Semisynthesis of S-Desoxybrevetoxin-B2 and Brevetoxin-B2, and

Publication Date (Web): March 13, 2008 ... NOAA-National Ocean Service. , # ... chain, causing intoxication of marine animals and people consuming sea...
0 downloads 0 Views 173KB Size
Download PDF
944

Chem. Res. Toxicol. 2008, 21, 944–950

Semisynthesis of S-Desoxybrevetoxin-B2 and Brevetoxin-B2, and Assessment of Their Acute Toxicities Andrew I. Selwood,† Roel van Ginkel,† Alistair L. Wilkins,‡ Rex Munday,| John S. Ramsdell,⊥ Dwayne J. Jensen,§ Janine M. Cooney,§ and Christopher O. Miles|,#,* Cawthron Institute, PriVate Bag 2, Nelson, New Zealand, Chemistry Department, The UniVersity of Waikato, PriVate Bag 3105, Hamilton 3240, New Zealand, HortResearch Ltd., Ruakura Research Centre, PriVate Bag 3123, Hamilton, New Zealand, AgResearch Ltd., Ruakura Research Centre, PriVate Bag 3123, Hamilton 3240, New Zealand, Marine Biotoxins Program, NOAA-National Ocean SerVice, 219 Fort Johnson Road, Charleston, South Carolina 29419, and National Veterinary Institute, PB 8156 Dep., NO-0033 Oslo, Norway ReceiVed December 17, 2007

Brevetoxins are neurotoxins associated with blooms of marine algae such as Karenia breVis and can accumulate in the marine food chain, causing intoxication of marine animals and people consuming seafood. Brevetoxin-B2 (5) is a toxic metabolite produced in shellfish exposed to algae that contain brevetoxin-B (1). S-Desoxybrevetoxin-B2 (4) has been proposed as a cometabolite produced during this transformation, and while LC-MS analyses suggest its presence in shellfish, it has not yet been isolated and characterized. Studies on these materials are severely constrained by the difficulty of obtaining and purifying them from natural sources. We have developed a convenient one-pot conversion of commercially available brevetoxin-B (1) into S-desoxybrevetoxin-B2 (4), and a simple method for converting 4 into brevetoxin-B2 (5). Full NMR and mass-spectral characterization of 4 and 5 confirmed their structures and showed that the ratio of diastereoisomers in the synthetic 4 and 5 was similar to that observed in naturally contaminated shellfish. The LD50 values for 4, 5, and dihydrobrevetoxin-B (6) by ip injection in mice were 211, 400, and 250 µg/kg, respectively. The methodology for synthesis of brevetoxin metabolites should greatly facilitate toxicological, biochemical and immunochemical studies of these substances, as well as the production of analytical standards. Introduction The toxic marine dinoflagellate alga Karenia breVis (formerly Gymnodinium breVe and Ptychodiscus breVis) is well known for producing neurotoxic polyether compounds known as brevetoxins (1), including brevetoxin-B (1) (2) (also known as PbTx-2 and brevetoxin-2) and dihydrobrevetoxin-B (6) (3) (also known as PbTx-3 and brevetoxin-3) (Figure 1), which can accumulate in filter-feeding shellfish. Consumption of shellfish contaminated with brevetoxins can cause poisoning of human consumers (4), a syndrome known as neurotoxic shellfish poisoning (NSP)1. Much of the toxicity associated with NSP incidents is now recognized as being due to the presence of metabolites of brevetoxin-B (1) (5, 6). One of the more abundant of the brevetoxin shellfish metabolites is the sulfoxide analogue of the cysteine conjugate, brevetoxin-B2 (5) (7–9). The corresponding sulfide analogue (4) has also been tentatively identified by LC-MS in shellfish (5, 8, 9), but the difficulty of purification has so far prevented its isolation from natural sources in sufficient quantity and purity for structural confirmation. Other brevetoxin metabolites from shellfish (6, 10–12) also appear to * To whom correspondence should be addressed. Phone: +64-7-8385041 and +47-2321-6226. Fax: +64-7-838-5189 and +47-2321-6201. E-mail: [email protected] and [email protected]. † Cawthron Institute. ‡ The University of Waikato. § HortResearch Ltd., Ruakura Research Centre. | AgResearch Ltd., Ruakura Research Centre. ⊥ NOAA-National Ocean Service. # National Veterinary Institute. 1 Abbreviations: Boc, t-butyloxycarbonyl; BTX, brevetoxin; HR-MS, high resolution mass spectrometry; NSP, neurotoxic shellfish poisoning.

Figure 1. Conversion of brevetoxin-B (1) into S-desoxybrevetoxin-B2 (4) and brevetoxin-B2 (5). Also shown are the structures of dihydrobrevetoxin-B (6); the minor product (7) from reaction of 1 with cysteine; the major product (8) from reaction of 1 with 2-hydroxythiepan; and the product (9) from reaction of 1 with 2-[(5-fluoresceinyl)aminocarbonyl]ethyl mercaptan. Structures 4 and 5 are based on full NMR and mass spectrometric characterization, while structures 7-9 are based on LC-MS/MS evidence and chemical reactivity.

be difficult to isolate so that good quality NMR data are scarce and few standards are available for quantitative analyses. We have developed an efficient semisynthetic procedure for converting commercially available 1 into 4 and 5. Material produced

10.1021/tx700441w CCC: $40.75  2008 American Chemical Society Published on Web 03/13/2008

S-DesoxybreVetoxin-B2 and BreVetoxin-B2

by a modification of this procedure has already provided valuable information on the in vitro toxicity of brevetoxin metabolites (13). The 1H and 13C NMR chemical shift assignments for semisynthetic S-desoxybrevetoxin-B2 (4) and brevetoxin-B2 (5) in CD3OD, obtained to confirm their structures, will aid future NMR structural studies of brevetoxin metabolites.

