.42 (2H, s, PhCH2/Dhpa), 3.89 (1H, m, α-CH/Lys),
4.51 (1H, m, α-CH/Asn), 4.96–5.06 (8H, PhCH2/Z × 4), 5.04
and 5.08 (each 2H, s, PhCH2/Bzl), 6.53 (1H, dd, C3-Ph /Dhpa),
6.68 (1H, d, C5-Ph /Dhpa), 7.08 (1H, d, C6-Ph /Dhpa), and
7.24–7.38 (30H, Ph/Z and Bzl × 6); HRFAB-MS: found m/z
1311.6325 (M+H)+ (calcd for C74H86N8O14 + H: 1311.6342);
[α]D19 −3.5° (c 1.0, MeOH).
5
a) Y. Aramaki, T. Yasuhara, K. Shimazaki, A. Kawai, and
N12-[Nα-(2,4-Dihydroxyphenylacetyl)asparaginyl]-N1-ly-
syl-4,8-diaza-1,12-dodecanediamine (NPTX-594) (1). To a
solution of compound 18 (50.0 mg, 0.0382 mmol) in MeOH
(10 mL) and AcOH (20 mL) was added Pd black (50 mg). The
reaction mixture was stirred under an atmosphere of hydrogen
for 2 h at r. t. The catalyst was filtered off, and the filtrate was
concentrated in vacuo. The crude product was dissolved in
water and filtered through Millipore® filter (Millipore Corp.,
Type: HA, pore size: 0.45 µm); the filtrate was then lyo-
philized. The crude product was finally purified by preparative
RPHPLC (10–40% CH3CN–0.1% TFA/8.0 mL min−1). The
fractions containing the desired compound were combined and
lyophilized. The thus-obtained powdery substance was dis-
solved in 0.1 M HCl (10 mL), and the solution was lyophilized
again to obtain 1 as hydrochloride19 (16.7 mg, 59.0%). 1H
NMR (500 MHz, D2O; auto-reference condition: HDO = 4.75
ppm) δ 1.37 (2H, m, γ-CH2/Lys), 1.40–1 52 (4H, m, C10H2 and
C11H2/Dada), 1.62 (2H, m, δ-CH2 /Lys), 1.83 (2H, m, β-CH2/
Lys), 1.85 (2H, m, C2H2/Dada), 1.97 (2H, m, C6H2/Dada), 2.64
and 2.70 (each 1H, dd, β-CH2 /Asn), 2.88 (2H, m, C9H2/Dada),
2.92 (2H, m, ε-CH2/Lys), 2.96 and 3.03 (each 2H, m, C5H2,
C7H2/Dada), 3.01 (2H, m, C3H2/Dada), 3.09 and 3.21 (each
1H, m, C12H2/Dada), 3.21 and 3.31 (each 1H, m, C1H2/Dada),
3.45 (2H, m, CH2/Dhpa), 3.89 (1H, m, α-CH/Lys), 4.49 (1H,
m, α-CH/Asn), 6.37 (1H, s, C3-Ph/Dhpa), 6.38 (1H, d, C5-Ph/
Dhpa), and 7.02 (1H, d, C6-Ph/Dhpa); FAB-MS: found m/z
595.3946 (M+H)+ (calcd for C28H50N8O6 + H: 595.3935);
[α]D19 −7.9° (c 1.0, H2O).
T. Nakajima, Biomed. Res., 8, 241 (1987). b) T. Toki, T. Yasuhara,
Y. Aramaki, Y. Hashimoto, K. Shudo, A. Kawai, and T. Nakajima,
Jpn. J. Saint. Zool., 41, 9 (1990).
6
a) Y. Aramaki, T. Yasuhara, T. Higashijima, T. Miwa, A.
Kawai, and T. Nakajima, Proc. Jpn. Acad., 62 (B), 359 (1986). b)
Y. Aramaki, T. Yasuhara, T. Higashijima, T. Miwa, A. Kawai, and
T. Nakajima, Biomed. Res., 8, 167 (1987).
7
E. V. Grishin, T. M. Volkova, and A. S. Arsesniev, Toxicon,
27, 541 (1989).
8
a) T. Toki, T. Yasuhara, Y. Aramaki, N. Kawai, and T.
