Hydroxypectenotoxin-2, a New Pectenotoxin ... - ACS Publications

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Chem. Res. Toxicol. 2006, 19, 310-318

Identification of Pectenotoxin-11 as 34S-Hydroxypectenotoxin-2, a New Pectenotoxin Analogue in the Toxic Dinoflagellate Dinophysis acuta from New Zealand Toshiyuki Suzuki,†,‡ John A. Walter,† Patricia LeBlanc,† Shawna MacKinnon,† Christopher O. Miles,§,| Alistair L. Wilkins,⊥ Rex Munday,§ Veronica Beuzenberg,†,O A. Lincoln MacKenzie,O Dwayne J. Jensen,# Janine M. Cooney,# and Michael A. Quilliam*,† Institute for Marine Biosciences, National Research Council, 1411 Oxford Street, Halifax, NS B3H 3Z1, Canada, Tohoku National Fisheries Research Institute, 3-27-5 Shinhama, Shiogama, Miyagi 985-0001, Japan, AgResearch Ltd., Ruakura Research Centre, PriVate Bag 3123, Hamilton, New Zealand, National Veterinary Institute, P.O. Box 8156, N-0033 Oslo, Norway, Chemistry Department, The UniVersity of Waikato, PriVate Bag 3105, Hamilton, New Zealand, Cawthron Institute, 98 Halifax Street, PriVate Bag 2, Nelson, New Zealand, and HortResearch Ltd., Ruakura Research Centre, PriVate Bag 3123, Hamilton, New Zealand ReceiVed September 4, 2005

A new pectenotoxin, which has been named pectenotoxin-11 (PTX11), was isolated from the dinoflagellate Dinophysis acuta collected from the west coast of New Zealand. The structure of PTX11 was determined as 34S-hydroxypectenotoxin-2 by tandem mass spectrometry and UV and NMR spectroscopy. PTX11 appears to be only the third pectenotoxin identified as a natural biosynthetic product from algae after pectenotoxin-2 and pectenotoxin-12. The LD50 of PTX11 determined by mouse intraperitoneal injection was 244 µg/kg. The LDmin of PTX11 in these experiments was 250 µg/kg. No signs of toxicity were recorded in mice following an oral dose of PTX11 at 5000 µg/kg. No diarrhea was observed in any of the animals administered with the test substance by either route of administration. Unlike pectenotoxin-2 (PTX2), PTX11 was not readily hydrolyzed to its corresponding seco acid by enzymes from homogenized green-lipped mussel (Perna canaliculus) hepatopancreas. Introduction Pectenotoxins are polyether macrolide toxins frequently associated with incidents of diarrhetic shellfish poisoning (DSP)1 (1, 2) (Figure 1). These compounds have been reported to be highly hepatotoxic and mildly diarrhetic (3, 4) and have also attracted attention due to their potent cytotoxicity against several human cancer cell lines (5, 6). Pectenotoxin-2 (PTX2) has been reported to be present in the toxic dinoflagellates Dinophysis fortii, Dinophysis acuminata, Dinophysis norVegica, Dinophysis rotundata, and Dinophysis acuta (7-17). It has been demonstrated that some of the other pectenotoxins such as pectenotoxin-1 (PTX1) are formed by metabolism of PTX2 in bivalve tissues (7, 8, 10, 18). Besides these pectenotoxin analogues, pectenotoxin-2 seco acid (PTX2sa) and its epimer 7-epi-pectenotoxin-2 seco acid * Corresponding author: Michael A. Quilliam, Institute for Marine Biosciences, National Research Council of Canada, 1411 Oxford Street, Halifax, NS B3H 3Z1, Canada. Phone, 1-902-426-9736; fax, 1-902-4269413; e-mail, [email protected]. † Institute for Marine Biosciences, National Research Council. ‡ Tohoku National Fisheries Research Institute. § AgResearch Ltd., Ruakura Research Centre. | National Veterinary Institute. ⊥ The University of Waikato. O Cawthron Institute. # HortResearch Ltd., Ruakura Research Centre. 1 Abbreviations: DSP, diarrhetic shellfish poisoning; ESI-MS, electrospray ionization mass spectrometry; LC-MS, liquid chromatography-mass spectrometry; LDmin, minimum lethal dose; MMW, monoisotopic molecular weight; MS/MS, tandem mass spectrometry; NMR, nuclear magnetic resonace spectroscopy; PTX, pectenotoxin; PTX1, pectenotoxin-1; PTX-2, pectenotoxin-2; PTX11, pectenotoxin-11.

(7-epi-PTX2sa) were isolated from New Zealand green-lipped mussels (Perna canaliculus) (19). It has been shown that PTX2sa is a bivalve metabolite of PTX2 (20, 21) that progressively epimerizes to the thermodynamically more stable 7-epiPTX2sa. Recently, a novel isomer of PTX1 was detected in D. acuta collected at Buller Bay on the west coast of the South Island of New Zealand (16), using liquid chromatography-mass spectrometry (LC-MS). This new pectenotoxin isomer was named pectenotoxin-11 (PTX11) (22). In the present study, the detailed structure of PTX11 was determined by tandem mass spectrometry (MS/MS) and UV and NMR spectroscopy. Acute mouse toxicity and susceptibility of PTX11 to enzymatic hydrolysis were also investigated.

