Preparation of calibration standards of N1-H paralytic shellfish toxin analogues by large-scale culture of cyanobacterium Anabaena circinalis (TA04)

Article abstract of DOI:10.3390/md9030466

Mouse bioassay is the official testing method to quantify paralytic shellfish toxins (PSTs) in bivalves. A number of alternative analytical methods have been reported. Some methods have been evaluated by a single laboratory validation. Among the different types of methods, chemical analyses are capable of identifying and quantifying the toxins, however a shortage of the necessary calibration standards hampers implementation of the chemical analyses in routine monitoring of PSTs in bivalves. In our present study, we studied preparation of major PST analogues as calibrants by large-scale cultivation of toxic freshwater cyanobacteria Anabaena circinalis TA04. The cells were steadily grown in 10 L bottle for 28 days. The primary N1-H toxins, C1/C2, were produced at a concentration of 1.3 ± 0.1 μmol/L. The intracellular and extracellular toxins occupied 80% and 20%, respectively. Over 220 μmol of the toxins was obtained from approximately 200 L of the culture over six months, demonstrating that it is sufficient to prepare saxitoxin analogues. The toxins were chemically converted to six N1-H analogues. Preparation of the analogues was carried out at relatively high yields (50-90%). The results indicate that our preparation method is useful to produce N1-H toxins. In our present study, detailed conditions for preparation of one of the rare N1-H analogues, gonyautoxin-5, were investigated.

Full text of DOI:10.3390/md9030466

Mar. Drugs 2011, 9, 466-477; doi:10.3390/md9030466
OPEN ACCESS
Marine Drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Preparation of Calibration Standards of N1-H Paralytic
Shellfish Toxin Analogues by Large-Scale Culture of
Cyanobacterium Anabaena circinalis (TA04)
1,
1
2
Ryuichi Watanabe *, Toshiyuki Suzuki and Yasukatsu Oshima
1
National Research Institute of Fisheries Science, 2-12-4 Fukuura, Kanazawa-ku,
Yokohama 236-8648, Japan; E-Mail: tsuzuki@affrc.go.jp
2
School of Marine Biosciences, Kitasato University, 160-4 Okirai-uto, Sanriku-cho,
Ofunato 022-0101, Japan; E-Mail: oshima.y@kitasato-u.ac.jp
*
Author to whom correspondence should be addressed; E-Mail: rwatanabe@affrc.go.jp;
Tel.: +81-45-788-7630; Fax: +81-45-788-5001.
Received: 9 February 2011; in revised form: 3 March 2011 / Accepted: 18 March 2011 /
Published: 22 March 2011
Abstract: Mouse bioassay is the official testing method to quantify paralytic shellfish
toxins (PSTs) in bivalves. A number of alternative analytical methods have been reported.
Some methods have been evaluated by a single laboratory validation. Among the different
types of methods, chemical analyses are capable of identifying and quantifying the toxins,
however a shortage of the necessary calibration standards hampers implementation of the
chemical analyses in routine monitoring of PSTs in bivalves. In our present study, we
studied preparation of major PST analogues as calibrants by large-scale cultivation of toxic
freshwater cyanobacteria Anabaena circinalis TA04. The cells were steadily grown in
10 L bottle for 28 days. The primary N1-H toxins, C1/C2, were produced at a concentration
of 1.3 ± 0.1 µmol/L. The intracellular and extracellular toxins occupied 80% and 20%,
respectively. Over 220 µmol of the toxins was obtained from approximately 200 L of the
culture over six months, demonstrating that it is sufficient to prepare saxitoxin analogues.
The toxins were chemically converted to six N1-H analogues. Preparation of the analogues
was carried out at relatively high yields (50–90%). The results indicate that our preparation
method is useful to produce N1-H toxins. In our present study, detailed conditions for
preparation of one of the rare N1-H analogues, gonyautoxin-5, were investigated.
Keywords: cyanobacteria; Anabaena circinalis; paralytic shellfish toxin; saxitoxin;
chemical derivatization; standard

