PREPARATION OF NEOSAXITOXIN

Abstract

A semisynthetic method of preparing neosaxitoxin from cultures of the dinoflagellate Gymnodinium catenatum is described. The scalable method includes the reductive desulfonation of an unresolved mixture of toxin C3 and toxin C4 and mild acid hydrolysis of the resulting gonyautoxin 6 (GTX6) to provide the neosaxitoxin.

Claims

1. A method of preparing neosaxitoxin comprising the mild acid hydrolysis of a quantity of GTX6 obtained by the reductive desulfonation of a quantity of C3,4 purified from an extract of a culture of an isolate of Gymnodinium catenatum where the reductive desulfonation is by contacting in a solution in a reaction solvent the quantity of C3,4 and a quantity of dithiol at a temperature and for a time sufficient to provide a reaction product in which greater than 97.5% (w/w) of the quantity of C3,4 has been converted to GTX6 to provide the quantity of GTX6.

2. The method of claim 1 where the method comprises the step of applying the reaction product to a silica based weak cation exchange sorbent and eluting with an aqueous weak acid to separate the GTX6 from the dithiol and provide the quantity of GTX6.

3. The method of claim 2 where the aqueous weak acid is an aqueous weak organic acid.

4. The method of claim 1 where the reaction solvent is buffered.

5. The method of claim 1 where the reaction solvent is buffered aqueous acetic acid.

6. The method of claim 1 where the solution has a pH in the range 7.2 to 7.8.

7. The method of claim 1 where the mild hydrolysis is by contacting the quantity of GTX6 with a dilute strong acid and heating to a temperature for a period of time sufficient to convert greater than 90% (w/w) of the GTX6 to neosaxitoxin.

8. The method of claim 7 where the strong acid is hydrochloric acid.

9. The method of claim 8 where the temperature is 80° C. ±2° C.

10. The method of claim 9 where the time is at least 30 minutes.

11. The method of claim 1 where the isolate of Gymnodinium catenatum is an isolate selected from the group of isolates consisting of: Gymnodinium catenatum CAWD101; Gymnodinium catenatum CAWD102; and Gymnodinium catenatum CAWD126.

12. The method of claim 11 where the isolate of Gymnodinium catenatum is the isolate designated CAWD102.

Description

BRIEF DESCRIPTION OF FIGURES

[0046] FIG. 1. A plan view of a hanging bag (1) formed from a length of tubular plastic for use in the bulk culture of isolates. Traversing single (3, 4, 5 and 6) and double solid lines (9 and 10) identify where two or more layers of the tubular plastic are heat welded together. Traversing broken lines identify where four (7 and 8) or two (12 and 13) layers of the tubular plastic are cut to provide a hanging loop (2) and cone, respectively.

[0047] FIG. 2. The profiles of toxins produced by isolates of the species Alexandrium minutum, Alexandrium ostenfeldii, Alexandrium pacificuma and Gymnodinium catenatum (Table 1) when cultured in vertical columns of aerated amended sea water and extracted without mild acid hydrolysis.

[0048] FIG. 3. Amounts of toxins determined to be produced by isolates of the species Alexandrium minutum, Alexandrium ostenfeldii, Alexandrium pacificuma and Gymnodinium catenatum (Table 1) when cultured in vertical columns of aerated amended sea water and extracted without mild acid hydrolysis.

DESCRIPTION

[0049] Extracts of cultures of isolates of Gymnodinium catenatum have been identified to produce relatively high amounts of toxins C3 and C4, the N-sulfocarbamoyl derivatives of GTX1 and GTX4 (Table 1). Extracts of cultures of these isolates are available from the Cawthron Institute, Nelson, New Zealand.

[0050] According to the present invention a quantity of C3,4 is purified from a concentrated extract of a culture of an isolate of Gymnodinium catenatum. The purified C3,4 is then converted to GTX6 by reductive desulfonation using a dithiol as the reducing agent (Scheme I, step a). Mild acid hydrolysis of the intermediate GTX6 yields neosaxitoxin (neoSTX) (Scheme I, step b) (Shimizu (1984)).

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[0051] In solution, toxin C3 and toxin C4, and gonyautoxin 1 (GTX1) and gonyautoxin 4 (GTX4), exist as pairs of epimers. For the former pair of epimers, the conversion of toxin C4 to toxin C3 is favoured (Hall et al (1984)). For the latter pair of epimers, GTX1 is the thermodynamically most favoured. Epimerisation is believed to occur under most conditions via keto-enol equilibration at C-12.