Experimental Procedures Chemicals. Analytical grade solvents (chloroform, hexane, methanol (MeOH)) were used for extraction of PTX11. HPLC grade solvent (acetonitrile (MeCN), MeOH) and analytical grade reagents (ammonium acetate, formic acid, ammonium formate) were used for isolation of PTX11 and LC-MS analysis. Distilled water was further purified with a Milli-Q water purification system (Millipore, Bedford, MA). Authentic PTX2 was available from previous work (23). Molecular modeling was performed with ChemBats3D Ultra version 6.0 (CambridgeSoft, Cambridge, MA). Authentic PTX1 was provided by Japan Food Research Laboratories (Tama, Tokyo, Japan) (24). Extraction of Toxins from Plankton Cell Concentrates. Phytoplankton cell concentrates were collected from various depths within the water column, approximately 3-4 km offshore in Buller Bay on the northwestern coast of the South Island of New Zealand,

10.1021/tx050240y CCC: $33.50 © 2006 American Chemical Society Published on Web 01/20/2006

Identification of Pectenotoxin-11

Chem. Res. Toxicol., Vol. 19, No. 2, 2006 311

Figure 1. Structures of pectenotoxins. PTX11, PTX11b, and PTX11c are new structures.

on February 15, 2002. Details of the sampling method have been described previously (16), and extraction of pectenotoxins was carried out essentially as reported previously (22). A condensed phytoplankton sample in seawater (ca. 3 L) was filtered through a GF/C filter. The harvested cells were rinsed with 5 mL of MeOH, which was combined with the aqueous filtrate. The filtrate was transferred (150 mL each time) to a 500 mg C18-silica solid-phase extraction (SPE) cartridge (Strata, Phenomenex, Torrance, CA) that had been preconditioned with 10 mL of MeOH followed by 10 mL of MeOH-water (1:9). The cartridge was washed with 15 mL of MeOH-water (3:7) followed by 15 mL of MeOH-water (6:4). The pectenotoxins were eluted with 15 mL of MeOH-water (8: 2). The combined pectenotoxin eluates were washed once with an equal volume of hexane. After adding water to reduce the MeOH concentration to 60%, the toxins were extracted twice with chloroform (1:1, v/v). The combined chloroform extracts were evaporated on a rotary evaporator and dissolved in MeOH for LCMS/MS and LC-UVD analysis. LC-MS/MS Analysis of Pectenotoxins. LC-MS/MS was performed using an Agilent 1100 (Palo Alto, CA) liquid chromatograph coupled to a PE-SCIEX (Thornhill, Ontario, Canada) API4000 mass spectrometer (25). Analytical separation of pectenotoxins was achieved on a Quicksilver cartridge column (50 mm × 2 mm i.d.) packed with 3 µm Hypersil-BDS-C8 (Keystone Scientific, Bellefonte, PA) maintained at 20 °C. The column was eluted isocratically with MeCN-water (19:21) containing 2 mM ammonium formate and 50 mM formic acid at a flow rate of 0.2 mL/ min. The injection volume was 3 µL (22). The LC effluent was introduced into a Turbo IonSpray interface without splitting. Highpurity air heated to 275 °C was used as a nebulizing gas. Mass spectra were acquired in either positive or negative ion mode by scanning Q1. Product-ion mass spectra were acquired in positive mode by colliding the Q1 selected precursor ion for [M + NH4]+ of pectenotoxins with nitrogen in Q2 operated in radio frequency (rf)-only mode and scanning the second quadrupole mass spectrometer, Q3, from m/z 50 to 940. Collision energies of 45 eV for CID experiments were used. LC-Diode Array Detection (DAD) Analyses. LC-DAD analysis was performed using an HP1090 liquid chromatograph equipped with a built-in photodiode array detector (Agilent, Palo Alto, CA). Ultraviolet (UV) spectra were acquired over the 200400 nm range. Separation was performed on the same column used for LC-MS with an MeCN-water (45:55) mobile phase, at a flow rate of 0.2 mL/min and an injection volume of 5 µL.

Isolation of PTX11. The processed extract of D. acuta (13.5 mg) was dissolved in 80% methanol and separated by flash chromatography (120 mm × 13 mm i.d.) on a JT Baker C18 (40 µm) column eluted with 80% MeOH. Fractions of ca. 1 mL were collected and analyzed by thin-layer chromatography (TLC) and LC-MS. Fractions that contained PTX11 were combined and evaporated. The resulting residue (4.6 mg) was further purified by semipreparative LC on a Spherisorb S5 ODS2 column (250 mm × 10 mm i.d.) with MeCN-water (57:43) containing 10 mM ammonium acetate at 4.0 mL/min. Peaks were monitored by UV absorbance at 235 nm. The PTX11 fraction from semipreparative LC was diluted with 2 vol of distilled water and applied to an OASIS HLB (500 mg) cartridge column (Waters) that had been preconditioned with 5 mL of MeOH followed by 5 mL of 10% MeCN. The cartridge column was washed with 5 mL of 10% MeCN; PTX11 eluted with 10 mL of MeOH and evaporated to give a sub-milligram quantity of PTX11. A similar quantity of PTX11 was also prepared by the method of Miles et al. (23). NMR Spectroscopy of PTX11. NMR spectra were obtained from solutions of PTX11 (3.19 mg) in CD3OD (99.8+ atom % D; Aldrich) and (CD3)2CO (99.9+ atom % D; Aldrich) with Bruker DRX-400 (8.45 T, sample temperature 30 °C) and DRX-500 (11.75 T, 20 °C) spectrometers. NMR assignments (Table 1) were obtained from examination of 1H, 13C, DEPT135, 1D-TOCSY, COSY, TOCSY, SELTOCSY, g-HSQC, g-HMBC, ROESY, SELROESY, and NOESY NMR spectral data. Chemical shifts, determined at 30 °C, are reported relative to internal CHD2OD (3.31 ppm) and CD3OD (49.0 ppm) for CD3OD, and at 20°C to internal (CHD2)COCD3 (2.04 ppm) and (CD3)2CO (29.85 ppm) for (CD3)2CO. Detailed experimental parameters are shown alongside spectra in the Supporting Information. Acute Toxicity Studies of PTX11. Female Swiss mice, of body weight between 18 and 22 g were used in all experiments. They were allowed laboratory chow and water ad lib throughout the experimental period. For intraperitoneal dosing, the required amounts of PTX11 (23) were dissolved in ethanol and then diluted 1:20 (v/v) with 1% Tween-60 in saline. A 1 mL volume of this diluted solution was used in all ip injections. For oral dosing, the test materials were dissolved in ethanol, and this solution was diluted 1:10 (v/v) with Tween-saline. The diluted solution (200 µL) was administered to the mice by gavage. Randomly selected groups of 5 mice were dosed intraperitoneally with PTX11 at 325, 250, 192, and 148 µg/kg body wt. A single group of 5 mice was dosed orally with PTX11 at 5000 µg/kg body wt. The mice were