Mar. Drugs 2011, 9
. Introduction
Paralytic shellfish toxins (PSTs), one of the most potent groups of neurotoxins in marine biotoxins,
467
1
are produced by toxic marine dinoflagellates and freshwater cyanobacteria. Around 30 analogues have
been reported (Figure 1). Some of these analogues are metabolic products of algae-derived toxins in
shellfish [1]. Approximately 20 analogues are found to show a wide range of toxicity [2]. Natural and
cultivated shellfish are contaminated with PSTs by feeding on toxin-producing algae, and are a
potential cause of paralytic shellfish poisoning (PSP) to humans. Although mouse bioassay (MBA),
which is the official testing method to determine shellfish toxicity in many countries, is effective in
protecting the consumers from PSP, alternative methods are required due to some drawbacks including
accuracy, reproducibility and ethical concerns [3].
A number of alternative analytical methods have been developed to date. Some have been evaluated
by a single laboratory validation [4,5]. Among different types of methods, high performance liquid
chromatography fluorescent detection (HPLC-FD) is one of the reliable methods to identify and
quantify individual toxins. It is reported that the toxicity obtained by HPLC-FD is usually consistent
with the total toxicity of MBA by measuring the major toxins C1–4, gonyautoxin (GTX) 1–6,
decarbamoylgonyautoxin (dcGTX) 2 and dcGTX3, neosaxitoxin (neoSTX), decarbamoylsaxitoxin
(dcSTX) and saxitoxin (STX) [2]. However, although the calibration standards are commercially
available from the National Research Council Canada (NRC), shortage of the calibration standards has
hampered implementation of HPLC-FD in routine monitoring of PSTs in bivalves.
In preparation of high amount of the calibration standards, selection of natural source materials is
important. Shellfish with a high level of toxicity [6,7] are one of the important natural sources but this
source has hardly been obtainable in Japan over the last decade due to very low levels of toxicity
(at most 10–50 MU/g). Therefore, some toxic dinoflagellates such as Alexandrium excavatum [8] and
A. tamarense [9] are used as appropriate source materials in terms of stable toxin source materials.
Some toxin-producing cyanobacteria such as Anabaena circinalis and Aphanizomenon flos-aquae
would also be ideal source materials in toxin production and cell growth.
Because primary toxins produced by toxic algae are insufficient to account for the complicated PST
analogues in shellfish, chemical conversion of the primary toxins to other analogues is used to produce
a variety of toxins. Some chemical or enzymatic reactions were summarized for each substituent
moiety; C11, C13 and N1 positions [10].
Figure 1. Structures of paralytic shellfish toxins.
−
R1
H
R2
R3
R4: –OCONH2 –OCONHSO3
–OH
dcSTX
H
H
H
H
H
H
STX
GTX5
GTX6
C3
OH
OH
H
neoSTX
GTX1
GTX2
GTX3
GTX4
dcneoSTX
dcGTX1
dcGTX2
dcGTX3
dcGTX4
−
−
OSO3
OSO3
H
C1
−
H
OSO3
OSO3
C2
−
OH
H
C4
STX: Saxitoxin; GTX: Gonyautoxin; dc-: decarbamoyl-.

