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42,43,44,45,46,47,55-Heptanor-41-oxohomoyessotoxin, a New Biotoxin from Mussels of the Northern Adriatic Sea Patrizia Ciminiello,*,† Ernesto Fattorusso,† Martino Forino,† and Roberto Poletti‡ Dipartimento di Chimica delle Sostanze Naturali, Universita` degli studi di Napoli “Federico II”, via D. Montesano 49, 80131, Napoli, Italy, and Centro di Ricerche Marine, via A. Vespucci 2, 47042 Cesenatico (FO), Italy Received December 21, 2000
The diarrhetic shellfish toxin composition in the digestive glands of mussels from the northern Adriatic sea was investigated. Along with known yessotoxins, identified by comparison of their chromatographic and spectral properties with those reported in the literature, we isolated a new analogue of yessotoxin, 42,43,44,45,46,47,55-heptanor-41-oxohomoyessotoxin, 1. Its structure was determined by 1H NMR spectroscopy and mass spectrometry.
Introduction The contamination of shellfish with marine biotoxins derived from micro-algae continues to be a problem for the shellfish industry and public health. Initially, where formerly a few regions were affected in scattered locations, now virtually every coastal state is threatened, in many cases over large geographic areas and by more than one harmful or toxic algal species. The number of toxic blooms, the economic losses from them, the types of resources affected, and the number of toxins and toxic species have therefore all increased dramatically in recent years all around the world (1-3). Recently, we focused our attention on biotoxin shellfish contamination in Italy, where, at the present, it appears to be almost exclusively due to Diarrhetic Shellfish Poisoning (DSP)1 toxins (4-9). Previous studies by Yasumoto’s group revealed the presence of three types of DSP toxins differing in basic skeletons and toxicological effects (10). The first (acidic toxins) consists of okadaic acid and related compounds, the second (neutral toxins) pectenotoxins, and the third yessotoxins. The scenario of shellfish toxicity in Italy seems quite uncommon and changeable. Recent work in our laboratory, carried out on mussels from the Adriatic Sea, the main producing area in Italy, resulted in the isolation and chemical characterization of a number of toxic compounds including DSP toxins. Some of them, representing moreover the major toxins, are new toxins, structurally related to yessotoxin (YTX) and not reported until now in any other country (7-9). As part of our study, we are continuing the characterization of the toxins from Adriatic mussels with the aim * To whom correspondence should be addressed. Phone: (39) 081 678 507. Fax: (39) 081 678 552. E-mail:
[email protected]. † Dipartimento di Chimica delle Sostanze Naturali. ‡ Centro di Ricerche Marine. 1 Abbreviations: CID, collision induced dissociation; COOHhomoYTX, carboxyhomoyessotoxin; COSY, correlation spectroscopy; DSP, diarrhetic shellfish poisoning; FAB-MS, fast-atom bombardment mass spectrometry; HOHAHA, homonuclear Hartmann Hahn; homoYTX homoyessotoxin; HRFABMS, high-resolution fast-atom bombardment mass spectrometry; NOE, nuclear Overhauser effect; NoroxohomoYTX, 42,43,44,45,46,47,55-Heptanor-41-oxohomoyessotoxin; 45-OHhomoYTX, 45-Hydroxyhomoyessotoxin; ROESY, rotating-frame Overhauser effect spectroscopy; YTX, yessotoxin.
Figure 1. Structure of noroxohomoyessotoxin.
to deepen our knowledge on the toxicological hazard of our seas and to obtain significant amounts of toxic material indispensable to evaluate the health risks due to these toxins. To emphasize once more the variety of the toxin composition peculiar to the Adriatic mussels, we have now isolated a new YTX analogue, 42,43,44,45,46,47,55heptanor-41-oxohomoyessotoxin (noroxohomoYTX, 1, Figure 1).