[0052] In the first step of the proposed 2-step reductive desulfonation according to SCHEME II a thiol group of the dithiol (R-SH) attacks the electrophilic C-12 of the keto form (I) to form a thiohemiketal (II). Conversion to a thioether (IV) occurs via an episulfonium ion intermediate (III) when the leaving group (O-sulfate) is oriented anti to the sulphur atom (as in the reactive epimer toxin C3 or GTX1).

[0053] In the second step of the proposed reaction mechanism the thiol group of the dithiol reacts with the sulphur of the thioether (IV) to form a disulfide thereby yielding an enolate that readily hydrates to GTX6 (Va) neoSTX (Vb).

[0054] The optimal pH for the conversion of GTX1,4 to neoSTX has been determined to be around 7.5 (cf. Laycock et al (1995)) and by analogy it is anticipated to be similar for the conversion of C3,4 to GTX6. Without wishing to be bound by theory it is believed a pH in this range ensures both (i) an optimal rate of epimerisation between the gonyautoxin epimers and (ii) optimal degrees of electrophilicity at C-12 and deprotonation of the dithiol used as the reducing agent.

[0055] The use of dithiols such as dithiothreitol (DTT) and dithiobutylamine (DTBA) is preferred over the use of monothiols such as glutathione (GSH) and mercaptoethanol (ME) (cf. Sakamoto et al (2000) and Sato et al (2000)). Higher rates of conversion are obtained when using the dithiols, rendering them more suitable for use in the production of GTX6 and neoSTX on a preparative scale.

[0056] The excess dithiol, sodium phosphate buffer and unreacted epimers have also been found to be most conveniently removed from the neoSTX containing conversion product by the use of cation exchange chromatography. The silica based weak cation exchange sorbent Sepra™ WCX has been determined to be a suitable sorbent as it has been determined not to retain the dithiol DTT. Trials of the polymeric based weak cation exchange sorbent Strata–X™ CW (Phenomenex) determined this sorbent to be unsuitable for purification of neoSTX from the conversion product on a preparative scale. The excess dithiol is retained by both an ion exchange and a reverse phase mechanism when using this sorbent. Although a portion of the excess dithiol is eluted with organic solvents such as acetonitrile/water a further portion is eluted with 1 M acetic acid frustrating the purification of the neoSTX when using this sorbent. Similar observations are anticipated to be made when the product of the reductive desulfonation is GTX6.

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EXAMPLE

A. Materials

[0057] 10 mM acetic acid (0.6 g/L in deionised water); amended seawater (7 mL/L L1 nutrient medium in seawater); deionised water (Milli-Q™, Merck-Millipore); 1 N hydrochloric acid (1 N HCl); L1 nutrient medium (1.25 g/L EDTA, 0.91 g/L FeCl.sub.3.6H.sub.2O, 0.29 mL/L trace metal stock solution, 21.6 g/L NaNO.sub.3, 1.44 g/L NaH.sub.2PO.sub.4 and 14.4 mL/L vitamin stock solution); Mobile Phase A (2.2 g/L sodium heptane sulphonic acid (sodium salt) and 0.31 g/L 85% phosphoric acid adjusted to pH 7.1 with 25% ammonium hydroxide); Mobile Phase B (0.1% acid in acetonitrile); Mobile Phase C (0.1% acetic acid in deionised water); roll of tubular (230 mm × 200 m × 250 .Math.m) low density poly(ethylene) (LDPE) plastic (Amcor Limited); seawater (30 to 37 ppt salinity); trace metal stock solution (2.5 g/L CuSO.sub.4.5H.sub.2O, 20 g/L Na.sub.2MoO.sub.4.2H.sub.2O, 23 g/L ZnSO.sub.4.7H.sub.2O, 11.9 g/L CoCl.sub.2.6H.sub.2O, 178 g/L MnCl.sub.2.4H.sub.2O, 1.3 g/L H.sub.2SeO.sub.3, 2.6 g/L NiSO.sub.4.6H.sub.2O, 1.8 g/L Na.sub.3VO.sub.4 and 1.9 g/L K.sub.2CrO.sub.4); vitamin stock solution (0.01 g/L biotin, 2 g/L thiamine and 0.01 g/L vitamin B12).

[0058] Vitamin stock solutions are filter sterilised and aseptically added through a 0.22 .Math.m syringe filter during preparation of amended seawater following the autoclaving of other ingredients.

