Brevisulcenal-F: A polycyclic ether toxin associated with massive fish-kills in New Zealand

Article abstract of DOI:10.1021/ja212116q

A novel marine toxin, brevisulcenal-F (KBT-F, from karenia brevisulcata toxin) was isolated from the dinoflagellate Karenia brevisulcata. A red tide of K. brevisulcata in Wellington Harbour, New Zealand, in 1998 was extremely toxic to fish and marine invertebrates and also caused respiratory distress in harbor bystanders. An extract of K. brevisulcata showed potent mouse lethality and cytotoxicity, and laboratory cultures of K. brevisulcata produced a range of novel lipid-soluble toxins. A lipid soluble toxin, KBT-F, was isolated from bulk cultures by using various column chromatographies. Chemical investigations showed that KBT-F has the molecular formula C₁₀₇H₁₆₀O ₃₈ and a complex polycyclic ether nature. NMR and MS/MS analyses revealed the complete structure for KBT-F, which is characterized by a ladder-frame polyether scaffold, a 2-methylbut-2-enal terminus, and an unusual substituted dihydrofuran at the other terminus. The main section of the molecule has 17 contiguous 6- and 7-membered ether rings. The LD₅₀ (mouse i.p.) for KBT-F was 0.032 mg/kg.

Full text of DOI:10.1021/ja212116q

Article
pubs.acs.org/JACS
Brevisulcenal-F: A Polycyclic Ether Toxin Associated with Massive
Fish-kills in New Zealand
Yuka Hamamoto,† Kazuo Tachibana,† Patrick T. Holland,‡ Feng Shi,§ Veronica Beuzenberg,‡
Yoshiyuki Itoh,∥ and Masayuki Satake*,†
†Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
‡Cawthron Institute, Private Bag 2, Nelson 7010, New Zealand
§Lincoln University, Lincoln, Canterbury 7647, New Zealand
∥MS Business Unit, JEOL Ltd., 3-1-2 Musashino, Akishima, Tokyo 196-8558, Japan
S
* Supporting Information
ABSTRACT: A novel marine toxin, brevisulcenal-F (KBT-F,
from karenia brevisulcata toxin) was isolated from the
dinoflagellate Karenia brevisulcata. A red tide of K. brevisulcata
in Wellington Harbour, New Zealand, in 1998 was extremely
toxic to fish and marine invertebrates and also caused
respiratory distress in harbor bystanders. An extract of K.
brevisulcata showed potent mouse lethality and cytotoxicity,
and laboratory cultures of K. brevisulcata produced a range of
novel lipid-soluble toxins. A lipid soluble toxin, KBT-F, was isolated from bulk cultures by using various column
chromatographies. Chemical investigations showed that KBT-F has the molecular formula C107H160O38 and a complex polycyclic
ether nature. NMR and MS/MS analyses revealed the complete structure for KBT-F, which is characterized by a ladder-frame
polyether scaffold, a 2-methylbut-2-enal terminus, and an unusual substituted dihydrofuran at the other terminus. The main
section of the molecule has 17 contiguous 6- and 7-membered ether rings. The LD50 (mouse i.p.) for KBT-F was 0.032 mg/kg.
spectra derived from the high molecular weights have
hampered elucidation of their structures. In this paper, we
report the structure of brevisulcenal-F (KBT-F, 1) which is one
of the most abundant toxins in K. brevisulcata.
INTRODUCTION
■
A widespread bloom of a red tide dinoflagellate was observed
on the central and southern east coast of the North Island of
New Zealand from January to March 1998.1 The red tide
resulted in massive kills of fish and invertebrates in Wellington
Harbour. Over 500 cases of human respiratory distress were
reported during the bloom with symptoms including a dry
cough, severe sore throat, runny nose, skin and eye irritations,
severe headaches, and facial sun-burn sensations. The causative
dinoflagellate was identified as the new algal species Karenia
brevisulcata.2 This species is similar to K. brevis and K.
mikimotoi, which produce brevetoxins3 and gymnocins,4
respectively. These toxins have structures characterized by a
trans-fused polycyclic ether ring backbone with an unsaturated
aldehyde terminus.
The cell extract from the K. brevisulcata exhibited strong
mouse lethality and cytotoxicity,5 and was partitioned between
chloroform and aqueous methanol under neutral and acidic
conditions.1 Each fraction contained different toxins. Lipophilic
toxins demonstrated strong mouse lethality and cytotoxicity,
and they were named brevisulcenals (initially called karenia
RESULTS
■
Extraction and Isolation of Brevisulcenal-F (1).