Experimental Procedures Isolation. NoroxohomoYTX was isolated from the hepatopancreas of toxic mussels Mytilus galloprovincialis collected in October 1998 (3.7 kg, dry weight) from one sampling site located along the Emilia Romagna coasts. The digestive glands were found to be toxic by the mouse bioassay method for DSP (11). The new toxin has been isolated with the procedure already described for the extraction of other DSP toxins (9). Digestive glands were extracted with acetone twice. After evaporating acetone, the aqueous concentrate was extracted thrice with EtOAc and then twice with BuOH. The EtOAc extracts were combined separately from the BuOH extracts. Isolation of noroxohomoYTX was carried out from the combined EtOAc extract. After removal of EtOAc, the extract was partitioned between 80% MeOH and hexane. The hydromethanolic layer was further partitioned between 40% aqueous methanol and methylene chloride. The dichloromethane soluble material was then chromatographed on a Develosil ODS column washing stepwise with MeOH-H2O (8:2 and 9:1) and MeOH in this
10.1021/tx000259v CCC: $20.00 © 2001 American Chemical Society Published on Web 04/26/2001
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Table 1. Comparison of 1H NMR Chemical Shift (δ) of HomoYTX with Those of NoroxohomoYTX in CD3OD position
HomoYTX
NoroxohomoYTX
position
HomoYTX
NoroxohomoYTX
1 2 2a CH3-3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 CH3-19 20 21 22 CH3-23 24
3.96; 4.00 1.78; 1.83 1.61; 1.78 1.19 4.32 1.70; 2.56 3.05 3.31 1.44; 2.22 3.17 3.15 1.45; 2.30 3.03 3.12 1.47; 2.34 3.35 3.26 1.84; 1.99 1.83; 1.89 1.29 3.46 1.80; 1.97 3.53 1.20 1.54; 1.77
3.96; 4.00 1.78; 1.83 1.61; 1.78 1.19 4.32 1.70; 2.56 3.05 3.31 1.44; 2.22 3.17 3.15 1.45; 2.30 3.03 3.12 1.47; 2.34 3.35 3.26 1.84; 1.99 1.83; 1.89 1.29 3.46 1.80; 1.97 3.53 1.20 1.54; 1.77
25 26 CH3-26 27 28 29 30 31 32 CH3-33 34 35 36 37 38 CH2)39 40 CH3-41 42 43 CH2)44 45 46 47
1.50; 1.75 1.73 1.07 2.81 3.34 1.58; 2.32 3.64 3.22 3.89 1.25 3.80 1.53; 2.14 4.09 3.43 2.47; 2.75 4.84; 5.05 3.92 1.43 5.86 6.35 5.01; 5.09 3.00; 3.00 5.91 5.10; 5.12
1.50; 1.75 1.73 1.07 2.81 3.34 1.58; 2.32 3.64 3.22 3.89 1.25 3.80 1.53; 2.14 4.09 3.43 2.18; 2.54 5.18; 5.22 4.71 1.38 2.22
matrix). NMR spectra were measured on a Bru¨ker AMX-500 spectrometer in CD3OD.
Results and Discussion
Figure 2. Structures of homoyessotoxins. order. Toxins eluted in the last fraction were passed through a Toyopearl HW-40 SF column with MeOH. The toxins were dissolved in MeOH-H2O 6:4 and further purified on a RP-8 column equilibrated with the same solvent. The column was then washed stepwise with MeOH-H2O (6:4 and 8:2) in this order. The presence of yessotoxins (YTXs) in the eluates was checked by TLC (silica gel 60, CHCl3,-MeOH-H2O 30:10:1) and by monitoring ultraviolet absorption at 230 nm. The final HPLC purification was carried out on a RP 18 column with CH3CNMeOH-H2O 1:2:2 as eluent, thus obtaining homoyessotoxin (6) [homoYTX, 2 (1,0 mg)], 45-hydroxyhomoyessotoxin (6) [45-OHhomoYTX, 3, (400 µg)], carboxyhomoyessotoxin (9) [COOHhomoYTX, 4, (600 µg)], and noroxohomoYTX, 1 (400 µg) in pure forms (Figures 1 and 2). NoroxohomoYTX, 1. FAB-MS (negative ion mode): m/z 1083 (M - Na)-, 1061 (M - 2Na + H)- and 981 (M - SO3Na Na + H)-. HRFABMS (negative mode): m/z 1803.3921 [calculated for C49H72O21S2Na (M - Na)- 1803.3905]; 1H NMR data (CD3OD) are reported in Table 1. MS and NMR Spectra Measurements. The fast-atom bombardment (FAB) mass spectrum was measured at 70 eV with a Kratos MS50 mass spectrometer (CsI ions, glycerol matrix). CID (collision induced dissociation) MS/MS experiments were carried out on a AUTOSPECTOF mass spectrometer (DHB