Materials and Methods General. Brevetoxin-B (1) was a gift from M. Poli (Integrated Toxicology Division, USAMRIID, Fort Detrick, MD 21702, USA), and dihydrobrevetoxin-B (6) was purchased from Calbiochem (San Diego, CA). Preparative separations were performed by stepwise gradient elution on 200-mg Strata C18-T widepore SPE (Phenomenex, Torrance, CA; part no 8B-S004FBJ) cartridges. Products were vacuum-dried to constant weight before weighing on a microbalance. L-Cysteine and N-Boc-Lcysteine were from Sigma (St Louis, MO), and 2-hydroxythiepan and 2-[(5-fluoresceinyl)aminocarbonyl]ethyl mercaptan were from Toronto Research Chemicals, North York, ON, Canada. All solvents were of analytical grade or better and MilliQ water was used throughout. LC-MS analysis (method 1) was used to monitor progress of preparative reactions and to characterize all intermediates. Preliminary Trial Thiolations. Brevetoxin-B (1) (25 µg) in 80% MeOH (100 µL) was added to vials containing (1) powdered L-cysteine (0.73 mg); (2) 2-hydroxythiepan (0.16 mg); and (3) 2-[(5-fluoresceinyl)aminocarbonyl]ethyl mercaptan (ca. 10 µg); and (4) an aliquot was kept as an unreacted control. After 2 h, the solutions were diluted by addition of 900 µL of 80% MeOH, filtered, and analyzed by LC-MS (method 2). S-Desoxybrevetoxin-B2 (4). N-Boc-L-cysteine (10 mg) was dissolved in water (2.5 mL), and 0.05 M Na2CO3 (1.5 mL) was added. This was added to brevetoxin-B (1) (1 mg) in MeCN (5 mL) with swirling, after which the pH was 8–8.5. The solution was left at room temperature for 30 min before analysis by LCMS, which showed nearly quantitative conversion to N-BocS-desoxy-42-oxobrevetoxin-B2 (2) (m/z 1116 ([M + H]+) and m/z 1016 ([M + H - Boc]+)). The reaction solution of 2 was cooled in ice–water, and precooled HCl (1 mL, 1 M, 4 °C) was added with swirling, affording a solution of pH 1.5–2. Then precooled NaBH3CN (1 mL, 1 M, 4 °C) was added with swirling. The pH remained at 1.5–2. LC-MS analysis of a sample taken less than 1 min after the addition of the reductant showed nearly quantitative conversion to N-Boc-S-desoxybrevetoxin-B2 (3) (m/z 1118 ([M + H]+) and m/z 1018 ([M + H - Boc]+)). HCl (10 mL, 5 M) was then added with swirling, resulting in some evolution of gas. LC-MS showed that complete deprotection of 3 occurred over 1.5–2 h to afford S-desoxybrevetoxin-B2 (4) (m/z 1018 ([M + H]+)). Extraction and purification of 4 was achieved with a 400-mg Strata C18-T widepore SPE (produced by combining the contents of two 200-mg Strata C18-T widepore SPE cartridges) preconditioned with MeCN followed by 10% MeCN, by applying the reaction solution (composed of ca. 10% MeCN in water) at ca. 1 mL/min under an argon blanket. The SPE column was then washed with 5 mL of 10% MeCN in water and eluted with a stepwise gradient of 50%, 80%, and 100% MeOH (10 mL each). S-desoxyBTXB2 (4) was found exclusively in the 80% MeOH fraction, and no oxidation (m/z 1034) or methylation (m/z 1032) products were detected. The solution was evaporated to dryness under a stream of argon at 40 °C to afford 4 as a colorless powder (1.06 mg, 93% overall yield from 1) and transferred to a vial for storage at -20 °C under argon. HR-MS: found m/z 1018.5176 for [4 + H]+ (C53H80NO16S requires 1018.5192, 1.6 ppm);