Brevisulcenals (KBTs) were extracted from mature cultures
using resin. Cells were lysed by addition of acetone. After
stirring for 1 h, the cultures were diluted with water and passed
through HP20 resin. Brevisulcenals were recovered with
acetone from the resin. The crude extract was dissolved in
55% MeOH with pH 7.2 phosphate buffer, and the lipophilic
toxins were extracted with CHCl3. The CHCl3 extract was
chromatographed on a diol−silica column with stepwise elution
and guided by cytotoxicity assay against mouse leukemia P388
cells. Final purification of KBTs was by two stages of reversed
phase chromatography using isocratic elution with 90%
MeOH/H2O followed by 85% MeOH/H2O and monitored
by UV absorbance at 230 nm. From 1450 L cultures, 3.1 mg of
1 was isolated. Similarly, 1.3 mg of 13C enriched 1 was isolated
from 250 L cultures with added 13C-NaHCO3 at about 35 mg/
L.
1
brevisulcata toxins (KBTs) in ref 1). Their H NMR spectra
contained large numbers of protons connected to oxygenated
carbons, and their sodiated molecular cations in MALDI MS
were observed at m/z over 2000. These observations indicated
that KBTs were large polycyclic ethers analogous to maitotoxin
(MTX)6 and prymnesins.7 However, their complex NMR
Received: January 6, 2012
Published: February 29, 2012
© 2012 American Chemical Society
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Figure 1. Structure and NMR interpretation of brevisulcenal-F (1). Bold lines indicated connectivities elucidated on the basis of the 1H−1H COSY
and TOCSY data. Double-headed arrows show the important NOE correlations observed in the NOESY spectra of 1. Broken lines depicted the key
HMBC correlations of 1.
Spectroscopic Measurements of Brevisulcenal-F. A
UV absorption maximum at 227 nm indicated that KBT-F
possessed a conjugated system in the molecule. A sodium
adduct ion peak in the positive ion MALDI mass spectrum was
observed at m/z 2076; the strong positive ion peak suggested
KBT-F does not have sulfate groups. The MALDI-SpiralTOF
accurate mass8 and 1H and 13C NMR spectra indicated that the
molecular formula of KBT-F was C107H160O38 ([M + Na]+
2076.0476, calcd 2076.0480).
and TOCSY spectra led to identification of spin systems
representing H-3 to H-7, H-9 to H-12, H-14 to H-15, H-17 to
H-18, H-20 to H-26, H-28 to H-30, H-32 to H-33, H-35 to H-
36, H-38 to H-40, H-42 to H-54, H-56 to H-65, H-66 to H-93,
and H-94 to Me-95 (Figure 1). A 2-methylbut-2-enal side
chain, originally suggested by the UV data, was confirmed by
COSY correlations, including a long-range correlation from
Me-96 to H-3 and H-3 to H-4, and by HMBC correlations
from Me-96 to C-1, C-2, and C-3. Although interproton
couplings between H-65 and H-66 and between H-93 and H-94
were not observed in the COSY spectra, the HMBC
correlations from H-67 to C-65 and from Me-107 to C-94
confirmed the carbon connections C-65−C-66 and C-93−C-
94, respectively. Other carbon connectivity was interrupted by
the oxygenated quaternary carbons bearing the methyls, so all
the partial structures were assembled by HMBC experiments.
Long range correlations from a methyl to the vicinal quaternary
carbon and neighboring carbons in the HMBC spectra made it
possible to trace the carbon skeleton from C-1 to C-95.
Interestingly, all the quaternary carbons are localized on the
ring A−Q assembly. Isotope shift experiments measured in
pyridine-d5/CD3OD (20:1) and pyridine-d5/CD3OH (20:1)
showed that 13 oxycarbons (C-20, C-36, C-42, C-60, C-61, C-
64, C-65, C-67, C-68, C-71, C-73, C-86, and C-94) were
bearing hydroxyl groups. The rest of the oxycarbons were
involved in ether linkages, so the number of ether rings in the
molecule was deduced to be 24. These observations matched
the r.+d.b. count of the molecular formula. The locations of
ether linkages were determined by NOE and HMBC
correlations (Figure 1) and the isotope shift experiments.