B. Inocula

[0059] Isolates are obtained from naturally occurring algal blooms in coastal waters. Species responsible for harmful algal blooms include Alexandrium minutum, Alexandrium pacificum (formerly referred to as Alexandrium catenella), Alexandrium tamarense and Gymnodium catenatum. Individual isolates have been evaluated for their production of toxins when cultured in bulk according to the following protocols. The concentrations of toxins detected in extracts of these cultures are provided in Table 1. The concentrations are expressed as picogram per cell and are determined for extracts that have not been subjected to mild acid hydrolysis. Representations of the structures of the toxins referred to in Table 1, FIG. 2 and FIG. 3 are provided below. The representations are of the structures of the toxins in their neutral (uncharged) form.

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TABLE-US-00001 Concentration (pg/cell) of toxins detected in extracts of cultures of isolates of the species Alexandrium minutum, Alexandrium pacificum, Alexandrium ostenfeldii and Gymnodinium catenatum. The relatively high concentrations of C3,4 present in cells of isolates of Gymnodinium catenatum are emphasised. Species Designation cells/mL GTX-1,4 C1,2 C3,4 dcGTX-2,3 GTX-2,3 GTX-5 GTX-6 dcSTX dcNEO STX NEO A. minutum CAWD011 55035 3.1 0.01 ND 0.02 0.57 0.01 ND ND ND 0.39 0.33 A. minutum CAWD012 25961 ND ND ND ND ND ND ND 0.01 ND ND ND A. pacificum CAWD044 5891 0.37 0.55 0.14 0.06 0 0.01 0.07 0 0 0 0.03 A. pacificum CAWD045 8004 0.59 0.67 0.13 0.04 0 0.19 0.15 0.01 0 0 0.18 A. pacificum CAWD046 5602 4.2 9.53 2.6 1.1 0.37 3 5.2 0.12 ND 0.12 0.21 A. pacificum CAWD047 7505 1.9 3.09 2.7 0.42 0.19 0.42 4.2 0.01 ND 0.03 0.17 A. pacificum CAWD049 2493 3.2 5.04 2.2 1.1 0.24 0.84 3.1 0.06 ND 0.12 ND A. pacificum CAWD050 6310 0.43 1.09 0.38 0.07 ND 0.44 0.04 ND ND 0.01 0.15 G. catenatum CAWD101 7788 0.49 170 48 8.4 3 1.3 0.25 0.02 ND 0.03 0.08 G. catenatum CAWD102 1953 1.3 9.3 85 3.2 ND 1.2 0.35 0.04 ND ND 0.32 A. pacificum CAWD121 9027 ND ND ND 0.02 ND 0.01 ND 0.01 ND ND ND G. catenatum CAWD126 17339 0.51 2.80 47 1.40 0.07 0.34 0.31 0.02 0.48 ND 0.08 A. ostenfeldii CAWD135 35571 ND 0.22 ND 0.02 0.77 ND ND ND ND 0.02 ND A. ostenfeldii CAWD136 2782 ND ND ND 0.07 0.49 ND ND ND ND 0.01 ND A. pacificum CAWD234 42662 5.6 3.25 1.6 0.18 0.17 1.5 0.2 0.04 ND 0.09 1.3 A. pacificum CAWD235 14680 1.5 2.88 1 0.2 0.14 2.5 0.13 0.09 ND 0.19 1.3 A. pacificum CAWD260 2136 0.06 ND ND 0.09 ND 0.06 ND 0.02 0.05 0.01 ND A. pacificum CAWD261 2502 ND ND ND 0.05 ND ND ND 0.01 ND ND ND A. pacificum CAWD262 1227 3.4 3.12 0.54 1.8 0.22 1.6 0.12 0.27 ND 0.18 0.35

C. Culture

[0060] Individual isolates are cultured in aerated vertical columns of amended seawater in a plurality of hanging bags. The bags are hung in an incubation room maintained at a temperature of 16 to 20° C. The bags are illuminated according to a 16-hour/8-hour light/dark cycle. Automated carbon dioxide (CO.sub.2) dosing is used to maintain the pH in the range 8 to 9 during the light phase of the light/dark cycle. Standard personal protection equipment is worn by operators to minimise the risk of exposure to toxins.

[0061] Reference is made to FIG. 1 of the accompanying drawings pages where a plan view of a cut and welded hanging bag (1) for use in the bulk culture of isolates is provided. The bags are formed from a roll of tubular plastic by cutting and heat welding according the following protocol.

[0062] After discarding the first two metres of a new roll of tubular plastic a 20 cm section is cut and the open ends of the excised section each sealed by a heat weld. The inner surfaces of the sealed section of tubular plastic roll are subjected to microbiological evaluation before the remainder of the new roll of tubular plastic is used for the fabrication of hanging bags.