Mar. Drugs 2011, 9
468
Among the known toxins, N-sulfocarbamoyl gonyautoxins, C1/C2, are useful to produce other
N1-H analogues by chemically removing the substituent moieties. Although adequate conditions for
conversion of C1/C2 to dcGTX2/dcGTX3, dcSTX, and GTX2/GTX3 were reported in previous
papers [11,12], detailed investigation of chemical conversion of other analogues (e.g., GTX5) that are
more dominant toxins in shellfish, has not been reported yet. In this paper, we focused on large-scale
cultivation of the cyanobacteria A. circinalis to prepare a variety of N1-H analogues from C1/C2 by
chemical conversion.
2
. Results and Discussion
2
.1. Large-Scale Culture of Cyanobacteria A. Circinalis (TA04)
The cultured A. circinalis (strain TA04) has been maintained in modified C medium since 1996 [13].
The culture showed dark-green color in mid-stationary phase, and turned to chrome yellow color in
late-stationary phase. The biomass and the toxin concentration also increased in the exponential growth
phase, followed by the short induction growth phase (~7 days, Figure 2). As the toxin concentration
declined when the culture reached the late-stationary phase, the cells were harvested at 28 days. The
cultured cyanobacteria produced C1/C2 (70–90%) as the dominant toxins. Although GTX2/GTX3 and
dcGTX2/dcGTX3 (5–15% each) were produced as the minor toxins, N1-hydroxylated toxins such as
GTX1/GTX4 were not found in the harvested culture. The toxin components determined in our present
study were different from those obtained in our previous study [13].
Figure 2. Growth curve (A) and toxin production (B) in a large-scale culture of
A. circinalis. (A) Fluorescent intensity shows the average level of chlorophyll a in cells
(n = 3); (B) Only C1/C2 produced by the cells were analyzed by high performance liquid
chromatography fluorescent detection (HPLC-FD). Solid line shows total toxin
concentration of C1/C2 in culture. Dashed line shows intracellular toxin concentration of
C1/C2. The difference corresponds to the extracellular toxin concentration.
1400
1200
1000
1600
(A)
(B)
1400
1200
1000
800
600
400
200
0
800
600
400
200
0
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Culture period (days)
Culture period (days)

Mar. Drugs 2011, 9
469
This difference is probably caused by physiological natural changes due to the long-term successive
culture over 15 years and the change of culture medium. On the other hand, the α/β-epimer ratio of
C-toxins was 1.1, which was close to previous data [14]. The toxin production of C1/C2 in large-scale
culture was 1.3 ± 0.1 µmol/L culture (7% RSD, n = 3) at 28 days. This value was somewhat lower than
that found in our previous study (1.8–2.0 µmol/L culture). Approximately 80% of the total toxins were
found inside the cells. Our result was similar to that obtained in another strain, MB06 [14], which was
collected in the same location as our material strain, TA04, in the ratio of epimers and
intra-/extracellular toxin in 1.2 L small scale culture, whereas the cell growth of strain TA04 was
slower than strain MB06.
The cyanobacteria were cultured under several modified conditions of Fitzgerald medium toward
efficient toxin production. The biomass and toxin production for 28 days were investigated in each
condition (Table 1). As a result, the concentration of nitrate and phosphate hardly affected the toxin
production and biomass of the cyanobacteria. Two different inoculation volumes were determined to
shorten the culture period, but the cell growth and the toxin production were the same as those of the
control. In PST biosynthesis of cyanobacteria, the precursors such as arginine and
S-adenosylmethionine were identified by the cultivation with stable isotopes [12] and a hypothetical
pathway was proposed. The in vitro experiments for PST biosynthesis suggested that pyridoxal
phosphate (PLP)-dependent enzymes were strongly involved in PST biosynthesis [15]. Therefore, the
toxin production in the cell was investigated in vivo by adding a common cofactor, PLP, to the medium.
Our study showed PLP hardly affected both cell growth and toxin production in our culture. PLP could
be insufficiently incorporated into cells to be available to enzymes.
Table 1. Biomass and toxin production of C1/C2 at 28 days of A. circinalis under modified
condition of Fitzgerald medium.
Toxin concentration of C1/C2 (µmol/L)
Entry Parameters
Biomass
Intracellular Extracellular
Total
(
%)
80
75
86
84
86
84
77
84
58
55
59
3
(%)
20
25
14
16
14
16
23
16
42
45
41
97
25
Control
2300
400
1.39
0.82
1.26
1.03
1.43
1.49
1.36
1.05
1.36
1.89
1.33
1.78
1.35
1
2
3
4
5
6
7
8
9
NaNO3
0 mM
2.9 mM
11.6 mM
0.14 mM
0.54 mM
0.5 L
2 L
10 µM
10 µM
100 µM
0.6 mM
1200
1600
1600
2000
2000
2500
2500
3100
2300
2700
K HPO
2
4
Inoculation
Pyridoxal phosphate
Glycerophosphate Na
1
1
0
1
Bicine
Average *
75
*
Average toxin concentration was calculated using the data from entry 1 to 10.
Moreover, the addition of organic phosphates, β-glycerophosphate sodium salt of 10 and 100 µM
each, was also investigated and no significant change was observed. Finally, Good’s buffer, bicine,