312 Chem. Res. Toxicol., Vol. 19, No. 2, 2006 Table 1. 1H and

Suzuki et al. 13C

NMR Assignments for PTX11 in CD3OD and (CD3)2CO

PTX2 (CD3OD, 30 °C)a atom

13C

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

172.1 49.2 77.4 30.5 22.7 35.4 108.7 33.4 22.9 81.9 76.1 82.6 44.7 214.6 80.2 72.0 36.9 81.6 34.4 28.9 110.2 80.2 30.1 38.2 86.1 51.1 31.5 141.1 131.8 136.1 122.3 84.1 76.1 33.8 82.6 98.6 71.7 30.6 28.2 61.8 15.9 23.3 26.1 26.7 23.8 12.7 17.9

1H

13C

2.32 3.46 1.52, 1.16 1.83, 1.57 1.68, 1.68 2.54, 1.55 2.07, 1.63 4.27 4.02 2.86, 1.97 3.80 4.25 2.08, 1.31 1.92, 1.68 2.19, 1.99 3.85 2.02, 1.67 1.69, 1.42 1.70,1.54 2.58 5.26 6.48 5.42 4.78 5.47 2.22, 2.10 4.50 3.29 2.12 1.66, 1.25 3.98, 3.68 1.09 1.19 1.34 1.22 0.96 1.70 0.95

PTX11 (CD3OD, 30 °C)

∼t 3.2 dd, 10.5, 5.8

d, 6.8

d, 6.7 d, 1.0 d, 6.9

172.3 49.6 77.6 30.4 22.6 35.2 108.7 33.4 22.8 81.9 76.2 82.6 44.7 214.5 80.2 72.0 36.9 81.6 34.4 28.8 110.1 80.2 30.2 38.3 86.1 51.1 31.5 141.2 131.8 136.6 121.7 81.1 76.0 72.8 84.7 98.3 71.4 30.4 28.4 62.0 15.9 23.4 26.1 26.7 23.7 12.7 18.0

1H

2.36 3.60 1.80, 1.15 1.80, 1.55 1.68, 1.68

multiplicity, J (Hz)b dd, 9.7, 6.8

2.55, 1.56 2.11, 1.66 4.28 4.01

br d, 1.5

2.85, 1.97

ABq, 16.2

3.80 4.25 2.08, 1.30

br d, 1.4

1.92, 1.68 2.18, 1.99 3.84 2.04, 1.66 1.65, 1.42 1.72, 1.56 2.60 5.28 6.48 5.38 4.81 5.31 4.50 4.16 3.55 2.17 1.67, 1.26 3.98, 3.70 1.13 1.20 1.33 1.22 0.96 1.70 1.00

br d, 10.6 dd, 15.7, 1.7 dd, 15.7, 3.3 dd, 4.2, 2.6 dd, 9.1, 4.2 d, 9.1 d, 2.7

d, 6.8

d, 6.7 d, 1.0 d, 6.9

PTX11 ((CD3)2CO, 20 °C) 13C

172.93 49.17 76.60 29.84 22.34 34.83 107.83 33.12 22.47 81.27 75.77 81.93 44.31 213.54 79.65 71.51 36.53 80.36 34.12 28.64 109.90 79.65 29.70 37.94 84.61 50.95 30.97 140.99 130.88 135.79 121.55 80.36 74.81 72.47 84.50 97.53 70.97 30.20 28.04 61.11 15.61 23.34 26.27 26.83 23.63 12.62 17.96

multiplicity, J (Hz)b

1H

2.29 3.57 1.76, 1.02 1.75, 1.47 1.60, 1.60

dq, 9.7, 6.9 ov

2.47, 1.49 2.13, 1.68 4.25 3.94c

bdd, 12.7, 7.2; ov

2.86, 1.92

ABq, 16.1

3.74 4.19 2.01, 1.29

d, 1.7 ddd, 12.1, 3.7, 1.7 dd, 12.5, 1.2; dd, 12.7, 3.8

ddd, 9.8, 6.5, 1.7 d, 1.8

1.88, 1.58 2.20, 1.87 3.82 2.04, 1.59 1.59, 1.37

dd, 10.1, 5.5

1.66, 1.55 2.61 5.32

m, 10.4, 6.5, 2.2 br d, 10.2

6.37 5.34 4.74 5.34 4.54c 4.14

ddd, 15.7, 2.1, 0.5 dd, 15.7, 3.2 bm, 3.0 dd, 4.3, 2.6 ddd, 9.0, 4.2, 4.2c d, 9.2