Chem. Res. Toxicol., Vol. 21, No. 4, 2008 945

found m/z 1016.5069 for [4 - H]- (C53H78NO16S requires 1016.5047, 2.2 ppm). Brevetoxin-B2. S-Desoxybrevetoxin-B2 (4), prepared and purified as above from 1 mg brevetoxin-B (1), was dissolved in methanol (0.5 mL), and 1% H2O2 (4 mL) was added. After 20 min at room temperature, LC-MS analysis indicated ca. 20% conversion to brevetoxin-B2 (5) (m/z 1034 ([M + H]+)). The mixture was stored at 4 °C and periodically analyzed by LCMS. After 2 days, conversion was ca. 95%, and after 3 days, 5 was the only detectable component in the solution, present as two partially separated peaks (ratio 43:57) by LC-MS. A 200mg Strata C18-T widepore SPE cartridge was preconditioned with 100% MeOH and then with 20% MeOH in water. The reaction solution was loaded at ca. 1 mL/min. The SPE column was then washed with 20% MeOH (5 mL) and eluted successively with 60%, 70%, and 80% MeOH (5 mL each). Virtually all of the 5 was present in the 70% MeOH fraction, which was evaporated under a stream of argon to give 5 as a colorless powder (1.00 mg, 86% overall yield from 1), which was stored at -20 °C under argon until required. HR-MS: found m/z 1034.5116 for [5 + H]+ (C53H80NO17S requires 1034.5141, 2.5 ppm); found m/z 1032.4974 for [5 - H]- (C53H78NO17S requires 1032.4996, 2.1 ppm). Analytical LC-MS. Method 1. LC-MS analyses of optimization trials and preparative reactions during the conversion of 1 into 4 and 5 were performed on a Waters 2695 Separations Module using a Phenomenex Luna 3 µm C18(2) 50 × 2 mm column held at 30 °C and connected to a Micromass Quattro Ultima triple-quadrupole mass spectrometer using electrospray ionization. Mobile phases were 50% MeOH (A), 90% MeCN (B), a buffer containing 33 mM ammonia and 500 mM formic acid in water (C), and 97.5% MeOH plus 2.5% propan-2-ol (D). A linear gradient was run at 0.2 mL/min from 90% A plus 10% C to 90% D and 10% C over 10 min, followed by 90% D plus 10% C for 7 min, after which the column was flushed with B. The MS was operated in positive scan mode (500–1600 amu). Method 2. LC-MS analyses of trial reactions of 1 with L-cysteine, 2-[(5-fluoresceinyl)aminocarbonyl]ethyl mercaptan, and 2-hydroxythiepan were performed with an LCQ Deca ion trap in positive and negative modes as described elsewhere (14). Stereoisomer Analysis of 4 and 5 by LC-MS: Method 3. A Waters 2695 liquid chromatograph (Milford, MA) was interfaced to a Waters-Micromass Quattro Ultima triple-quadrupole mass spectrometer with a Z-Spray electrospray ionization (ESI) source (Manchester, United Kingdom) in positive mode. Two BDS hypersil C8 3 µm 50 × 2.1 mm (Thermo, CA) columns were connected in series in a column oven set at 30 °C. The injection volume was 10 µL. The columns were eluted with methanol–water–formic acid (62.5:37.5:0.1 v/v) at 0.2 mL/ min. The ESI source was operated at 100 °C with nitrogen gas for nebulization, desolvation (350 °C), and cone control. The source voltages were capillary 3.0 kV and cone 100 V. Data was acquired on two multiple reaction monitoring (MRM) channels, 1034.6 > 929.6 (for 5) and 1018.6 > 204.2 (for 4) with collision energies 35 and 45 eV, respectively, and the collision cell was operated at 3 mbar argon. HR-MS. High resolution mass spectrometry (HR-MS) was performed in positive and negative ion modes on a Bruker Daltonics MicrOTOF spectrometer. The samples were dissolved in 4:1 methanol–water containing 1% formic acid and infused via a syringe pump at 4 µL/min. Cluster ions from sodium formate (2 mM) were used for mass-calibration. Mass spectra were acquired with a time-of-flight analyzer over m/z 500–1500.

946

Chem. Res. Toxicol., Vol. 21, No. 4, 2008

Table 1. 1H and

13

Selwood et al.

C NMR Chemical Shift Assignments for Semisynthetic 4 and 5 in CD3ODa,b and Natural 5 in CD3CN–D2O (1:1)c BTX-B2 (5) in CD3OD

S-desoxyBTX-B2 (4) in CD3OD atom 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 1′ 2′ 3′

13

1

C

166.1 116.1 164.0 69.6 78.2 31.1 80.2 76.0 45.8 84.7 86.4 36.6 34.4 89.0 84.7 30.4 38.9 79.4 89.5 29.9 75.4 75.4 42.2 76.3 81.2 40.3 127.7 137.0 81.2 77.6 38.4 70.6 78.3 31.7 64.5 75.9 72.6 34.4 71.8 34.8 39.2; 39.5 63.4; 64.9 17.3 16.1 18.8 22.1 20.4 18.4 14.5 33.9; 35.6 34.6; 34.9 55.1; 55.2 172.8

H

5.73 4.42 4.07 1.86, 2.22 3.22 1.55, 3.35 3.18 1.62, 2.02 3.43 3.63 1.70, 1.62,

2.12 1.75

2.08 1.75

3.18 1.70, 1.75 3.41 1.47, 1.78 4.02 2.28, 2.52 5.75 5.77 3.93 3.34 1.52, 2.16 3.41 3.06 1.55, 1.97 4.01 3.76 1.65, 2.25 4.01 1.40, 2.50; 1.38, 2.52 1.89; 1.85 3.58 (2H); 3.64, 3.56 1.96 1.31 1.03 1.29 1.19 1.28 1.21 2.74 (2H); 2.58, 2.84 2.92; 3.16 3.69

1

13

H

166.0 116.1 164.0 69.6 78.2 31.1 80.2 75.9 45.8 84.7 86.4 36.7 34.4 89.1 84.7 30.3 38.9 79.4 89.5 29.9 75.4 75.4 42.2 76.3 81.2 40.3 127.7 137.0 81.2 77.8 38.5 70.6 78.3 31.6 64.5 75.9 72.6 34.4 71.8 34.5–35.5 35.3 63–64 17.3 16.1 18.8 22.1 20.5 18.4–18.5 14.5–14.6 56–57 53.5–54.5 51.8–53.4 nd

C

5.73 4.40 4.07 1.86, 2.20 3.21 1.54, 2.12 3.35 3.17 1.62, 1.75 2.03 3.43 3.63 1.68, 2.07 1.65–1.80 3.18 1.68, 1.74 3.40 1.48, 1.78 4.02 2.29, 2.51 5.75 5.77 3.93 3.32 1.51, 2.14 3.40 3.07 1.54, 2.00 3.96 3.76 1.65, 2.25 4.02–4.04 1.4–1.5; 2.4–2.8 2.20–2.25 3.5–3.7(2H) 1.97 1.32 1.03 1.30 1.20 1.28 1.24 2.7–3.1 (2H) 3.0–3.5 (2H) 3.98–4.05

BTX-B2 (5)c in CD3CN–D2O 1

13

H

165.2 114.3 162.7 67.7 76.1 28.9 78.2 74.4 43.5 82.7 84.5 34.2 32.2 86.9 83.4 28.2 36.9 78.1 87.1 27.6 73.2 73.9 39.9 74.6 79.9 38.1 126.7 134.6 79.0 75.6 36.1 68.4 76.1 29.2 62.7 74.4 70.3 31.9 69.2–70.2 33.2–33.5 32.6 61.3–63.7 16.2 14.7 17.5 20.3 18.7 17.2 13.1 53.1–55.0 51.2, 51.4 49.9, 50.9 170.5, 170.8