[0063] A two-metre length (A+G) is dispensed from the tubular plastic roll and a hanging loop (2) formed at the first end. The hanging loop (2) is formed by folding back a 10 cm section (A) of the tubular plastic and heat welding together the four layers of plastic to provide a first heat weld (3) proximal to the cut first end. A second heat weld (4) is formed within the folded back section (A) parallel and spaced apart from the first heat weld (3) by about 2 cm followed by diagonal heat welds (5,6) traversing the region between the horizontal first and second heat welds (3,4). The corners of the folded back section distal from the welds are then cut (7,8) to provide a hanging loop (2) of a width (B) capable of supporting the length of tubular plastic when filled with a volume of amended seawater.

[0064] The second end of the length of tubular plastic is sealed by double diagonal heat welds (9,10) converging to a point (11) proximal to the centre of the second end. The integrity of each double diagonal heat weld (9,10) is inspected visually before each triangle of plastic outside the conical sealed end is cut (12,13) away. The conical portion of the sealed end has a depth (C) of around 20 cm. Each hanging bag (1) is capable of containing a culture volume of approximately 24 L.

[0065] Prior to filling with inoculum and amended seawater a hanging bag (1) is hung in position and the top outside corner surface sterilised by wiping with isopropanol. A downward pointing first hole is formed in the surface sterilised region using a sterile pick. The tip of a sterilised air vent inserted into the hole and taped to the outside of the bag using PVC insulation tape (50 mm width). A region of the outer surface of the conical sealed end is also surface sterilised by wiping with isopropanol. A second hole is formed in the surface sterilised region using a sterile pick. The downward pointing tip of a sterile inoculation line is inserted into the hole and the line taped to the outside of the bag using PVC insulation tape (50 mm width).

[0066] Inoculum is fed into the hanging bag from a parent culture via the inoculation line. The line from the parent culture and the inoculation line to the hanging bag are each connected to a manifold. Air is purged from the lines by pumping amended seawater into both. The parent culture is then allowed to flow into the hanging bag. Pressurised air is introduced into the hanging bag containing the parent culture via its sterilised air vent. Equal volumes of the parent culture are transferred to multiple hanging bags. The inoculum containing hanging bags are then filled with the amended seawater to a fill line (D). Once the hanging bags are filled, the inoculation lines are disconnected from the manifold and connected to an air line via a sterilised air filter. The culture volume is aerated via the air line.

[0067] To monitor the pH of the medium during the culture the surface of a pH probe, including the glass bulb, is washed with deionised water and 70% (w/w) ethanol. A region of the outer surface of the hanging bag around 4 cm above the fill line is surface sterilised by wiping with isopropanol and a hole made in the bag within this region using a sterilised 10 mL pipette tip. The pH probe is inserted via the hole and into the culture volume and held flat against the inner side wall of the bag.

[0068] Under these incubation conditions the isolates have a doubling time of about two days and are harvested when the cell density has reached 7 × 10.sup.4 to 10.sup.5 cells/mL as determined by microscopic image cytometry.

D. Harvest

[0069] With aeration maintained the pH probe is removed and a volume of about 9 mL glacial acetic acid introduced into the culture volume. After about 10 to 20 minutes a volume of about 100 mL of a suspension of hydrated bentonite clay is added to the culture to provide a dosage of about 4 mL/L of culture. After a further 5 to 10 minutes the inoculation line is clamped and disconnected from the air filter. Settling of cells occurs over a period of time of at least 2 hours. The settled cells are drained into a centrifuge bottle and the collected volume (300 to 400 mL/bag) centrifuged in balanced bottles at a force of 1,500 × g for a period of time of 5 minutes. The supernatant is discarded, and the harvested cells weighed before storing frozen at -20° C.

E. Extraction

[0070] Extracts for (i) determining toxin profiles and monitoring toxin production, or (ii) preparation of C3,4 are prepared according to the following methods.

[0071] For monitoring toxin production, 10 mL of the culture volume is transferred to a polypropylene centrifuge tube and the cells pelleted by centrifugation at a force of 1,500 × g for a period of time of 5 minutes. The supernatant is discarded, and the pellet resuspended in a volume of 250 .Math.L 1 mM acetic acid. The volume is sonicated for a period of time of 2 minutes and heated to 80° C. for a period of time of 10 minutes. The cellular material is pelleted by centrifugation at a force of 3,220 × g for a period of time of 5 minutes and 10-fold and 20-fold dilutions of the supernatant then prepared in 80% (v/v) acetonitrile/0.25% (v/v) acetic acid.