c

3.54c 2.13 1.64, 1.20 3.89, 3.58 1.12 1.17 1.25 1.15 0.95 1.68 0.95

dd, 3.2, 5.7c ddd, 10.9, 8.4, 2.6; ov d, 6.9

d, 6.9 d, 1.2 d, 6.9

a Data for PTX2 is from Miles et al. (23). b Multiplicities and magnitudes of 1H-1H couplings J (Hz) are shown where peaks are clearly defined; s ) singlet, d ) doublet, t ) triplet, q ) quartet, m ) multiplet, br ) broad, ov ) overlap. Magnitudes of JHH were measured from well-resolved first-order multiplets. c 1H resonances were detected for OH groups situated at these positions, as follows: 11-OH, δH 3.35 brs; 34-OH, two doublets of unequal intensity, δH 5.06 d, 5.08 d, 2JHH 4.2 Hz; 36-OH, two singlets of unequal intensity δH 4.49 s, 4.50 s; 37-OH, δH 3.73 d, 2JHH 5.7 Hz.

examined closely for the first 4 h after dosing, then at least 3 times a day for 14 days. Deaths and physiological changes were recorded. LD50 values and confidence intervals were calculated by the method of Weil (26). The protocols for these studies were reviewed by the institutional animal care and use committee. Conversion of PTX2 and PTX11 with Green-Lipped Mussel Hepatopancreas. To a homogenized supernatant of hepatopancreas from green-lipped mussels (20, 23) (500 µL) was added a solution of PTX2 and PTX11 (1.0 µg of each) in MeOH (25 µL), and the stirred mixture was held at 20 °C. Aliquots of the reaction mixture were periodically taken for analysis by LC-MS as described elsewhere (23), except that hydrolysis was quenched by addition of the aliquots to MeCN instead of MeOH to avoid enzymatic formation of seco acid methyl esters. High-Resolution Mass Spectrometry. Accurate mass measurement on PTX11 was kindly performed by Dr. M. Evans at the Universite´ de Montre´al using a VG Micromass ZAB with fast atom bombardment ionization.

Results and Discussion PTX2 and PTX11 were detected by LC-MS as the predominant PTXs in a D. acuta sample collected from New Zealand in a previous study (16, 22). Figure 2 shows the analysis of the plankton extract by LC with UV diode array and electrospray ionization mass spectrometry detection, both operated in spectrum scanning modes. The UV absorbance chromatogram (235 nm) (Figure 2a) showed two major peaks at 5.0 and 6.2 min. The UV spectra acquired at the apex of both these peaks (Figure 2d) both showed a maximum at 235 nm, a characteristic of all pectenotoxins due to a 1,3-dienyl function. Examination of the positive ion mass spectra of these peaks showed abundant [M + NH4]+ ions at m/z 876 for the peak at 6.2 min (data not shown) and m/z 892 for the peak at 5.0 min (Figure 2e), indicating compounds with molecular weights of 858 and 874, respectively. Reconstructed mass chromatograms

Identification of Pectenotoxin-11

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Figure 2. Results from the analysis of the D. acuta (New Zealand) extract by LC with detection by UV diode array detection and mass spectrometry. (a) UV absorbance (235 nm) chromatogram; (b) m/z 876 mass chromatogram; (c) m/z 892 mass chromatogram with dotted lines indicating retention times of PTX1, PTX4, and PTX8 if they had been present; (d) UV spectra of PTX2 (dotted line) and PTX11 (solid line); (e) positive ion mass spectrum of PTX11; (f) negative ion mass spectrum of PTX11.

for these two ions are shown in Figure 2b,c. The UV spectrum, MS/MS spectrum (data not shown), and the retention time of the peak at 6.2 min matched those of PTX2. The peak at 5.0 min was deemed a novel pectenotoxin and called PTX11 (22), since it had a different retention time than PTX1 (3.0 min), PTX4 (4.3 min), and PTX8 (6.6 min) (22), the only known pectenotoxins of molecular weight 874. Additional evidence of the molecular weight of PTX11 was also provided by its negative ion mass spectrum (Figure 2f), which showed a formate attachment ion, [M + HCOOH - H]-, at m/z 919 and an [M - H]- ion at m/z 873. Figure 3a shows the product ion spectrum of the [M + NH4]+ ion (m/z 892) of PTX11 acquired on a triple quadrupole MS/ MS. The spectrum of a PTX1 standard is shown for comparison in Figure 3b. Both compounds showed many common fragment ions, but a few of the ions were shifted up or down in mass by 16 amu. Detailed analysis of the fragmentations provided information on the location of the hydroxyl in PTX11. Figure 4 presents our proposed assignment of fragment ions deduced from examination of the structure and through comparison of the spectra of PTX1 and PTX11, as well as PTX2 (spectrum not shown). The higher mass regions of both spectra show a very low abundance [M + H]+ ion at m/z 875, due to an initial elimination of ammonia from [M + NH4]+, and a series of ions, [M + H - nH2O]+ (n ) 1-5), due to sequential water losses from [M + H]+. Fragment ions in the rest of the spectrum are most easily explained by an initial opening of the macrocyclic ring at the lactone site as shown in Figure 4. This is supported by the fact that the product ion spectra of the [M + NH4]+ ions of the seco acid of PTX2 were almost identical to those of PTX2 (data not shown), with only an additional loss of water fragment at high mass. The base peak in both spectra was m/z 213, a fragment ion observed in the spectra of all pectenotoxins. This appears to be