1
The H NMR spectrum showed two methyl doublets, 11
methyl singlets, five olefinic protons, and an aldehyde in
addition to aliphatic methylenes and methines bearing oxygen.
The 1H decoupled 13C and DEPT, and HSQC spectra showed
signals of 13 methyls, one methine and 25 methylene aliphatic
carbons, 51 oxgenated methine and 10 oxygenated quaternary
carbons, five methine and a quaternary olefinic carbons, and an
aldehyde carbon. The number of methyl singlets was equal to
the number of the quaternary carbons. This means that the
quaternary carbons, except the olefinic carbon, bear a singlet
methyl and an oxygen atom. On the other hand, the number of
methyl doublets was not equal to the number of aliphatic
methines. Therefore, the doublet methyl observed at δH 1.37 is
attached to an oxymethine carbon and located at one terminus.
These facts revealed that the scaffold of KBT-F consisted of a
95-carbon chain substituted with 12 branched methyls but
lacking alicycles or longer carbon branches.
The structural elucidation of KBT-F was mainly carried out
using both the intact and 13C-enriched KBT-F molecules by
analyses of extensive 2D NMR spectra measured with 400, 500,
1
and 800 MHz instruments. Detailed analysis of H−1H COSY
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Table 1. 1H and 13C NMR Assignments (δ) of Brevisulcenal-
F (1) in Pyridine-d5
Although the close chemical shifts H-24/H-28, Me-104/Me-
105, and H-75/H-79 hampered confirmation of rings G, K, and
T, the proton chemical shifts and signal shapes of methylenes
CH2-26, CH2-39, and CH2-77 were typical for methylenes
residing in a six-membered ether ring. Judging from NOE
correlations and interproton coupling constants shown in 2D
spectra, the polycyclic ethers of rings A−Q and S−W were
deduced to be trans−cisoid fused, as is the case with other
polycyclic ether compounds such as the brevetoxins. The left-
hand portion of the molecule consisted of the 2-methylbut-2-
enal side chain and 17 contiguous ether rings A−Q including
seven contiguous six-membered ether rings C−I. The right-
hand portion has an unprecedented terminal structure
consisting of dihydrofuran X and six-membered ether ring W
that was deduced as follows. A strong HMBC correlation H-
88/C-84 and NOE correlations between H-84/H-89 were
observed. The equatorial proton H-88 gave rise to the strong
HMBC correlation indicating a dihedral angle H−C-88−O−C-
84 of nearly 180°. Therefore, ring W was a six-membered ether
ring and C-89 resided on an axial orientation. Moreover, NOE
correlations between H-88/H-93 and the 13C chemical shifts of
C-89 (δ 87.3) and C-92 (δ 91.2) observed in the lower field
indicated that ring X was a dihydrofuran (Table 1). Therefore,
an alternative regio-isomer, a fused 7/6 ether ring structure, was
ruled out and the sequence for rings W-X was determined.
High energy collision induced dissociation MS/MS has been
shown to be a practical method for structural verification of
polycyclic ethers. Studies on yessotoxin,9 maitotoxin,6 and the
gymoncins4 revealed that fused polycyclic ethers give rise to
characteristic ring cleavages with product ions that enable
deduction of ether ring sizes and position and type of
substituents directly. Therefore, MALDI-SpiralTOF-TOF was
used for structural confirmation of KBT-F.10
In CID MS/MS analysis of YTX and MTX, the sulfate esters
at the terminus of the molecules played an important role to
simplify the product ion spectra because a negative charge is
localized at that position, resulting in charge remote
fragmentation. However, KBT-F does not contain a functional
group for charge remote fragmentation. To introduce a charge
site into the molecule, KBT-F was reacted with 3-
(hydrazinecarbonyl)benzene sulfonate in 80% pyridine (Figure
2). Negative ion MALDI MS of a KBT-F benzene sulfonate
derivative (2) revealed a molecular ion at m/z 2250 [M −
Na]−.
The MALDI-SpiralTOF-TOF produced prominent product
ions generated by bond cleavage at the characteristic sites of
ether rings (Figure 3). A mass difference of 56 Da between
prominent ions indicated the presence of a six-membered ether
ring. A mass difference of 70 Da could indicate either a seven-
membered ether ring or a six-membered ether ring bearing a
methyl; however, the NMR analyses enabled distinguishing the
absence or presence of a methyl on an ether ring by the
NOESY and HMBC experiments as mentioned above.
no.
1H
13C
no.