Mar. Drugs 2011, 9
470
was also used to control the pH of the culture medium during culture. The toxin production was
slightly improved compared to the control, but the cell harvest was hampered and the intracellular
toxins leaked outside in harvest because of the increased viscosity of the culture.
Figure 2 indicates that approximately 140 µmol of C1/C2 could be produced from 100 L culture. It
was demonstrated that over 220 µmol of C1/C2 was obtained from 200 L of the culture which was
produced over 6 months cultivation. This amount of toxins would be sufficient to prepare the
calibration standards of STX analogues.
2
.2. Preparation of Paralytic Shellfish Toxin Analogues
C1/C2 are one of the most modified STX analogues among the known toxins. C1/C2 are also
known as the lowest toxicity group among STX analogues [2]. Figure 3 shows the preparation scheme
of PST analogues by chemical conversion from C1/C2. N-sulfocarbamoyl group on C1/C2 is
hydrolyzed in neutral pH, and this reaction leads C1/C2 to dcGTX2/dcGTX3. Subsequently, the
sulfate ester group of the toxins is reductively eliminated by 2-mercaptoethanol (2-ME) to prepare
dcSTX [11]. Thus, our route to produce dcSTX is useful compared to other routes through STX [16]
because production of STX is legally restricted in Japan and many countries.
Figure 3. Preparation scheme of paralytic shellfish toxin analogues. (A) Phosphate buffer
(
pH 7.0), 70 °C, 60 min; (B) Phosphate buffer (pH 6.0), 2-mercaptoethanol, 60 °C, 60 min;
C) Acetate buffer (pH 5.2), 2-mercaptoethanol, 50 °C, 2 h; (D) 0. 13 M HCl aq., 100 °C,
(
1
5 min; (E) 2-mercaptoethanol, 100 °C , 15 min.; (F) 0.05 M HCl, 100 °C, 30 min [17];
G) 7.5 M HCl, 100 °C , 3 h [18,19]. Solid arrows, this study; dashed arrows,
previous studies.
(