due to breakage of the C10-C11 bond adjacent to ring B. Losses of one and two water molecules from m/z 213 produced ions at m/z 195 and 177. Another low mass ion (m/z 161), observed in both spectra, is explained by fragmentation of ring F. Fragmentation of ring C followed by a series of water eliminations gives rise to one group of ions at m/z 311, 293, 275, and 257 and another group at m/z 591, 573, and 555. All of these were observed in both spectra, suggesting that the hydroxyl in PTX11 was not in rings A, B, C, or G. Fragmentation of the bridging ring connected to ring D with water eliminations gave rise to one set of ions at m/z 371, 353, and 335 and another set at m/z 473, 455, and 437 in the spectrum of PTX11. These ions do not appear in PTX1, presumably due to the influence of the hydroxymethyl function at C18. The presence of corresponding series of ions at 16 amu less (m/z 457, 439, and 421) in the spectrum of PTX2 (data not shown) confirms this notion. A set of ions at m/z 551, 533, and 515 in the PTX11 spectrum is very clearly shifted 16 amu lower than the equivalent set (at m/z 567, 549, and 531) in the spectrum PTX1. These ions can be explained through fragmentation of the C25-C26 bond and two subsequent losses of water. Finally, the ion at m/z 273 in PTX11 can be explained by a scission of the C26-C27 bond to form a low abundance ion at m/z 309 which loses water to give ions of m/z 291 and 273. The corresponding ions in PTX1 would be isobaric with the previously discussed ion series at m/z 293, 275, and 257, and high-resolution measurements would be required to confirm this proposal. These results indicated that PTX11 is a structural analogue of PTX2 containing an extra hydroxyl group on a carbon between C27 and C34. PTX11 was preparatively isolated from extracts of the D. acuta sample as a white solid. High-resolution FAB-MS data for PTX11 were consistent with an elemental composition of C47H70O15Na ([M + Na]+ m/z 897.4586 vs 897.4612 (calcd), -2.9 ppm), the same as PTX1.

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Suzuki et al.

Figure 3. LC-MS/MS product ion spectra obtained for the [M + NH4]+ ions (m/z 892.5) of PTX11 (a) and PTX1 (b). All m/z values have been rounded down. 1H and 13C NMR spectra of PTX2 and PTX11 recorded in two solvents (CD3OD and (CD3)2CO) at different temperatures (Table 1 and Supporting Information) showed close correspondences in chemical shifts and coupling constants (JHH) for all resonances of PTX2, except those for positions 3, 4, and 32-37. Analysis of COSY and TOCSY spectra (Figures S8, S9, S16, S17, S24, S25, S33, and S34 in Supporting Information) confirmed that the connectivities of the 1H spin systems were identical for both compounds apart from the loss of one

proton at position 34 and a change of δH for the remaining H34 in PTX11, consistent with a hydroxyl substituent at this position. The corresponding 13C resonances determined from gHSQC spectra (Figures S12, S20, S27, and S37 in Supporting Information) also showed changes consistent with this substitution. Resonances of OH groups were detectable in the 1H spectra recorded in acetone-d6 (Supporting Information, Figures S22 and S23), and one of these peaks (δH 5.06) showed coupling (4.2 Hz) to H34. Other OH groups also showed coupling when

Identification of Pectenotoxin-11

Chem. Res. Toxicol., Vol. 19, No. 2, 2006 315

Figure 4. Proposed fragmentations observed in positive ion MS/MS spectra of PTX1 and PTX11. Ions in bold text arise from PTX11; ions in italic text arise from PTX1, and ions in parentheses are of very low abundance. All transitions shown involve loss of water (18 amu) unless otherwise indicated.

vicinal protons were present. It is noteworthy that OH resonances for 34-OH and 36-OH of PTX-11 in acetone-d6 were split into two separate peaks in an approximately 2:1 intensity ratio, the separation being greater at 34-OH (see footnote to Table 1). This effect is consistent with slow exchange between two OH-conformers involved to differing degrees in intramolecular H-bonding. The atomic connectivity and resonance assignments of PTX11 were confirmed by 1H/13C gHMBC spectra (Figures S11, S19, S28, S29, S38, and S39 in Supporting Information). Figures S1 and S2 in the Supporting Information summarize HMBC for solutions in acetone-d6 at 20 °C, in which all observed twoand three-bond 1H-13C correlations were consistent with the proposed structure. All methyl protons showed correlations to all carbons two- and three-bonds removed, and several correlations associated with rings and spiro junctions were observed in both PTX11 and PTX2. The close agreement of chemical shifts and HMBC patterns between PTX11 and PTX2, except for the differences noted above, suggested that the relative stereochemistries of the two compounds were likely to be the same in all regions other than position 34. Confirmation of this was provided by close correspondences in the pattern of NOESY and/or ROESY connectivities (Supporting Information, Figures S10, S18, S26, S35, and S36) and coupling constants (JHH, Table 1) for the two compounds. Figures S3 and S4 in Supporting Information summarize ROESY connectivities for compounds in acetoned6. The orientation of H32, H33, and H35 methine protons of PTX2 (R-, R-, and β-, respectively) corresponded to those of the equivalent protons in PTX11. The ROESY spectrum of PTX11 (solvent CD3OD, Figure S18 in Supporting Information) included correlations from H32 (4.81 ppm) to H-31 (5.38 ppm) and from H-33 (5.31 ppm) to H-30 (6.48 ppm) and H34 (4.50 pm).