1H
13C
no.
1H
13C
1
2
3
4
9.51
197.1
143.3
152.0
37.1
36
37
38
39
4.13
78.6
82.9
80.3
33.1
71
72
73
74
5.13
4.35
4.61
2.54
2.83
3.38
3.81
1.80
2.65
3.51
3.36
2.21
2.59
3.38
3.46
1.73
2.49
4.29
3.26
4.40
2.00
2.60
3.77
6.00
6.19
6.45
5.28
1.70
4.37
1.37
1.74
1.47
1.58
1.48
1.49
1.45
1.64
1.56
1.41
1.44
1.43
1.05
67.5
82.6
67.3
41.7
6.61
2.45
2.55
4.37
5.45
5.98
4.33
2.14
2.29
4.18
5
6
76.6
127.2
140.1
78.8
40
41
42
43
78.3
82.7
75.9
42.2
75
76
77
80.2
78.8
38.6
7
4.04
2.30
2.68
3.84
4.09
2.13
2.46
1.95
2.01
3.27
3.26
1.90
2.55
3.44
3.19
1.67
1.86
1.76
2.02
8
9
3.86
1.98
2.04
1.89
2.05
4.08
80.5
78
79
80
79.6
80.2
37.9
10
26.7
44
45
46
84.4
86.4
33.3
11
27.1
81
82
83
80.5
79.7
39.0
12
13
14
75.7
81.9
44.1
47
30.4
2.05
2.08
3.91
48
49
50
85.4
85.2
42.6
84
85
86
87
66.9
83.5
67.5
34.4
15
16
17
73.0
76.9
41.9
1.99
2.24
4.54
51
52
53
82.3
82.7
31.2
18
19
20
21
22
23
74.5
80.1
75.4
82.7
75.7
38.2
88
89
79.0
87.3
132.3
132.9
91.2
49.0
69.0
23.8
11.7
24.6
23.8
17.6
18.0
24.8
23.8
23.3
21.5
24.6
19.9
12.4
4.40
3.68
4.20
1.89
2.73
3.43
3.59
1.75
2.39
54
40.6
90
91
55
56
57
80.1
86.0
35.9
92
3.40
2.17
2.33
4.02
3.72
4.48
4.13
4.85
2.67
2.73
4.98
4.60
4.30
4.52
4.56
2.17
3.17
4.70
93
94
24
25
26
82.2
80.9
46.7
95
58
59
60
61
62
63
73.2
75.9
72.7
75.4
76.2
38.5
96
97
98
27
28
29
76.7
86.2
30.4
99
3.45
2.01
2.06
3.67
100
101
102
103
104
105
106
107
30
31
32
76.5
77.5
45.2
64
65
66
67
68
69
70.8
77.5
77.0
75.5
72.4
35.9
1.89
2.35
4.86
33
34
35
81.4
82.1
48.7
2.31
2.59
70
74.3
The prominent product ions showed the sequence of cyclic
ethers for rings A−Q and S−W. The sequence of seven
contiguous six-membered ether rings C−I was clearly
confirmed by differences of 70/70/72/56/70/70/70 Da
(Figure 3a). The differences of 70, 70, and 56 Da between
m/z 727 and 797, 1037 and 1107, and 1868 and 1924
confirmed the positions of rings G, K, and T. The differences of
100 Da between m/z 937, 1037, and 86 Da between m/z 1107
and 1193 indicated the location of seven-membered ether rings
(rings J and L) bearing a hydroxyl group. As depicted in Figure
Figure 2. Preparation of KBT-F benzene sulfonate derivative (2).
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characteristic cleavage of a six-membered ether ring bearing a
hydroxyl group, confirming the size of ring W. Thus, all the
prominent ions observed in the product ion spectra supported
the planar structure deduced from the NMR data.