Mar. Drugs 2011, 9
471
GTX5 was directly converted from C1/C2 by reductive elimination of the sulfate. C1/C2 were
reacted with 2-ME to give 48% of GTX5, 18% of the remaining C1/C2, and 34% of unknown
reactants or the loss. Of the reaction temperatures tested (50–80 °C), a good yield (50%) of GTX5 was
obtained at 50 °C. The yield reached 30% within 30 min and slowly increased with time, and then
gradually declined over 3 h. The remaining substrate was decreased to 20–40% within 30 min and then
slightly declined. HPLC analysis of the reactant mixture revealed that dcGTX2/dcGTX3 and dcSTX,
as by-products in the reaction, were not produced, indicating that the reaction specifically proceeded
without hydrolyzing the N-sulfocarbamoyl chain of C1/C2. Interestingly, HPLC analysis of the
reaction mixture revealed that the relative proportion of C1 to C2 decreased after the reaction,
implying that the α-epimer reacted selectively with a thiol compound compared to the β-epimer.
Chromatographic behavior of the product was the same as that of the authentic GTX5 (Figure 4).
Figure 4. Chromatograms of the authentic GTX5 (upper) and the product (bottom). The
authentic GTX5 was used as a mixture of GTX1–5.
GTX1
.67 min
GTX5
12.24 min
GTX2
17.43 min
9
GTX4
.48 min
GTX3
13.97 min
8
GTX5
12.24 min
0
2
4
6
8
10
Time (min)
12
14
16
18
20
Figure 5 shows LC-MS/MS spectra of the authentic GTX5 and the product converted from C1/C2.
The MS/MS spectrum of the authentic GTX5 gave ion peaks at m/z 282, and 300 corresponding to
+
+
[
M − H O − SO + H] and [M − SO + H] , respectively (Figure 5A). Basically the same fragment
2 3 3
ions were obtained for the product (Figure 5B), demonstrating that the product was GTX5. GTX5 was
prepared in NaHCO solution containing dithiothreitol (DTT) in a previous study [20]. They also
3
attempted preparation of GTX5 by the reaction of C1/C2 with 2-ME in place of DTT, but GTX5 was
not obtained. The fact that our method was successful to produce GTX5 with 2-ME was probably
because of acidic condition in which the thioester intermediate obtained in neutral pH [21] was not
formed. Conversion efficiency of GTX5 from C1/C2 prepared in our present method was better than
that (36%) reported in the previous study [20].
The toxins with an N-sulfate group such as C3/C4, GTX5 and GTX6 are easily converted to the
corresponding carbamate toxins by hydrolyzing in diluted mineral acids [12,19,22]. In our present
study, this hydrolysis reaction was applied to prepare GTX2/GTX3 from C1/C2. The products showed
the same chromatographic behavior with the authentic GTX2/GTX3 in HPLC-FD. The products,

Mar. Drugs 2011, 9
472
GTX2/GTX3, were obtained as an equilibrium mixture (ratio: 3.0 to 3.5), although C1/C2 used for the
reaction were not an equilibrium mixture.
Figure 5. MS/MS spectra of the authentic GTX5 (A) and the product (B). Both toxins
were analyzed at declustering potential of 11 V and collision energy of 21 V. The
precursor ion was set to 380.
(A)
(B)
In LC-MS/MS analysis, epimers such as GTX2/GTX3 show different fragmentation patterns.
+
GTX2 as α-epimer gives a predominant ion peak at m/z 316 corresponding to [M − SO + H] , whereas
3
GTX3 as β-epimer gives several major peaks at m/z 298, 316 and 378 corresponding to
+
[
M − H O + H] . The products were confirmed to show the same fragmentation pattern in each epimer.
2
In the process of preparation of PST analogues, it was a requirement to avoid generation of STX
because production of STX is illegal in Japan and many countries. In our method, STX was not
detected in any processes by HPLC-FD. Although STX is not prepared in this study as mentioned
above, STX could be prepared from GTX2/GTX3 or GTX5 as described in the previous
reports [12,17], if necessary. Thus, C1/C2 as starting materials could be converted to six analogues and
STX, giving a relatively high yield in each reaction (Figure 3). The starting materials of C1/C2 can be
obtained at a yield of 50% from the extract of the cell by purifying them with several chromatography
steps such as activated charcoal, gel filtration, and ion exchange. The purified C1/C2 were converted
to dcGTX2/dcGTX3 (step A), GTX5 (step C), and GTX2/GTX3 (step D), and subsequently a portion
of the resulting dcGTX2/dcGTX3 were converted to dcSTX (step B). At least 10 µmol of the toxin
calibrants could be prepared from 200 L of culture of the toxic cyanobacteria (Table 2).