C

5.69 4.37 4.03 1.80, 2.14 3.16 1.41, 3.37 3.09 1.48, 1.88 3.49, 3.58 1.49, 1.60,

2.04 1.69 2.55 2.04 1.69

3.12 1.59, 1.69 3.35 1.39, 1.76 3.93 2.21, 2.43 5.68 5.69 3.97 3.49 1.45, 2.09 3.39 3.09 1.48, 1.91 3.85 3.73 1.57, 2.18 3.98 1.32–2.44 2.06 3.48–3.59 1.87 1.20 0.92 1.19 1.07 1.18 1.13 2.82–3.07 3.04, 3.35 4.05, 4.07

a

Chemical shifts for 13C and 1H are reported relative to CD3OD at 49.0 ppm and CHD2OD at 3.31 ppm, respectively. b Signals attributable to diastereoisomers of 4 are separated by a semicolon. 1H and 13C shifts for the dominant isomer are underlined where sufficiently resolved in 1H, 1D-SELTOCSY or 2D-TOCSY, or HSQC NMR spectra. c Data from ref 7.

Shellfish Extraction. Brevetoxin-contaminated New Zealand green-lipped mussels (Perna canaliculus) were harvested in January 1993 near Coromandel, New Zealand and stored at -20 °C. This batch of mussels was previously used for isolation of brevetoxin-B2 (7). Mussel homogenate (2 g) was extracted twice with 4:1 methanol–water (9 mL), at 60 °C for 20 min. The extract was washed with hexane, and 5 mL of the defatted extract was diluted with 15 mL of water. The diluted extract was loaded onto a 60-mg Strata-X SPE column (Phenomenex, Torrance, CA) and washed with methanol–water (1:3; 4.5 mL). Brevetoxins were eluted with methanol (5 mL), and an aliquot was evaporated to dryness and dissolved in acetonitrile-water (1:9) for stereoisomer analysis by LC-MS (method 3).

NMR Spectroscopy. NMR spectra of 4 and 5 were obtained from solutions in CD3OD (99.8+ atom% D; Aldrich, USA) using a Bruker DRX 400 MHz spectrometer fitted with a 5 mm dual, gradient shielded, inverse probe. NMR assignments (Table 1) were obtained from an examination of 1H, 13C, DEPT135, 1D-SELTOCSY, COSY, TOCSY, g-HSQC, g-HMBC, NOESY, and ROESY NMR spectra. Chemical shifts, determined at 30 °C, are reported relative to internal C HD2OD (3.31 ppm) and CD3OD (49.0 ppm) (15). Toxicology. Female Swiss albino mice, of initial body weight 18–22 g, were used in all experiments. The animals were allowed free access to food (Laboratory Chow, Sharpes Animal Feeds, Carterton, NZ) and tap water throughout the experimental

S-DesoxybreVetoxin-B2 and BreVetoxin-B2

Chem. Res. Toxicol., Vol. 21, No. 4, 2008 947

period. Acute toxicities were determined according to the principles of OECD Guideline 425 (16) and LD50 values and 95% confidence limits calculated using the AOT 425 Statistical Program (17). Test materials (4-6) were dissolved in ethanol and aliquots diluted with 1% Tween-60 in saline. The diluted solution (1 mL), containing 0.5% v/v ethanol, was injected ip. The mice were observed intensively throughout the day of dosing (for ca. 8 h). Survivors were subsequently weighed each day for the next 7 days, after which time they were killed. All mice were examined macroscopically after death.

Results and Discussion We anticipated that it might be possible to derivatize brevetoxin-B (1) with cysteine via Michael addition to form S-desoxy-42-oxobrevetoxin-B2 (7) and then to reduce the resulting aldehyde to form S-desoxybrevetoxin-B2 (4) (Figure 1). Similar chemistry would be also useful for conjugating brevetoxins to, for example, proteins for immunoassay development and for preparation of fluorescently or isotopically labeled brevetoxin analogues. Small-scale trials were conducted to test the feasibility of this approach with a range of thiols, including L-cysteine. LC-MS analysis (methods 1 and 2) of attempts to react cysteine directly with brevetoxin-B (1) revealed a mixture of two brevetoxin derivatives in a 3:1 ratio. The minor derivative showed m/z 1016 and appropriate fragmentation for S-desoxy42-oxobrevetoxin-B2 (7). The major component (m/z 1119) corresponded to the addition of two cysteinyl residues to 1 with loss of H2O, possibly due to intramolecular reaction of the aldehyde group of 7 with the cysteinyl amino group together with the addition of a second cysteine. Consistent with this, LC-MS analysis indicated that reaction of 1 with 2-hydroxythiepan (which lacks an amino group), a reaction chemistry that has been used with variations to produce brevetoxin-protein conjugates (unpublished observations) during development of immunoassays for brevetoxins (18, 19), proceeded rapidly and without complication to afford expected products (mainly 8) (m/z 1076 ([M + H]+)). Similarly, LC-MS analysis of the reaction of 1 with 2-[(5-fluoresceinyl)aminocarbonyl]ethyl mercaptan indicated that it proceeded slowly, but without complications, to give the expected product (9) (m/z 1330 ([M + H]+)). These observations suggested that Michael addition of an N-protected derivative of L-cysteine to 1 might result in a clean reaction to give the desired cysteinyl adduct which, after deprotection, would yield 4. Controlled oxidation of 4 would give brevetoxin-B2 (5). S-Desoxybrevetoxin-B2 (4). LC-MS analysis (method 2) of trial reactions showed that N-Boc-cysteine added efficiently and without complications to 1 to afford a single product with mass spectral characteristics corresponding to the expected derivative (2). The reaction was complete with 7 min. A preparative procedure based on this reaction was therefore developed and optimized, involving reduction of aldehyde-2 and subsequent deprotection to afford the desired sulfide (4). Reduction of 2 with NaBH4 led to a complex mixture of products, but reduction with NaBH3CN in acid proceeded cleanly to give 3. It was essential that the reducing agent was added after acidification to ca. pH 2, as otherwise a brevetoxin derivative of m/z 1043 (presumably the cyanohydrin of 2) was formed. Deprotection of 3 was performed simply by acidification after completion of the reduction step. The deprotection reaction proceeded steadily and without the formation of byproduct to afford 4. However, if MeOH was used instead of acetonitrile as the cosolvent for the reaction, a product with m/z 1032 (presumably the methyl ester of 4) was also formed. Because of the high yield and lack