[0072] For preparation of C3,4, the frozen pellet of harvested cells is defrosted before resuspending in an equal volume of 0.5% acetic acid and leaving at room temperature for a period of time of 30 to 60 minutes. The suspension in acetic acid is then heated and maintained at a temperature of 85±2° C. in a water bath for a period of time of 10 to 15 minutes. The heated suspension is then cooled in an ice slurry before centrifugation at a force of 3,990 × g for a period of time of 2 minutes and the first supernatant decanted to a collection vessel. An equal weight of 0.5% acetic acid is added to the pelleted cellular material, mixed well, and centrifuged at a force of 3,990 × g for a period of time of 5 minutes. The second supernatant is decanted to the collection vessel and the total volume of collected supernatants reduced by rotary evaporation under vacuum (less than 15 mBar) at a temperature of 30° C. (For example, portions of a total volume of 10 litres of supernatant are transferred from the collection vessel to a weighed 5 litre round bottom flask and the volume reduced by rotary evaporation until an extract having a weight of 800±50 g and a density between 1.08 to 1.12 g/mL is obtained.) The concentrated extract is stored frozen at -20° C.

F. Analysis

[0073] A sample of extract is prepared for analysis using activated carbon solid phase extraction (SPE). A Supelclean™ ENVI-Carb™ SPE tube (bed wt. 250 mg, volume 6 mL) is conditioned with a volume of 3 mL of 20% acetonitrile/1% acetic acid followed by a volume of 3 mL of 0.025% ammonia. Following elution to the top frit the tube is loaded with a total volume of 400 .Math.L consisting of 10 to 400 .Math.L of extract and quantum sufficit deionised water. The cartridge is eluted to the top frit using a vacuum of -15 to -20 kPa and washed with a volume of 3 mL of deionised water before elution with a volume of 5 mL of 20% acetonitrile/1% acetic acid. The eluate is collected in a polypropylene tube and a volume of 10 .Math.L diluted 4-fold with the addition of acetonitrile in a polypropylene autosampler vial. The contents of the vial are mixed on a vortex mixture with further dilution as required.

Method 1

[0074] Aqueous extracts and fractions enriched for the presence of C3,4 are analysed by high pressure liquid chromatography (HPLC) (Shimadzu Prominence) with ultraviolet (UV) detection according to this Method 1. Samples are diluted with 10 mM acetic acid to provide a nominal concentration of 200 .Math.g/mL. A volume of 40 .Math.L of diluted sample is injected onto a column (4.6 × 150 mm) of 3.5 .Math.m Zorbax Bonus RP eluted with Mobile Phase A at a flow rate of 1 mL/min for a period of time of 30 minutes while being maintained at a temperature of 20° C. The absorbance of the eluate is monitored at wavelengths of 210 nm (purity) and 245 nm (quantity) using a photodiode array detector (PAD). The purity of samples is calculated based on the percentage area at 210 nm and retention time with reference to a standard at a concentration of approximately 200 .Math.g/mL C3,4. Samples are analysed in triplicate with 10 mM acetic acid used as a blank and the chromatogram obtained subtracted from that obtained for all samples.

Method 2

[0075] Extracts and fractions enriched for the presence of C3,4 are analysed and quantified by liquid chromatography-mass spectrometry (LC-MS) (Shimadzu 8050) according to this Method 2. Samples are diluted to an appropriate concentration using 80% acetonitrile/0.25% acetic acid. A mixed standard containing a number of reference paralytic shellfish toxins (PSTs) is used. A maximum volume of 2 .Math.L of diluted sample is injected by means of an auto sampler maintained at 4° C. onto a column (2.1 × 100 mm) of 1.7 .Math.M Waters Acquity UPLC BEH amide eluted at a flow rate of 0.4 mL/min while being maintained at a temperature of 20° C. The column is eluted stepwise with 75% Mobile Phase B/25% Mobile Phase C for a period of time of 5 minutes following injection, followed by 55% Mobile Phase B/45% Mobile Phase C for a period of time of 0.50 min before reverting to 75% Mobile Phase B/25% Mobile Phase C. The eluate is monitored by mass spectrometry using selective ion monitoring in ESI- and ESI+ ionisation modes.