Mar. Drugs 2011, 9
Table 2. Amounts of toxins prepared from large-scale culture (200 L) of cyanobacteria
473
A. circinalis. The amounts of C1/C2 as substrates are calculated as 110 µmol, because the
toxins are isolated at 50% yield from the extract of the cyanobacteria.
Reaction
Remaining Residual Residual
¶
Substrates Amounts Products Amounts Yields
§
†
steps
substrates Amounts ratio
(
µmol)
70
(µmol)
35
(%)
50
(µmol)
(%)
A
B *
C
C1/2
dcGTX2/3
dcSTX
C1/2
C1/2
21
30
dcGTX2/3
C1/2
15
14
90
25
GTX5
12
48
5
18
D
C1/2
15
GTX2/3
14
90
§
¶
The steps correspond to Figure 3; * dcGTX2/3 were used as a part of the products in step A; (Amount of
†
Product)/(Amount of Substrate) × 100; (Residual Amounts)/(Amount of Substrate) × 100.
3
. Experimental Section
3
.1. Large-Scale Culture of Cyanobacteria and the Harvest
The cyanobacterium A. circinalis used in this study is a non-axenic strain TA04. The field sample
of A. circinalis was collected at Tullaroop reservoir, Victoria, Australia, and strain TA04 was one of
single-trichome isolates prepared by Negri et al. [14]. Strain TA04 was cultured in Fitzgerald medium
in 2 L culture, and then scaled-up to 10 L volume (Thermo Fisher Scientific, Rochester, NY, USA).
2
The cyanobacteria were cultured on a 16 L/8 D cycle at 17 °C and 25 µmol/m /s, passing air through a
disc filter (0.20 µm pore size, Advantec, Japan). The bottle cap was equipped with three pores, where
one was used as air-inlet with the disc filter, one was as air-outlet with the disc filter and one was as a
sampling tube with a cock. The cyanobacteria were maintained by inoculating 1 L of the culture in
exponential growth phase (two weeks later) to 9 L of the sterilized medium. The 10 L batch culture
was carried out in quintuplicate, of which one was used for inoculation of seed population, and
repeated three times. Four weeks later after inoculation of the seed population, the A. circinalis cells
were harvested by a continuous centrifugation or filtration (No. 2, Advantec, Japan). The cell-free
culture filtrate and the cell fractions were treated according to our previous procedure [11].
3
.2. Cell Growth Measurement and Toxin Analysis
Cell growth was estimated from fluorescent intensity derived from chlorophyll a in the cell. The
culture was taken in axenic through a sampling tube. Culture samples (200 µL) were collected, and
added to wells of a 96-well microplate in triplicate. The cell-free culture filtrate (200 µL) as blank was
prepared by filtering the culture with a syringe filter (Dismic-25cs, 0.45 µm, Advantec, Japan) or a
glass fiber paper (GA-100, 25 mm, Advantec, Japan). It is subtracted from the culture fluorescence
using a fluorescence plate reader (Ex: 460 nm; Em: 645 nm, FL600, BioTek).
Toxin content (C1/C2) of the cell and the cell-free filtrate was analyzed with HPLC-FD [2]. The
culture of 10 to 30 mL at each growth phase was passed through a glass fiber paper (GA-100, 25 mm,
Advantec, Japan) to collect cells. The paper was dissected and suspended into 1 mL of 0.5 M acetic
acid. The suspension was centrifuged, following ultrasonication, to obtain the supernatant as analyte.
On the other hand, the filtrate was adjusted to become acidic by adding 0.5 M acetic acid. The filtrate