The stereochemistry at C34 was revealed by analyses of the JHH couplings that H35 and H33 exhibited with the 34-CH2 or 34-CHOH protons of PTX2 and PTX11, respectively. Couplings discussed in the following are those measured in CD3OD, but similar values were measured in acetone-d6. The appearance of the H35 signal of PTX2 as a doublet of doublets (J ) 10.5, 5.8 Hz) was consistent with a conformation of PTX2 in which the 34-methylene protons were oriented pseudoaxially (H34R) and pseudoequatorially (H34β), respectively, with respect to H35. The observed coupling constants are in accord with those predicted by the Karplus equation (27) for moderate (30-60°) and large (150-180°) dihedral angles, respectively. On the other hand, H33R of PTX2 appeared as a triplet (J ) 3.2 Hz) due to small couplings with H32R and H34R, but not H34β, since the dihedral angle between H34β and H33, as revealed in molecular modeling studies, is close to 90°. The calculated solution conformation of the C32-C35 ring of PTX2 was very similar to the solid-state conformation determined by X-ray crystallography for PTX1 (1). In PTX11 (solvent CD3OD), the H-34 methine (CHOH) signal (4.50 ppm) appears as a well-defined doublet of doublets (J ) 9.1, 4.2 Hz), consistent with a pseudo-trans-1,2-diaxial relationship between H35 (4.16 ppm) and H34, and a pseudoequatorial/axial relationship between H33 (5.31 ppm) and H34. It follows from these observations that H34 is pseudoequatorially oriented on the R-face (upper face) of the C32-C35 ring, and that PTX11 is therefore the 34β-hydroxy analogue of PTX2 (Figure 1). The appearance of the H33 signal of PTX11 as a doublet of doublets (J ) 4.2, 2.6 Hz) (couplings with H34R and H32, respectively) is also consistent with the presence in PTX11 of a pseudoequatorial 34β-OH group, rather than a pseudoaxial 34R-OH group, since the latter would be expected to lead to H32 appearing as a doublet (i.e., coupled only to H31) due to

316 Chem. Res. Toxicol., Vol. 19, No. 2, 2006

Figure 5. Cross-section (H-34) from the ROESY NMR spectrum of PTX11 in CD3OD.

the dihedral angle between H33 and H34β approaching 90°. Correlations observed in ROESY, SELROESY, and SELNOESY spectra of PTX11 (Figure 5 and Figure S21 in Supporting Information) were also consistent with a pseudoaxially oriented 34R-proton. For example, mutual ROESY and NOESY correlations were observed for the R-face H32 (4.81 ppm), H33 (5.31 ppm), and H34 (4.50 ppm) oxymethine (-CH-O-) protons of PTX11. H34R also showed a ROESY correlation (Figure 5) to H37 (3.55 ppm), together with a TOCSY-like artifact (antiphase) correlation to the strongly coupled adjacent H35 signal (4.16 ppm, J ) 9.1 Hz). A TOCSYlike H35 artifact peak was also seen in the corresponding SELROESY spectrum, but the corresponding SELNOESY peak (Figure S21 in Supporting Information) showed relatively little antiphase character indicating a positive NOE. Because the modeled internuclear distance between H35 and H34R is 3.0 Å, we cannot exclude the possibility that a moderate ROESY response might have been superimposed on the TOCSY-like artifact peak seen in these spectra. ROESY spectra recorded under differing conditions of magnetic field, solvent, temperature, concentration, mixing time, and spinlock were consistent except where ambiguities existed owing to overlap. The ROESY correlation that H34R showed with H37 was analogous to that observed for these protons in ROESY and NOESY spectra of PTX2 and demonstrates that the preferred solution conformations of the C32-C35 and C36-C40 rings of PTX2 and PTX11 are similar. It is also noteworthy that, while the C4 and C37 resonances of PTX11 (30.4 and 71.4 ppm) corresponded closely to those determined for PTX2 (30.4 and 71.7 ppm, respectively), the resonance of one of the H4 methylene protons, and of the H37 methine proton, differed significantly (1.80 and 3.55 ppm, respectively in PTX11, compared to 1.52 and 3.29 ppm, respectively, in PTX2). This difference can be attributed to the orientation of the 34β-OH group toward these protons, as revealed by molecular modeling of PTX11 (Figure 6). The above NMR data therefore establishes the structure of PTX11 as 34S-hydroxyPTX2, based on the absolute configuration previously established for the pectenotoxins (28). PTX11 appears to be only the third pectenotoxin identified as a natural biosynthetic product from algae, after PTX2 and PTX12. Interestingly, three small peaks were observed in the m/z 892 chromatogram of the original plankton extract (Figure 2c) that

Suzuki et al.