Figure 2. LC-MS MRM chromatograms obtained with enhanced resolution for stereoisomer analysis of (A) a mixture of semisynthetic 4 and 5 and (B) an extract from mussels (P. canaliculus) naturally contaminated with brevetoxins.

of side-reactions, the product (4) was readily and conveniently purified by gradient elution on an SPE column to afford 4 as a high-purity colorless solid in 93% overall yield from 1. MS/MS fragment ions (Supporting Information) and HR-MS were entirely consistent with the proposed structure, and the fragmentations were essentially identical to those reported (5) for putative 4 during LC-MS/MS analysis of brevetoxincontaminated oysters. LC-MS analysis (methods 1 and 2) indicated that 4 was composed of two closely eluting partially resolved compounds (ratio ca. 4:3) displaying m/z 1018 ([M + H]+), presumed to be the two 41-diastereoisomers. An LC-MS/ MS method (method 3) was developed to resolve the stereoisomers of 4 and 5 by omitting ammonium formate from the eluent, using isocratic elution, and increasing the column length. Analysis performed with this method indicated that the semisynthetic 4 was composed of approximately equal amounts of two stereoisomers, which were also present in a similar ratio in extracts of naturally contaminated mussels (Figure 2). Analyses of one- and two-dimensional NMR data showed that the semisynthetic specimen was a ca. 55:45 mixture of two diastereoisomeric forms of 4. 1H NMR signals were correlated using COSY, TOCSY, and 1D-SELTOCSY spectral data. 13C NMR signals were determined using a 70° excitation pulse with a pulse repetition rate of 1.68 s. Thereafter, 13C NMR signal multiplicities were defined in a DEPT135 experiment, while 1 H-13C correlations were defined using HSQC and HMBC spectral data. The 1H and 13C NMR signal assignments for the C-1 to C-40 portion of 4 reported in Table 1 correspond closely to those reported elsewhere for brevetoxin B (1) and its metabolites with intact ring systems (7, 10–12). A notable feature of the spectral data for 4 (Table 1) was pairs of 1H signals attributable to two diastereoisomers (at C-41) in the ratio 55: 45, and the occurrence of the C-50 and C-1′ signals of the two diastereoisomeric forms of 4 at 33.9 and 35.6 ppm (C-50) and at 34.6 and 34.9 ppm (C-1′). The greatest difference in 13C

948

Chem. Res. Toxicol., Vol. 21, No. 4, 2008 Table 2. Selected 2J and/or 3J HMBC Correlations Observed for 4 in CD3OD

1

H (δ ppm)

1.03 1.19 1.21 1.28 1.29 1.31 1.96 2.28 2.58 2.74 3.16 3.22 3.69 a

(H-45) (H-47) (H-49) (H-48) (H-46) (H-44) (H-43) (H-26) (H-50)b (H-50)c (H-1′) (H-7) (H-2′)

13

C (δ ppm)

34.4 (C-13), 36.6 (C-12), 89.0 (C-14) 42.2 (C-23), 75.4 (C-21 and C-22) 64.5 (C-35), 72.6 (C-37), 75.9 (C-36) 40.3 (C-26), 76.3 (C-24), 81.2 (C-25), 127.7 (C-27)a 38.9 (C-17), 79.4 (C-18), 89.5 (C-19) 45.8 (C-9), 76.0 (C-8), 80.2 (C-7) 69.6 (C-4), 116.1 (C-2), 164.0 (C-3) 81.2 (H-25), 127.7 (H-27), 137.0 (H-28) 34.9 (C-1′), 39.2 (C-41), 63.4 (C-42) 34.6 (C-1′), 39.5 (C-41), 64.9 (C-42) 172.8 (C-3′) 16.1 (C-44), C-8 (75.4), C-11 (86.4) 172.8 (C-3′), 34.6–34.9 (C-1′)

Weak 4J correlation. b Major isomer. c Minor isomer.