Stereochemistry of KBT-F. The stereochemistry of KBT-F
was determined by combining NOE data and proton coupling
constants (Figure 4). The NOE correlations from angular
methyls to hydroxy bearing protons Me-103/H-36 and Me-
105/H-42 were assigned the β orientation of C-36-OH and C-
42-OH on rings J and L. The NOE due to Me-100/H-20 and
the small coupling constant (2 Hz) H-20/H-21 indicated C-20-
OH on ring D was orientated in an axial direction. The NOE
correlations H-59/H-60 and H-59/H-61 and the small coupling
constants H-59/H-60 and H-60/H-61 suggested that C-60-OH
and C-61-OH on ring Q were orientated in axial and equatorial
directions, respectively. The NOE correlation H-73/H-75
indicated an equatorial direction of C-73-OH on ring S. The
proton coupling constants of H-66/H-67 and H-68/H-69 were
8.6 and 9.7 Hz, respectively. Additionally, NOESY cross peaks
were observed between H-66/H-68, H-66/H-71, and H-68/H-
71. Thus, ring R has a chair conformation, and the orientations
of C-67-OH and C-68-OH on ring R both were determined to
be equatorial. Configurations of other stereogenic centers (C-
64, C-65, C-93, and C-94) on acyclic moieties and the
diastereomeric relationships between rings Q−R, R−S, and
W−X remain unknown because signal overlapping and small
coupling constants prevented application of J-based conforma-
tional analysis.11
Biological Properties of KBT-F. The details of the
biological properties of KBTs are reported in ref 1. The
mouse lethality of KBT-F was estimated to be 0.032 mg/kg.
KBT-F was also toxic against mouse leukemia P388 cells at 2.7
nM.
Figure 3. Product ion mass spectrum of 2. The spectrum was taken for
the precursor ion [M − Na]− at m/z 2250 and is shown in two parts:
(a) m/z 250−1550 (C1−C63); (b) m/z 1500−2200 (C63−C95).
CONCLUSION
■
The NMR and MS/MS analyses led to elucidation of the
structure of brevisulcenal-F (KBT-F) and its partial config-
urations. KBT-F has 24 ether rings including an unusual
dihydrofuran, 13 hydroxyl groups, 13 methyl groups, and a 2-
methylbut-2-enal terminus. KBT-F contains 17 contiguous
ether rings A−Q, which is the longest of known polycyclic
ethers. The long contiguous ether ring assembly with the 2-
methylbut-2-enal terminus is reminiscent of the gymnocins.
Unlike the brevetoxins, KBT-F does not have unsaturated
3b, the observed product ions at m/z 1798, 1738, 1651, 1592,
and 1531 well matched the position of ring R, the hydroxyl
groups at C-64 and C-65, and an acyclic structure. Rings W−X
were also confirmed. The product ions at m/z 2176 and 2108
were generated by the bond cleavage between C-92 and C-93,
and C-89 and C-88, respectively. The difference of 68 Da
corresponded to the cleavage of the dihydrofuran X. The
difference of 72 Da between m/z 2108 and 2036 was the
Figure 4. Relative configurations determined independently for rings A−G, ring R, rings S−W, and ring X.
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Co., Japan) with isocratic elution (90% MeOH/H2O followed by 85%
MeOH/H2O) and guided by UV absorbance at 230 nm.
middle sized ether rings. The molecule consists of two parts:
one is the rigid ether ring assembly, rings A−Q, and the other is
a flexible acyclic part and the short ether ring assembly, rings
S−X. The majority of the 13 hydroxyl groups reside in the
flexible part of the molecule from C-60 to C-94, while all the
angular methyls reside on the rigid ring assembly A−Q.
Although the pharmacological mode of action of KBTs has not
been elucidated, these structural features must be related to
their potent biological activities, which are much higher than
those of the gymnocins.
From a biosynthetic point of view, the terminal dihydrofuran
X is also unusual among the marine polyethers. Formation of
the fused ether rings in biosynthesis of marine ladder-frame
polyethers is proposed to proceed by cascade endo-tet closure
of a polyepoxide intermediate12 whereas biosynthesis of
dihydrofurans in terrestrial organisms is proposed to be by
exo epoxide opening of an epoxide precursor.13
Preparation of the Benzene Sulfonate Derivative (2). KBT-F
(20 μg) was treated with an excessive amount of 3-
(hydrazinecarbonyl)benzene sulfonate sodium salt for 2 h in 80%
pyridine. After solvent elimination, the reaction mixture was
partitioned between CHCl3 and H2O. The chloroform fraction was
used for the MS/MS experiments (MALDI-SpiralTOF-TOF). The
product ion spectra were analyzed using mMass software.15
MALDI-SpiralTOF MS and SpiralTOF-TOF Measurements.