Method 3

[0076] Purified products of extraction, fractionation and conversion are analysed for quantity and purity according to this Method 3. Isolated products (C3,4 or GTX6 or neoSTX) are diluted to a concentration of 200 .Math.g/mL in 10 mM acetic acid. The diluted sample is then further diluted 100-fold in 8% acetonitrile/0.25% acetic acid to provide a solution of product at a concentration of 20 mg/mL for quantitative analysis. A mixed standard containing a number of reference paralytic shellfish toxins (PSTs) is also prepared in the same solvent. A solution of 2 .Math.L of the diluted product (20 ng/mL) is injected by means of an autosampler maintained at a temperature of 40° C. onto a column (2.1 × 100 mm) of 1.7 .Math.m Waters Acquity UPLC BEH amide eluted at a flow rate of 0.6 mL/min while being maintained at a temperature of 60° C. The column is eluted stepwise with 80% Mobile Phase B/20% Mobile Phase C for a period of time of 6 minutes following injection, followed by 55% Mobile Phase B/45% Mobile Phase C for a period of time of 0.50 minutes before reverting to 80% Mobile Phase B/20% Mobile Phase C. The eluate is monitored by mass spectrometry monitoring in ESI- and ESI+ ionisation modes.

G. Purification

[0077] The concentrated extract is thawed at room temperature. The extract is divided between two balanced 500 mL conical centrifuge bottles and centrifuged at a force of 4000 × g for a period of time of 10 minutes. The supernatant is decanted and filtered under reduced pressure through a series of 110 mm diameter hardened ashless and glass microfiber filter papers (Whatman™ grade 540, Whatman™ grade 542 and Whatman™ grade GF/A).

[0078] The filtered extract is then subjected to crossflow ultrafiltration by recirculation through two filters (VivaFlow 200) connected in parallel with an outlet pressure no greater than 2.5 bar until the volume of the extract has been reduced to a volume of 10 to 20 mL of retentate.

[0079] The retentate is transferred to a 100 mL bottle and made up to a total volume of 100 mL with deionised water. The diluted retentate is similarly subjected to rounds of crossflow ultrafiltration reducing the volume of retentate to 10 to 20 mL before making up to a total volume of 100 mL with deionised water.

[0080] A 32 mm 5 .Math.m syringe filter (Pall Corp.) is installed on the inlet of a 50 g Sepabeads™ SP-270 (Supelco) SPE cartridge conditioned with 500 mL of deionised water at a flow rate of 30 mL/minute. The washed retentate is passed through the conditioned SPE cartridge at a flow rate of 30 mL/minute and the effluent collected. The SPE cartridge is then eluted with 200 mL of deionised water at a flowrate of 30 mL/minute and the effluent collected.

[0081] The volume of the combined effluents is reduced by rotary evaporation under vacuum (less than 15 mBar) at a temperature of 30±2° C. The volume is reduced to provide a weight of 500±50 g, the density determined gravimetrically, and the total reduced volume calculated on this basis.

[0082] Prior to purification of the C3,4 from the reduced volume on a preparative scale a sample of the reduced volume is prepared and analysed as described (F. Analysis). A 10,000-fold dilution of the sample should provide a concentration of C3,4 in the range 20 to 50 ng/mL.

[0083] The total reduced volume is desalted by loading on a 100 g 25 × 450 mm carbon column (Enviro-Clean™ Graphitized Carbon Non-Porous, UCT) conditioned with 1 L of deionised water. The reduced volume is loaded at a flow rate of 30 mL/min using a Mini-Flash Pump (Sorbent Technologies, Inc.) and eluted at a flow rate of 15 mL/min with a stepwise gradient of deionised water for a period of time of 40 minutes followed by 0.2% (v/v) acetic acid/30% (v/v) acetonitrile for a period of time of 40 minutes.

[0084] The eluate is monitored at 205 nm using an inline UV detector and sequential volumes of 10 mL of eluate collected as fractions. Volumes of 5 .Math.L of selected fractions are diluted to a total volume of 10 mL with 0.25% (v/v) acetic acid/80% (v/v) acetonitrile and submitted to LC-MS analysis as described (F. Analysis). The C3,4 containing fractions are combined.

[0085] The pH of the desalted combined fractions collected from the carbon column is adjusted to 7.8 using concentrated ammonium hydroxide at a rate of approximately 2 .Math.L/mL. The pH adjusted volume is then loaded on a 35 × 460 mm column of 280 g Sepra™ WCX-NH.sub.4.sup.+ conditioned using a volume of 1 L of 50 mM ammonium bicarbonate at a flow rate of 30 mL/min. The column is eluted with a gradient of 10 to 80% 0.5 M ammonium bicarbonate over a period of time of 100 minutes. The eluate is monitored at wavelengths of 205 nm and 245 nm using an inline UV detector. Sequential volumes of 25 ml are collected as fractions and combined according to the UV monitoring and LC-MS analysis where required (F. Analysis). Fractions containing greater than 2% of the total amount of C3,4 are combined and the pH of the combined fractions reduced to 6.5 by the dropwise addition of glacial acetic acid. A 5,000-fold dilution of the acidified volume is subjected to LC-MS analysis as described (F. Analysis) and the total quantity and yield of C3,4 and the ratio to GTX6 calculated.