chemical shifts between the two diastereoisomers of 4 (1.5 ppm) was found for C-42 (63.4 and 64.9 ppm) (7). A SELTOCSY experiment performed with excitation of one of the H-50 methylene resonances (2.58 ppm) of the more abundant isomer using a mixing time of 80 ms showed correlations to the other H-50 signal (2.84 ppm) and to the H-42 (3.56 and 3.64 ppm), H-41 (1.85 ppm), and H-40 (1.38 and 2.52 ppm) signals of this isomer, whereas excitation of the H-50 signal of the less abundant isomer (2.74 ppm, 2H) showed correlations to the H-42 (3.58 ppm, 2H), H-41 (1.89 ppm), and H-40 (1.40 and 2.50 ppm) signals. Other isomer signal assignments for the pair (major/minor) of diastereoisomers (Table 1) should be regarded as tentative due to moderate signal-to-noise ratio of the 13C spectrum and/or partial signal overlap in 1H and HSQC spectra. We have not determined the configuration at C-41 of the two isomeric forms of 1, nor have other authors reported the C-41 configuration of any of the four diastereoisomers of 5. Selected HMBC correlations observed for 4, including correlations between H-50 and C-1′ observed for each of the diastereoisomers, are presented in Table 2. Brevetoxin-B2. Several oxidants were tested for the conversion of sulfide-4 into its sulfoxide derivative, brevetoxin-B2 (5), previously isolated from shellfish (7). LC-MS analysis showed that hydrogen peroxide oxidized 4 at an acceptable rate, without the formation of detectable amounts of the sulfone or other byproduct. A second batch of 4 was, therefore, prepared as described above and oxidized with dilute H2O2 on a preparative scale. Progress was monitored by LC-MS, and when the reaction was complete, the product was purified by chromatography on an SPE column to afford brevetoxin-B2 (5) as a high-purity colorless solid in 86% overall yield from 1. The MS/MS fragmentations (Supporting Information) and HR-MS were consistent with the proposed structure. LC-MS analysis (method 3) indicated that semisynthetic-5 was composed of approximately equal amounts of four stereoisomers, which were also present in a similar ratio in extracts of naturally contaminated mussels (Figure 2). Ratios of stereoisomers varied slightly from sample-to-sample and with species in naturally contaminated New Zealand shellfish (unpublished observations). Assignments obtained from analysis of NMR spectra for the semisynthetic brevetoxin-B2 (5) in CD3OD were similar to those reported for 5 isolated from natural sources (7) in CD3CN-D2O (1:1), other than for the consistent occurrence of 13C NMR signals in the latter solvent at shifts 1–2.5 ppm lower than in CD3OD (Table 1). 1H NMR solvent shift effects (CD3OD compared to CD3CN-D2O (1:1)) varied by ( 0.15 ppm. The 1 H and 13C resonances for C-1 to C-40 of 5 were identified from a combination of 1H, COSY, TOCSY, 13C, DEPT135, HSQC, and HMBC NMR data. In general, the resonances of

Selwood et al. Table 3. LD50 Values and 95% Confidence Intervals for S-Desoxybrevetoxin-B2 (4), Brevetoxin-B2 (5), and Dihydrobrevetoxin-B (6) compound

LD50 (µg/kg)

confidence interval

4 5 6

211 400 250

200–250 400–525 176–328

these atoms corresponded closely to those determined for the equivalent resonances of 4. Because of the limited amount of semisynthetic 5 available for NMR analysis, the signal-to-noise ratio of the 13C and DEPT135 spectra were such that the 13C resonances of the four sets of diastereoisomeric side chain carbons could not be identified with certainty. The 1H NMR resonances of side-chain protons were, however, readily defined in COSY and TOCSY spectra. In general, 2–4 sets of partially overlapped signals were observed for the four sets of diastereoisomeric side-chain protons. Comparison of the COSY spectra of 4 and 5 showed that conversion of the sulfide linkage to a sulfoxide linkage resulted in the nearby H-41, H-50, H-1′, and H-2′ signals experiencing shifts of 0.1–0.4 ppm (Table 1). A knowledge of the range of 1H NMR shifts exhibited by the four sets of side-chain protons of 5 enabled identification of the 13C resonances of side-chain carbon atoms at a resolution of the order 0.5–1.0 ppm via low-intensity correlations observed in the HSQC spectrum of 5. A notable aspect of the 13C NMR assignments established for 5 was the marked downfield shift (ca. 20 ppm) experienced by the C-50 and C-1′ atoms of 5, compared to the shifts of these atoms in 4 (Table 1). Previous studies suggested that naturally produced brevetoxinB2 (5) (7) and its fatty-acid derivative, brevetoxin-B4 (6) were composed of a mixture of the four possible diastereoisomers (at C-41 and S). Confirmation of this was provided by the LCMS (method 3) profile for mussels naturally contaminated with 5 (Figure 2), which was very similar to the profile for semisynthetic 5. Thus, the semisynthetic 5 appears to correspond to the natural form of brevetoxin-B2 from isolated shellfish. The NMR data presented here and the clear evidence for the presence of diastereoisomers at C-41 should assist in characterization of related brevetoxin metabolites (5) tentatively identified by LC-MS/MS when these are isolated for NMR analysis. The LD50 of dihydrobrevetoxin-B (6) by ip injection was 250 µg/kg in mice (Table 3). Abdominal breathing was noted soon after injection, with respiration rates higher than normal. At lethal doses, mice became immobile after ca. 15 min, and their respiration rates dropped precipitately shortly afterward. At this time, exophthalmia was noted, and the hind legs of the mice became splayed. Rapid flicking movements of the hind legs occurred immediately before death. No macroscopic lesions were observed at necropsy. At sublethal doses, the respiration rate again declined, and the mice remained immobile and hunched for a prolonged period, when their limbs appeared to be completely paralyzed. Movement was regained after 3–5 h, although one mouse dosed with 6 remained in a very weak condition for 24 h, when it was humanely killed. Other mice receiving a sublethal dose of 6 were of normal behavior and appearance after 24 h, although they had lost 2–3 g in body weight at this time. The weight loss was regained over the next few days, and the mice remained in apparent good health. No gross pathological abnormalities were recorded in mice killed at the end of the 7-day observation period. The LD50 of S-desoxyBTX-B2 (4) was 211 µg/kg, similar to that of 6. Clinical signs of intoxication were similar to those recorded with 6, although the recovery time after sublethal doses

S-DesoxybreVetoxin-B2 and BreVetoxin-B2

was shorter. Again, no abnormalities were seen in any of the mice at necropsy. The LD50 of BTX-B2 (5) was 400 µg/kg, higher than that for 4 and 6, although the symptoms observed after injection were similar to those recorded with 4 and 6. The recovery time after sublethal doses of 5 was greater than that observed with 4, and similar to that in animals dosed with 6. The LD50 values determined for 5 and 6 in this study are in reasonable agreement with literature values. Baden and Mende (20) reported an ip LD50 of 170 µg/kg for 6 in mice that had been fasted for 16 h before dosing. Initial experiments in this laboratory indicate that fasting may lead to an increase in the severity of the toxic effects of brevetoxins. The minimum lethal dose of 5 in mice was reported as 306 µg/kg (7), though whether the mice in this study were fed or fasted was not stated. The potency of 4 and 5 in sodium channel inhibition assays was about an order of magnitude less than that of 6 (13), whereas the ip toxicities of 4 and 6 were similar, and 5 was ca. 2-fold less toxic. This indicates that factors other than sodium channel affinity (e.g., bioavailability, metabolism, disposition, or excretion) also contribute to brevetoxin toxicity in vivo. The mouse bioassay is a standard method for determining when shellfish harvesting can be resumed after brevetoxin-containing algal blooms in the USA (21), and knowledge of the relative ip potencies of 4-6 permits calibration of mouse bioassay results with results from analytical methods such as LC-MS, ELISA, or receptor binding assays.