Mar. Drugs 2011, 9
474
was evaporated to dryness and then dissolved in 500 µL of 0.5 M acetic acid. The toxin solution was
ultrafiltered (Ultracell YM-10, 10000 MWCO, Millipore, Billerica, MA, USA) to obtain the resulting
filtrate as analyte.
3
.3. Serial Preparation of dcGTX2/dcGTX3 and dcSTX from C1/C2
The preparation of dcGTX2/dcGTX3 and dcSTX from C1/C2 was carried out according to the
method of Watanabe et al. [11]. C1/C2 as starting materials were purified from the harvested cell
fraction in the cyanobacteria A. circinalis (TA04) culture. Typically, dcGTX2/dcGTX3 were obtained
by the hydrolysis of C1/C2. C1/C2 were heated in 100 mM potassium phosphate buffer at pH 7.0 for
6
0 min, stirring at 70 °C, and then cooled on ice. The resulting dcGTX2/dcGTX3 were isolated by gel
filtration chromatography (Bio Gel P-2, 10 mm × 400 mm, Bio-Rad) and weak-cation exchange
chromatography (Bio-Rex 70, 200–400 mesh, 10 mm × 400 mm, Bio-Rad) at overall yield of 50%.
The purified dcGTX2/dcGTX3 was reacted under stirring conditions at 60 °C in 100 mM potassium
phosphate buffer at pH 6.0 containing an equal volume of 2-ME for 60 min to quantitatively give the
resulting dcSTX.
3
.4. Preparation of GTX5 from C1/C2
C1/C2 (1.4 µmol) were evaporated to dryness and dissolved in 2 mL of 100 mM acetate buffer at
pH 5.2 containing equal volume of 2-ME in a 4 mL glass vial. The toxin solution was reacted at 50 °C
for 2 h. The reactant was evaporated and then diluted with acetonitrile to be a final concentration of
9
0% (v/v) acetonitrile solution and then the diluted solution was passed through a ZIC-HILIC cartridge
(3 mL, 200 mg, Merck SeQuant, Umeå, Sweden) preconditioned with 6 mL of distilled water and
6
3
1
mL of acetonitrile in order. The cartridge was washed with 3 mL of acetonitrile and then eluted with
mL of distilled water as toxic fraction. The toxic fraction was purified by Bio-Rex 70 (200–400 mesh,
0 mm × 450 mm, Bio-Rad) to give GTX5 at an overall yield of 48%. The product was confirmed and
identified by the comparison with the authentic standard that was previously prepared by Oshima and
confirmed in structure and purity with NMR [2]. Chromatographic behavior of the toxin was
determined with HPLC-FD. The parameters on LC-MS instrument (3200 Q TRAP LC/MS/MS system,
AB-SCIEX, Concord, Canada) were optimized with the authentic GTX5, prior to the analysis. The
flow injection analysis was performed in mass range of m/z 200–500 and positive ion mode at 500 °C.
The eluents were flowed at 0.2 mL/min with 65% B, where eluent A was distilled water and eluent B
was acetonitrile, both containing 20 mM formic acid. The MS/MS analysis was carried out in mass
range of m/z 50–450 at declustering potential of 11 V and collision energy of 21 V. The fragmentation
pattern between the product and the authentic was compared and identified.
3
.5. Preparation of GTX2/GTX3 from C1/C2 by Acidic Hydrolysis
Preparation of GTX2/GTX3 was carried out according to a previous method [12]. C1/C2 (1.4 µmol)
were evaporated to dryness and dissolved in 1 mL of 0.13 M HCl aq. The solution was heated at
00 °C , stirring for 15 min and then cooled on ice. The products, GTX2/GTX3, were recovered using a
1

Mar. Drugs 2011, 9
475
ZIC-HILIC cartridge as described above. The reaction quantitatively proceeded. The structure was
confirmed as described above.
4
. Conclusions
In this study, major N1-H toxins were prepared from C1/C2 as starting material. In particular, this is
the first report of the detailed conditions for preparation of GTX5 from C1/C2 analogues. The
chemical conversion allows us to prepare the toxins systematically. The toxin yield, of over 220 µmol
of C1/C2 extracted from 200 L of culture of the cyanobacteria, was estimated to be capable of
preparing at least 10 µmol of each toxin, based on our reaction yields (Table 2). We previously
reported a method using NMR to quantify concentrations of PST standards [23]. Based on the
preparation technique of PSTs and the quantitative NMR, large amount of the N1-H toxin standards
could be prepared and supplied constantly for shellfish safety monitoring programs using chemical
analyses. The N1-OH toxins such as GTX1/GTX4 are also essential for the chemical analyses. The
preparation technique of these toxins is currently being investigated, and will be reported elsewhere.
Acknowledgements
The authors are greatly appreciative to Blackburn, CSIRO, Australia, for providing cyanobacteria
A. circinalis (TA04). This study was funded by Ministry of Health, Labour and Welfare in Japan.
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