[0086] The combined fractions from the weak cation exchange chromatography are loaded onto a 35 × 480 mm column of 175 g Sepra™ ZT-WCX-H.sup.+ form conditioned using 1 L of deionised water at a flowrate of 30 mL/min. The loaded column is eluted with a continuous gradient of 0 to 100% 1 M acetic acid in water over a period of time of 100 minutes and sequential volumes of 25 mL of eluate collected as fractions while monitoring at a wavelength of 205 nm using an inline UV detector.

[0087] The C3,4 containing fractions are collected from baseline to baseline and the total volume reduced to a volume of 10 to 20 mL by rotary evaporation at 30° C. under reduced pressure of less than 15 mBar. A 500,000-fold dilution of the reduced volume is subjected to LC-MS analysis as described (F. Analysis) and the total quantity and yield of C3,4 calculated on this basis.

[0088] The C3,4 containing reduced volume is loaded on a 50 × 500 mm column of P2 gel conditioned with 2 L of 100 mM acetic acid at a flowrate of 50 mL/min and eluted isocratically with the same conditioning mobile phase. The eluate is monitored at a wavelength of 205 nm for a period of time of 200 minutes using an inline UV detector and sequential volumes of 10 mL of eluate collected as fractions. The C3,4 containing fractions are collected from baseline to baseline and the total volume reduced by rotary evaporation at a temperature of 30° C. under reduced pressure of less than 15 mBar.

[0089] The reduced volume of combined fractions obtained by gel filtration is transferred to a pre-weighed 100 mL round bottom flask and evaporated to dryness by rotary evaporation at a temperature of 30° C. under a reduced pressure of less than 15 mbar followed by drying in a freeze dryer at a shelf temperature of 10° C. and pressure of 0.05 mbar for 24 hours. The open mouth of the round bottom flask is securely covered with an air permeable, lint free tissue before placing in the freeze dryer. The yield of purified C3,4 is determined gravimetrically. The purified C3,4 is dissolved in a known volume of deionised water to provide a solution containing 70 to 100 mg/mL of C3,4.

[0090] A 500-fold dilution of the solution in 10 mM acetic acid is prepared and analysed as described (F. Analysis). The final volume required to provide a concentration of 40 to 45 mg/mL of C3,4 is calculated and the solution transferred via a filter to a pre-weighed 10 mL amber glass vial, rinsing with deionised water to provide a transferred volume having the target concentration of 40 to 45 mg/mL. A 200-fold dilution of the transferred solution is prepared in 10 nM acetic acid and analysed as described (F. Analysis). The dilution is diluted a further 10-fold in 10 mM acetic acid and analysed for purity as described (F. Analysis).

H. Desulfonation

Analytical Scale

[0091] Preliminary studies were performed to confirm the viability of the proposed conversion of C3,4 to GTX6. A volume of 400 .Math.L of phosphate buffer (pH 7.3) at a concentration of 200 mM and a volume of 9.6 .Math.L of a solution of dithiothreitol (DTT) at a concentration of 421 mM were added to a volume of 100 .Math.L of a solution of C3,4 at a concentration of 3.6 mM to provide a reaction mixture containing molar equivalents in the ratio 20:10:1.

[0092] The reaction mixture was heated in a water bath to a temperature of 50° C. for a period of time of 60 minutes before being cooled in ice water to provide a solution of reaction product. A volume of 10 .Math.L of the solution of reaction product was diluted 10,000-fold with 80% (v/v) acetonitrile/0.25% acetic acid and analysed [F. Analysis].

TABLE-US-00002 Calculation of yield of GTX6. Toxin Concentration (.Math.g/mL) 0 60 min C3, 4 354 - GTX6 - 238

[0093] A yield of 67% (w/w) was obtained (Table 2), i.e., a molar yield of greater than 80%. It is anticipated that further improvements in yield and reduced reaction times (less than one hour) will be achievable when using higher concentrations (greater than 80 mM of C3,4). Reduced reaction times are also anticipated to reduce the formation of decarbamoylneosaxitoxin (dcNEO) attributed to the hydrolysis of GTX6 at the elevated pH of the reaction mixture.