Conclusions The methodology developed here conveniently gives 4 and 5 in high yield and purity, and the same procedure works efficiently on a scale of at least 5 mg (unpublished observations). The structures of semisynthetic 4 and 5 were established by NMR analysis and comparison with NMR data for natural 5 reported in an earlier study (7). LC-MS analysis performed with enhanced resolution (Figure 2) and LC-MS/MS spectra indicated that the semisynthetic 4 and 5 corresponded closely in retention time, mass spectral fragmentation pattern, and isomer ratios to 4 and 5 in shellfish naturally contaminated with brevetoxins. This confirms the presence of 4 in naturally contaminated shellfish, the identity of which was previously inferred from mass spectral fragmentation. These results indicate that semisynthetic 4 and 5 can be used in place of the natural metabolites, which are very difficult to isolate in sufficient quantity and purity from shellfish, for toxicological studies (13) and production of analytical standards. In mice, the acute ip toxicity of 4 was similar to that of 6, while 5 was less toxic. Brevetoxin-B reacted rapidly and efficiently with thiols, via Michael addition to its R,β-unsaturated aldehyde moiety, and would be expected to undergo similar conjugation reactions with cysteine-containing peptides and proteins. Subsequent proteolysis of BTX-containing conjugates in vivo could be expected to release covalently linked brevetoxin-peptide conjugates. Brevetoxin metabolites can be detected over a greatly extended period in organisms exposed to brevetoxin-B (1), when compared to dihydrobrevetoxin-B (6, which lacks the reactive R,βunsaturated aldehyde moiety present in 1) (8, 9, 22). The conjugation chemistry of 1, together with the processing of amino acids from brevetoxin-protein conjugates via proteolysis, could, therefore, be contributing factors in the extended lifetimes of brevetoxin-B metabolites in vivo. The synthetic procedure developed here was also suitable for producing the γ-Glu-Cys-, Cys-Gly-, and GSH-analogues of 4 (unpublished observations), and we anticipate that the methodology will, after optimization of reaction conditions, also prove useful for the preparation of

Chem. Res. Toxicol., Vol. 21, No. 4, 2008 949

other derivatives of brevetoxin, including natural metabolites (e.g., BTX-B4) and analogues labeled with tracers such as radioisotopes or fluorescent tags. Acknowledgment. We are grateful to M. Poli (Integrated Toxicology Division, USAMRIID, Fort Detrick, MD 21702, USA) for providing brevetoxin-B, R. W. Dickey (Gulf Coast Seafood Laboratory, USFDA, Dauphin Island, AL 36528, USA) for helpful advice on LC-MS methods, J. I. Loader (AgResearch) for obtaining some of the preliminary NMR spectra, P. Gread (University of Waikato Spectrometry Facility) for obtaining HR-MS spectra, and A. D. Hawkes (AgResearch) for accurate weighing of the products. This study was supported by New Zealand Foundation for Research, Science and Technology Grants CAWX0301 and CAWX0703. Disclaimer: This publication does not constitute an endorsement of any commercial product or intend to be an opinion beyond scientific or other results obtained by the National Oceanic and Atmospheric Administration (NOAA). No reference shall be made to NOAA, or this publication furnished by NOAA, to any advertising or sales promotion which would indicate or imply that NOAA recommends or endorses any proprietary product mentioned herein, or which has as its purpose an interest to cause the advertised product to be used or purchased because of this publication. Supporting Information Available: 1H NMR (with and without solvent suppression), 13C, DEPT135, COSY (with expansion), TOCSY, HMBC, HSQC and ROESY NMR spectra of 4; MS/MS spectra of 4 (positive and negative modes); 1H NMR (with solvent suppression), 13C, DEPT135, COSY (with expansion), TOCSY, HMBC expansion, and HSQC NMR spectra of 5; MS/MS spectrum of 5 (positive mode); LC-MS analyses of trial reactions of 1 with L-cysteine, 2-hydroxythiepan, and 2-[(5-fluoresceinyl)aminocarbonyl]ethyl mercaptan; dose rates and time-to-death for 4, 5, and 6 administered i.p. to mice. This material is available free of charge via the Internet at http:// pubs.acs.org.