Nakajima, Biomed. Res., 9, 75 (1988). b) T. Toki, T. Yasuhara, Y.
Aramaki, K. Osawa, A. Miwa, N. Kawai, and T. Nakajima,
Biomed. Res., 9, 421 (1988).
9
a) Y. Itagaki, T. Fujita, H. Naoki, T. Yasuhara, M.
Andriantsiferana, and T. Nakajima, Natural Toxins, 5, 1 (1977).
b) M. S. Palma, Y. Itagaki, T. Fujita, M. Hisada, H. Naoki, and T.
Nakajima, Natural Toxins, 5, 47 (1977). c) M. Hisada, T. Fujita,
H. Naoki, Y. Itagaki, H. Irie, M. Miyashita, and T. Nakajima, Tox-
icon, 36, 1115 (1998).
10 a) V. J. Jasys, P. R. Kelbaugh, D. M. Nason, D. Phillips, K.
J. Rosnack, N. A. Saccomano, J. G. Stroh, and R. A. Volkmann, J.
Am. Chem. Soc., 112, 6696 (1990). b) G. B. Quistad, S.
Suwanurupha, M. A. Jarema, M. J. Shapiro, W. S. Skinner, G. C.
Jamieson, A. Lui, and E. W. Fu, Biochem. Biophys. Res. Com-
mun., 169, 51 (1990). c) G. B. Quistad, C. C. Reuter, W. S.
Skinner, P. A. Dennis, S. Suwanurupha, and E. W. Fu, Toxicon, 29,
329 (1991). d) K. D. McCormich and J. Meinwald, J. Chem.
Ecol., 19, 2411 (1993). e) W. S. Skinner, P. A. Dennis, A. Lui, R.
L. Carney, and G. B. Quistad. Toxicon, 28, 541 (1990).
11 a) T. Teshima, T. Wakamiya, Y. Aramaki, T. Nakajima, N.

T. Wakamiya et al.
Bull. Chem. Soc. Jpn., 74, No. 9 (2001) 1749
Kawai, and T, Shiba, Tetrahedron Lett., 28, 3509 (1987). b) T.
Teshima, T. Matsumoto, M. Miyagawa, T. Wakamiya, T, Shiba, N.
Narai, M. Yoshioka, and T. Nakajima, Tetrahedron, 46, 3819
(1990). c) T. Teshima, T. Matsumoto, T. Wakamiya, T, Shiba, Y.
Aramaki, T. Nakajima, and N. Kawai, Tetrahedron, 47, 3305
(1991). d) T. Chiba, M. Akizawa, M. Matsukawa, N. Nishi, N.
Kawai, and M. Yoshioka, Chem. Pharm. Bull., 44, 972 (1996).
12 Recentry, Miyashita et al. developed the azide strategy for
the preparation of polyamine parts. The synthesis of NPTXs
based on this strategy was reported in the following paper: H.
Saito, E. Yuri, M. Miyazawa, Y. Itagaki, T. Nakajima, and M.
Miyashita, Tetrahedron Lett., 39, 6497 (1998). Many references
related to the synthesis of other spider toxins are also cited in this
paper.
Kusumoto, H. Kobayashi, Y. Sai, I. Tamai, and A. Tsuji, Bull.
Chem. Soc. Jpn., 71, 699 (1998).
14 P. G. Mattingly, Synthesis, 1990, 366.
15 We obtained similar result in the case of hydrogenation of
BocNH(CH2)5N3 or THP-O(CH2)8N3, and the details will be re-
ported elsewhere.
16 F. Chillemi, Gazz. Chim. Ital., 93, 1079 (1963).
17 Although the data are not shown, FAB-MS/MS spectrum
of synthetic compound coincided completely in all mass field with
that of natural one; sodium attached molecular ion, (M + Na)+,
was used for the measurement.
18 Details about the biological activity of NPTX-594 and its
analogs will be reported soon elsewhere.
19 Although elemental analysis was not carried out, we as-
sume that the synthetic compound is obtained as tetrahydrochlo-
ride.
13 a) J. B. Hansen, U. E. Nielson, and O. Buchrdt, Synthesis,
1982, 404. b) G. L. Stahl, R. Walter, and C. W. J. Smith, J. Org.
Chem., 43, 228 (1978). c) T. Wakamiya, M. Kamata, S.