Figure 6. Molecular model of PTX11, showing calculated structure, atom numbering for the F-ring, and selected NOE interactions observed in the NMR spectrum.

had UV and mass spectra almost identical with those of PTX11. These peaks have been labeled PTX11b, PTX11c, and PTX13. It was previously reported that PTX1 and PTX6 undergo acidcatalyzed isomerization at the spiroketal carbon of the A/B ring system by ring opening and re-closure (29). The isomers of PTX1 were named PTX4 and PTX8, while those of PTX6 were named PTX7 and PTX9 (Figure 1). In our previous study, both PTX2 and PTX11 were shown to convert to two analogous isomers under acidic conditions, which we named PTX2b/ PTX2c and PTX11b/PTX11c (22), respectively. The observed retention times of PTX11b and PTX11c, relative to that of PTX11, closely matched the relative retention times of the corresponding isomers of PTX1, PTX6 and PTX2. Therefore, PTX11b and PTX11c are presumed by analogy to be spiroketal isomers of PTX11 with the structures shown in Figure 1. These structural assignments are only tentative since insufficient quantities were available for NMR. The peak labeled PTX13 (previously PTX11x in Suzuki et al. (22)) in Figure 2c is another hydroxylated PTX that is also produced by the D. acuta, and its structure will be reported separately. The LD50 of PTX11 by intraperitoneal injection was 244 µg/ kg, with 95% confidence limits between 214 and 277 µg/kg. The LDmin of PTX11 in these experiments was 250 µg/kg. Deaths occurred in most cases between 4 and 15 h after dosing, although two animals dosed at 250 µg/kg were killed in extremis after 22 and 23 h. Preliminary studies showed that PTX11, like PTX2, was much less toxic orally than by intraperitoneal injection, and we used the highest dose that we could, consonant with the amount of material available for testing, for the definitive experiment on oral toxicity. This was 5000 µg/kg. No diarrhea was observed in any of the animals dosed with the test substance by either route of administration. The acute toxicity of PTX11 by intraperitoneal injection was similar to that of PTX2 (23), and the symptoms of intoxication were identical. Mice given sublethal doses of PTX11 showed no longterm effects of treatment and remained in good health until the end of the experiment, 14 days after dosing. Figure 7 shows changes in concentration of PTX2, PTX2sa, and PTX11 during incubation with supernatant from centrifuged green-lipped mussel hepatopancreas. Although PTX2 was rapidly (t1/2 ) 35 min) and cleanly converted to PTX2sa by enzymes from the mussel hepatopancreas as described previously (20, 23), hydrolysis of PTX11 to its seco acid in the same

Identification of Pectenotoxin-11

Chem. Res. Toxicol., Vol. 19, No. 2, 2006 317 and in CD3OD and at 11.7 T, 20 °C in acetone-d6. This material is available free of charge via the Internet at http://pubs.acs.org.

References

Figure 7. Concentrations of PTX2, PTX2sa, and PTX11 versus time during incubation with homogenized green-lipped mussel hepatopancreas.

sample was not detectable even after 16 h. PTX11 is therefore at least 2 orders of magnitude less easily hydrolyzed than PTX2 by the enzymes in the mussel hepatopancreas. This suggests that because of steric hindrance by the 34-hydroxyl group, and possibly hydrogen bonding between the 34-OH and the carbonyl oxygen of PTX11 (Figure 6), the latter is not a favored substrate for the enzyme(s) responsible for hydrolysis of the lactone ring of PTX-2. As a consequence, PTX11 is expected to accumulate to a much greater extent than PTX2 (which is rapidly detoxified to PTX2sa (23)) in mussels. This is consistent with the previous observation that there was a large difference between the ratios of PTX11 and the putative PTX11sa (0.63) and PTX2/PTX2sa (0.04) in naturally contaminated green-lipped mussels collected in New Zealand (16). In conclusion, the structure of PTX11 isolated from the toxic dinoflagellate D. acuta collected from the coast of New Zealand was elucidated as 34S-hydroxy-PTX2. Although the toxicity of PTX11 is similar to that of PTX2 when injected intraperitoneally into mice, no diarrhea was observed after oral or ip administration of PTX11, and no adverse effects were observed upon oral administration of PTX11 at a dose that would equate to 300 mg for a 60-kg human. Similar results have recently been reported for PTX2 and PTX2sa (23). This confirms that the pectenotoxins are wrongly included within the DSP group, although more detailed studies of the toxicity of pectenotoxins, particularly with regard to their chronic toxicity, are required. Acknowledgment. The authors gratefully acknowledge the technical assistance of W. Hardstaff, Ian Burton, A. D. Hawkes, and A. I. Selwood. Thanks to K. Solley and J. Knight for assistance with plankton sampling. We thank the Japan Food Research Laboratories for providing a sample of PTX1. Financial support for a visit of T. Suzuki to Halifax was provided by the Asia-Pacific Economic Cooperation. This study was also supported by New Zealand Foundation for Research, Science and Technology Contract Numbers CAWX0301, and by Norwegian Research Council Grant 139593/140. This publication is NRCC No. 2005-42555. Supporting Information Available: HMBC for PTX2 and PTX11 in acetone-d6; ROESY correlations for PTX2 and PTX11 in acetone-d6; and spectra recorded at 9.4 T, 30 °C, in acetone-d6