Preparative Scale

[0094] A quantity of 183 mg (as the free base) of C3,4 purified according to the preceding steps from an extract of a culture of an isolate of Gymnodinium catenatum is dissolved in a total volume of 5 mL of dilute acetic acid and mixed with a volume of 45 mL of 0.2 M phosphate buffer at a pH of 7.5 in a 100 mL round bottom flask. The mixture is placed on ice and the pH adjusted from 6.8 to 7.5 with the addition whilst stirring of solid sodium carbonate. A quantity of 1.5 g of dithiothreitol (DTT) is added to the pH adjusted mixture and its dissolution promoted by placing the reaction mixture containing round bottom flask in an ultrasonic bath before transferring to a water bath maintained at a temperature of 50° C. Aliquots of a volume of 10 .Math.L are removed from the reaction mixture and transferred to the water bath (T=0) and periodically (every 15 minutes) thereafter. Aliquots are diluted 50-fold by the addition of a volume of 490 .Math.L 80% acetonitrile 0.25% acetic acid immediately following removal from the reaction mixture and analysed by LC-MS as described (F. Analysis) to monitor the progress of the reaction in near real time. After incubation for 45 minutes at 50° C. the reaction mixture is chilled by transferring the round bottom flask to an ice slurry.

I. Isolation

[0095] The desulfonation product is loaded onto a quantity of 39 g Sepra™ WCX packed in an empty flash cartridge (Grace) and preconditioned with a volume of 250 mL of 50% (w/w) acetonitrile followed by a volume of 250 mL of deionised water. The conversion product is loaded onto the packed cartridge with rinses of deionised water with collection of the effluent (about 200 mL). The dissolution of any crystals formed during storage of the conversion product at 4° C. is achieved by the addition of a minimal amount of deionised water. The loaded packed cartridge is then eluted at a rate of 50 mL/min with a total volume of 1.5 L, followed by elution with a continuous gradient to 1 M acetic acid over 20 minutes and the collection of sequential volumes of 10 mL of eluate as fractions while monitoring UV absorbance at 205 nm and 254 nm.

[0096] A volume of 5 .Math.L of fractions demonstrating UV absorbance at 205 nm is diluted 100,000-fold in 80% acetonitrile 0.25% acetic acid and analysed by LC-MS. Fractions confirmed to comprise GTX6 are combined, frozen at -70° C. and lyophilised. The dried GTX6 is dissolved in a small volume of 10 mM and transferred to a pre-weighed 10 mL glass vial and a volume of 10 .Math.L analysed.

J. Hydrolysis

[0097] To provide neosaxitoxin the purified GTX is subjected to mild acid hydrolysis (Shimizu (1984)).

Analytical Scale

[0098] Preliminary studies were performed to confirm the viability of the proposed conversion of GTX6 to neosaxitoxin. A volume of 2 .Math.L of concentrated hydrochloric acid (HCl) was added to a volume of 118 .Math.L of a solution of GTX6. The acidified solution (0.2 M HCl, pH 0.97) was then heated in a boiling water bath for a period of time of 10 minutes before cooling in iced water.

[0099] A volume of 10 .Math.L of the cooled acidified solution was diluted 1,000-fold with 80% (v/v) acetonitrile/0.25 % acetic acid and analysed [F. Analysis].

TABLE-US-00003 Calculation of yield of neosaxitoxin. (* Mean of duplicate samples.) Toxin Concentration* (.Math.g/mL) 0 min 60 min GTX 6 16.74 - Neosaxitoxin - 14.05

[0100] A yield of 84% (w/w) was obtained (Table 3), i.e., a qualitative conversion of GTX6 to neosaxitoxin.

Preparative Scale

[0101] Larger amounts of purified GTX6 are subjected to mild acid hydrolysis by dissolving in a volume of 1 N HCl in a round bottom flask and heating to 80° C. for at least 30 min. The hydrolysate is chilled by transferring the round bottom flask to an ice slurry.

K. Purification

[0102] The pH of the cooled acidified solution is adjusted to about 6.6 before being loaded onto a Sepra™ WCX column and eluting with approximately three column volumes of water followed by approximately five column volumes of 1 M acetic acid. Neosaxitoxin containing fractions are combined and evaporated to dryness before being transferred to a final vessel in 0.1 % (w/v) acetic acid. The product neosaxitoxin similarly isolated from the hydrolysate [I. Isolation] .

[0103] Although the invention has been described with reference to embodiments or examples it should be appreciated that variations and modifications may be made to these embodiments or examples without departing from the scope of the invention. Where known equivalents exist to specific elements, features or integers, such equivalents are incorporated as if specifically referred to in this specification. Variations and modifications to the embodiments or examples that include elements, features or integers disclosed in and selected from the referenced publications are within the scope of the invention unless specifically disclaimed. The advantages provided by the invention and discussed in the description may be provided in the alternative or in combination in these different embodiments of the invention.

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