References (1) Baden, D. G. (1989) Brevetoxins: unique polyether dinoflagellate toxins. FASEB J. 3, 1807–1817. (2) Lin, Y.-Y., Risk, M., Ray, S. M., Van Engen, D., Clardy, J., Golik, J., James, J. C., and Nakanishi, K. (1981) Isolation and structure of brevetoxin B from the “red tide” dinoflagellate Ptychodiscus breVis (Gymnodinium breVe). J. Am. Chem. Soc. 103, 6773–6775. (3) Chou, H.-N., and Shimizu, Y. (1982) A new polyether toxin from Gymnodinium breVe Davis. Tetrahedron Lett. 23, 5521–5524. (4) Kirkpatrick, B., Fleming, L. E., Squicciarini, D., Backer, L. C., Clark, R., Abraham, W., Benson, J., Cheng, Y. S., Johnson, D., and Pierce, R. (2004) Literature review of Florida red tide: implications for human health effects. Harmful Algae 3, 99–115. (5) Wang, Z., Plakas, S. M., El Said, K. R., Jester, E. L. E., Granade, H. R., and Dickey, R. W. (2004) LC/MS analysis of brevetoxin metabolites in the Eastern oyster (Crassostrea Virginica). Toxicon 43, 455–465. (6) Morohashi, A., Satake, M., Naoki, H., Kaspar, H. F., Oshima, Y., and Yasumoto, T. (1999) Brevetoxin B4 isolated from greenshell mussels Perna canaliculus, the major toxin involved in neurotoxic shellfish poisoning in New Zealand. Nat. Toxins 7, 45–48. (7) Murata, K., Satake, M., Naoki, H., Kaspar, H. F., and Yasumoto, T. (1998) Isolation and structure of a new brevetoxin analog, brevetoxin B2, from Greenshell mussels from New Zealand. Tetrahedron 54, 735– 742. (8) Plakas, S. M., Wang, Z., El Said, K. R., Jester, E. L. E., Granade, H. R., Flewelling, L., Scott, P., and Dickey, R. W. (2004) Brevetoxin metabolism and elimination in the Eastern oyster (Crassostrea Virginica) after controlled exposures to Karenia breVis. Toxicon 44, 677–685. (9) Plakas, S. M., El Said, K. R., Jester, E. L. E., Granade, H. R., Musser, S. M., and Dickey, R. W. (2002) Confirmation of brevetoxin

950

(10)

(11)

(12)

(13) (14)

(15)

Chem. Res. Toxicol., Vol. 21, No. 4, 2008 metabolism in the Eastern oyster (Crassostrea Virginica) by controlled exposures to pure toxins and to Karenia breVis cultures. Toxicon 40, 721–729. Ishida, H., Nozawa, A., Hamano, H., Naoki, H., Fujita, T., Kaspar, H. F., and Tsuji, K. (2004) Brevetoxin B5, a new brevetoxin analog isolated from cockle AustroVenus stutchburyi in New Zealand, the marker for monitoring shellfish neurotoxicity. Tetrahedron Lett. 45, 29–33. Ishida, H., Nozawa, A., Totoribe, K., Muramatsu, N., Nukaya, H., Tsuji, K., Yamaguchi, K., Yasumoto, T., Kaspar, H., Berkett, N., and Kosuge, T. (1995) Brevetoxin B1, a new polyether marine toxin from the New Zealand shellfish AustroVenus stutchburyi. Tetrahedron Lett. 36, 725–728. Morohashi, A., Satake, M., Murata, K., Naoki, H., Kaspar, H. F., and Yasumoto, T. (1995) Brevetoxin B3, a new brevetoxin analog isolated from the greenshell mussel Perna canaliculus involved in neurotoxic shellfish poisoning in New Zealand. Tetrahedron Lett. 36, 8995–8998. Bottein Dechraoui, M.-Y., Wang, Z., and Ramsdell, J. S. (2007) Intrinsic potency of synthetically prepared brevetoxin cysteine metabolites BTX-B2 and desoxyBTX-B2. Toxicon 50, 825–834. Miles, C. O., Wilkins, A. L., Hawkes, A. D., Jensen, D. J., Cooney, J. M., Larsen, K., Petersen, D., Rise, F., Beuzenberg, V., and MacKenzie, A. L. (2006) Isolation and identification of a cis-C8-diolester of okadaic acid from Dinophysis acuta in New Zealand. Toxicon 48, 195–203. Miles, C. O., Wilkins, A. L., Hawkes, A. D., Selwood, A., Jensen, D. J., Aasen, J., Munday, R., Samdal, I. A., Briggs, L. R., Beuzenberg, V., and MacKenzie, A. L. (2004) Isolation of a 1,3-enone isomer of heptanor-41-oxoyessotoxin from Protoceratium reticulatum cultures. Toxicon 44, 325–336.

Selwood et al. (16) OECD (2001) OECD Guideline for Testing of Chemicals 425. Acute Oral ToxicitysUp and down Procedure, Organisation for Economic Co-operation and Development, Paris, France. http://www.epa.gov/ oppfead1/harmonization/docs/E425guideline.pdf (accessed 2 November 2007). (17) EPA (2002) User Documentation for the AOT425StatPgm Program Prepared for the US Environmental Protection Agency by Westat, May 2001; updated by USEPA Sept. 2002. http://www.oecd.org/dataoecd/ 19/57/1839830.pdf (accessed 2 November 2007). (18) Woofter, R., Dechraoui, M.-Y. B., Garthwaite, I., Towers, N. R., Gordon, C. J., Córdova, J., and Ramsdell, J. S. (2003) Measurement of brevetoxin levels by radioimmunoassay of blood collection cards after acute, long-term, and low-dose exposure in mice. EnViron. Health Perspect. 111, 1595–1600. (19) Maucher, J. M., Briggs, L., Podmore, C., and Ramsdell, J. S. (2007) Optimization of blood collection card method/enzyme-linked immunoassay for monitoring exposure of bottlenose dolphin to brevetoxinproducing red tides. EnViron. Sci. Technol. 41, 563–567. (20) Baden, D. G., and Mende, T. J. (1982) Toxicity of two toxins from the Florida red tide marine dinoflagellate Ptychodiscus breVis. Toxicon 20, 457–461. (21) Subcommittee on Laboratory Methods for Examination of Shellfish (1970) in Recommended Procedures for the Examination of Seawater and Shellfish (Taylor, D., and Siegler, H. H., Eds.) pp 61–66, American Public Health Association, Washington, DC. (22) Radwan, F. F. Y., Wang, Z., and Ramsdell, J. S. (2005) Identification of a rapid detoxification mechanism for brevetoxin in rats. Toxicol. Sci. 85, 839–846.

TX700441W