(1) Yasumoto, T., Murata, M., Oshima, Y., Sano, M., Matsumoto, K., and Clardy, J. (1985) Diarrhetic shellfish toxins. Tetrahedron 41, 1019-1025. (2) Yasumoto, T., and Murata, M. (1993) Marine toxins. Chem. ReV. 93, 1897-1909. (3) Terao, K., Ito, E., Yanagi, T., and Yasumoto, T. (1986) Histopathological studies on experimental marine toxin poisoning. I. Ultrastructural changes in the small intestine and liver of suckling mice induced by dinophysistoxin-1 and pectenotoxin-1. Toxicon 24, 1141-1151. (4) Ishige, M., Satoh, N., and Yasumoto, T. (1988) Pathological studies on the mice administrated with the causative agent of diarrhetic shellfish poisoning (okadaic acid and pectenotoxin-2). Rep. Hokkaido Inst. Public Health 38, 15-19. (5) Zhou, Z. H., Komiyama, M., Terao, K., and Shimada, Y. (1994) Effects of pectenotoxin-1 on liver cells in vitro. Nat. Toxins 2, 132-135. (6) Jung, J. H., Sim, C. S., and Lee, C. O. (1995) Cytotoxic compounds from the two-sponge association. J. Nat. Prod. 58, 1722-1726. (7) Lee, J. S., Murata, M., and Yasumoto, T. (1989) Analytical methods for determination of diarrhetic shellfish toxins. In Mycotoxins and Phycotoxins ‘88 (Natori, S., Hashimoto, K., and Ueno, Y., Eds.) pp 327-334, Elsevier, Amsterdam. (8) Lee, J. S., Igarashi, T., Fraga, S., Dahl, E., Hovgaard, P., and Yasumoto, T. (1989) Determination of diarrhetic shellfish toxins in various dinoflagellate species. J. Appl. Phycol. 1, 147-152. (9) Draisci, R., Lucentini, L., Giannetti, L., Boria, P., and Poletti, R. (1996) First report of pectenotoxin-2 (PTX2) in algae (Dinophysis fortii) related to seafood poisoning in Europe. Toxicon 34, 923-935. (10) Suzuki, T., Mitsuya, T., Matsubara, H., and Yamasaki, M. (1998) Determination of pectenotoxin-2 after solid-phase extraction from seawater and from the dinoflagellate Dinophysis fortii by liquid chromatography with electrospray mass spectrometry and ultraviolet detection: Evidence of oxidation of pectenotoxin-2 to pectenotoxin-6 in scallops. J. Chromatogr., A 815, 155-160. (11) James, K. J., Bishop, A. G., Draisci, R., Palleschi, L., Marchiafava, C., Ferretti, E., Satake, M., and Yasumoto, T. (1999) Liquid chromatographic methods for the isolation and identification of new pectenotoxin-2 analogues from marine phytoplankton and shellfish. J. Chromatogr., A 844, 53-65. (12) Draisci, R., Palleschi, L., Giannetti, L., Lucentini, L., James, K. J., Bishop, A. G., Satake, M., and Yasumoto, T. (1999) New approach to the direct detection of known and new diarrhoeic shellfish toxins in mussels and phytoplankton by liquid chromatography-mass spectrometry. J. Chromatogr., A 847, 213-221. (13) Sasaki, K., Takizawa, A., Tubaro, A., Sidari, L., DellaLoggia, R., and Yasumoto, T. (1999) Fluorometric analysis of pectenotoxin-2 in microalgal samples by high performance liquid chromatography. Nat. Toxins 7, 241-246. (14) Pavela-Vrancic, M., Mestrovic, V., Marasovic, I., Gillman, M., Furey, A., and James, K. J. (2001) The occurrence of 7-epi-pectenotoxin-2 seco acid in the coastal waters of the central Adriatic (Kastela Bay). Toxicon 39, 771-779. (15) Vale, P., and Sampayo, M. A. (2002) Pectenotoxin-2 seco acid, 7-epipectenotoxin-2 seco acid and pectenotoxin-2 in shellfish and plankton from Portugal. Toxicon 40, 979-987. (16) MacKenzie, L., Holland, P., McNabb, P., Beuzenberg, V., Selwood, A., and Suzuki, T. (2002) Complex toxin profiles in phytoplankton and Greenshell mussels (Perna canaliculus), revealed by LC-MS/MS analysis. Toxicon 40, 1321-1330. (17) Miles, C. O., Wilkins, A. L., Samdal, I. A., Sandvik, M., Petersen, D., Quilliam, M. A., Naustvoll, L. J., Rundberget, T., Torgersen, T., Hovgaard, P., Jensen, D. J., and Cooney, J. M. (2004) A novel pectenotoxin, PTX12, in Dinophysis Spp. and shellfish from Norway. Chem. Res. Toxicol. 17, 1423-1433. (18) Yasumoto, T., Murata, M., Lee, J. S., and Torigoe, K. (1989) Polyether toxins produced by dinoflagellates. In Mycotoxins and Phycotoxins ‘88 (Natori, S., Hashimoto, K., and Ueno, Y., Eds.) pp 375-382, Elsevier, Amsterdam. (19) Daiguji, M., Satake, M., James, K. J., Bishop, A., Mackenzie, L., Naoki, H., and Yasumoto, T. (1998) Structures of new pectenotoxin analogs, pectenotoxin-2 seco acid and 7-epi-pectenotoxin-2 seco acid, isolated from a dinoflagellate and Greenshell mussels. Chem. Lett. 7, 653-654. (20) Suzuki, T., Mackenzie, L., Stirling, D., Adamson, J. (2001) Pectenotoxin-2 seco acid: A toxin converted from pectenotoxin-2 by the New Zealand Greenshell mussel, Perna canaliculus. Toxicon 39, 507-514. (21) Suzuki, T., Mackenzie, L., Stirling, D., and Adamson, J. (2001) Conversion of pectenotoxin-2 to pectenotoxin-2 so acid in the New Zealand scallop, Pecten noVaezelandiae. Fish. Sci. 67, 506-510.

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