MALDI-SpiralTOF MS spectra were recorded on a JEOL-S3000
instrument using 2,5-dihydroxybenzoic acid (DHB, Wako) or α-
cyano-p-hydroxycinnamic acid (CHCA, Wako) as a matrix. The KBT-
F derivative was dissolved in MeOH/CHCl3 and mixed with
norharmane matrix and subjected to MALDI-SpiralTOF-TOF
measurements in a negative mode. The product ion spectra were
recorded with a laser irradiation at 349 nm, a laser frequency at 250
Hz, and −20 kV of acceleration voltage in the first TOF stage. The
collision energy was 20 keV to induce high energy-collision induced
dissociation. Product ions formed by collision induced dissociation
were reaccelerated by 9 kV for the analysis in the second TOF stage.
Brevisulcenal-F (1). Isolated as a colorless amorphous solid:
Structural elucidation of other analogues and configurational
analyses of unelucidated stereostructures are now underway.
19
[α]D 50.9 (c 0.05, CHCl3); UV maxima (λ) 227 (ε 7900) nm. The
high resolution MALDI-TOF MS of 1 gave [M + Na]+ at m/z
EXPERIMENTAL SECTION
■
1
2076.0476 (calcd 2076.0480 for C107H160O38Na). H and 13C NMR
General Methods. All solvents were purchased at highest
commercial grade and used as supplied unless otherwise noted.
Optical rotations were obtained on a JASCO DIP-350 polarimeter in
CHCl3. UV−visible absorption spectra were measured on a JASCO V-
550 UV-spectrometer. NMR spectra were recorded on three NMR
data were described in Table 1.
ASSOCIATED CONTENT
■
S
* Supporting Information
1
1
instruments with H for 500 MHz (13C for 125 MHz), H for 400
1H−1H COSY, TOCSY, NOESY, HSQC, and HMBC spectra
of KBT-F. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
MHz (13C for 100 MHz), or H for 800 MHz (13C for 200 MHz).
Chemical shift values are reported in ppm (δ) referenced to internal
signals of residual protons [1H NMR; C5HD4N (7.21); 13C NMR,
C5D5N (125.8)].
AUTHOR INFORMATION
Corresponding Author
*msatake@chem.s.u-tokyo.ac.jp.
Culture Growth and Harvesting. K. brevisulcata (CAWD82) was
collected from the Wellington Harbour in 1998 and is held at the
Cawthron Institute Culture Collection of Microalgae (CICCM),
Cawthron Institute, Nelson. Bulk cultures (150−250 L batches) were
grown in 12 L carboys using 100% GP+Se media14 under a 12/12 h
day/night timed cool white fluorescent lighting regime and 25 min
aeration every 30 min. Starter culture (14−21 days old) was added to
100% GP + Se media at a ratio of 1:10 to 1:15. Cultures were
maintained for up to 21 days. Aliquots of culture were assessed for
cells numbers with an inverted microscope. For 13C enrichment,
cultures were augmented at 0 and 7 days with NaH13CO3 (0.25 g per
12 L). Production of toxins was assessed by liquid chromatography−
mass spectrometry (LC-MS) following SPE using a 50 mL aliquot of
culture extracted with Strata-X (60 mg, Phenomenex Inc., CA),
washed with Milli Q water and 20% methanol, and they were eluted
with methanol or methanol followed by acetone (3 mL each).
Toxins were extracted from mature cultures using Diaion HP20
resin. The prewashed resin was packed in a polypropylene column. K.
brevisulcata cultures were transferred to a 200 L barrel, and cells were
lysed by addition of acetone to 7% v/v. The cultures were settled for
one hour and diluted with reversed osmosis purified water (RO water)
to 5% v/v acetone before pumping at 0.3 L/min through a filter
system followed by the HP20 resin column. The column was then
washed with water, and the HP20 resin was transferred to a 2 L flask.
Toxins were recovered by soaking the resin with AR acetone (1 L) and
decanting (3×). The combined acetone extract was rotary evaporated
to produce a dried crude extract.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Prof. M. Murata, Osaka University, for his
generous gift of 3-(hydrazinecarbonyl)benzene sulfonate
sodium salt and to Prof. Y. Oshima, Tohoku University, and
Dr. B. Keyzers, Victoria University, for information from their
initial research on KBTs. We also thank Mr. C. Kurosaki and
Ms. M. Yoshida (Yokohama Institute, RIKEN) for measuring
the NMR spectra. This work was financially supported by a
bilateral program from JSPS and FRST, CAWX0703 and
CAWX0804 from NZ-MSI, KAKENHI (22404006), ERATO
from JST, and Global COE Program for Chemistry Innovation,
the University of Tokyo.
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