From the digestive glands (3.7 kg), 1.0 mg of homoYTX, 2 (6), 400 µg of 45-OHhomoYTX, 3 (6), 600 µg of COOHhomoYTX, 4 (9), and 400 µg of noroxohomoYTX, 1, were obtained. HomoYTX, 45-OHhomoYTX, and COOHhomoYTX were identified by comparison of their chromatographic behavior, as well as of their spectral properties, with authentic samples previously isolated in our laboratory. Negative FAB mass spectrum of noroxohomoYTX (1) gave a pattern of fragmentation typical of a sulfated compound with ion peaks at m/z 1083 (M - Na)-, 1061 (M - 2Na + H)-, and 981 (M - SO3Na - Na + H)-. Preliminary analysis of 1H NMR spectrum of 1 showed a close resemblance to those of YTXs, strongly suggesting that it possessed the same basic polycyclic ether skeleton as YTXs. There were two basic differences, however, in the proton spectrum of 1: first of all, the lack of the characteristic signals of the side chain at C-40 in the olefinic region of spectrum; second, the presence of an additional methyl signal at relatively lowfield (δ 2.22). The polycyclic skeletal structure was mainly confirmed on the basis of homonuclear 2D NMR data, obtained from COSY (correlation spectroscopy) and HOHAHA (homonuclear Hartmann Hahn) experiments. In addition, the analysis of these spectroscopic data suggested that the toxin under investigation was of homoyessotoxin type (6, 9), namely with an extra methylene to the structure of YTX in the western side chain of the molecule. In fact, the characteristic signals relative to the protons on C-1, C-2, and C-2a were present in the 1H NMR spectrum of 1 (see Table 1) and the proton connectivities from H2-1 to H2-2a were easily determined by COSY and HOHAHA experiments. These experiments revealed, in addition, 1H NMR chemical shifts for positions 1-37 in 1 perfectly superimposable to the corresponding shifts in a typical homoYTX; remarkable differences, however, were observed for the resonance of the protons located on the last ring at the east part of the molecule. The structure
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Figure 3. Characteristic fragment ions observed in the tandem mass spectrum of noroxohomoYTX, 1. In the CID spectrum for the molecular ion at m/z 1083 as a precursor, all the fragmentations were accompanied by loss of sulfonate (SO3) which denotes that fragment ions linked one of the sulfate esters.
Figure 4. Significant NOEs confirming the ring conformations of 1. (a) Conformers and stereostructure around the last two rings of the eastern part of the molecule.
of this ring was deduced from further consideration of 2D COSY and HOHAHA data, which allowed the extension of the skeletal spin system from H-37 to H2-38, whose resonances at δ 2.54 and 2.18 were upfield shifted, compared to the corresponding resonances in the 1H NMR spectrum of homoYTX. The location of the exomethylene group at position C-39, as well as the closure of the last ether ring was made possible on the basis of both a long-range coupling between H-38 (δ 2.18) and H2-53 (δ 5.18 and 5.22) and a NOE (nuclear Overhauser effect) between H-53 (δ 5.18) and H-40 (δ 4.71). At this point, to ultimate the structural determination of the toxin it remained to introduce in the molecule a
side chain of 43 mass units at C-40. It had to be a COCH3 group because of the presence in the 1H NMR spectrum of 1 of the characteristic downshifted resonance of a methyl adjacent to a ketone functionality (δ 2.22) and a strong NOE between this methyl and H-40, which in turn became greatly deshielded (δ 4.71) compared to the corresponding resonance in the 1H NMR spectrum of homoYTX (δ 3.92). The whole of the above data indicated structure 1 for compound under investigation, apart from the stereochemistry. Negative ion FAB MS/MS provided essential information (Figure 3) to confirm the above structure. Collisionally induced dissociation (CID) MS/MS experi-
NoroxohomoYTX from Adriatic Mussels
ment was carried out on the molecular-related ion at m/z 1083 (M - Na)-. The validity of this methodology was well established in previous structural studies on yessotoxin (12), maitotoxin (13), and brevetoxin B3 (14). All the fragment ions arose from the “western” part of the molecule where a negative charge is localized on a sulfate group. All prominent ions in the MS/MS spectrum of 1 generated by bond cleavage at the sites characteristic of ether rings provided invaluable information regarding sizes of ether rings and/or substituents on rings, that were consistent with the proposed structure. The ROESY (rotating-frame Overhauser effect spectroscopy) spectrum further confirmed the above planar structure; in addition, it revealed relative stereochemistry of the molecule to be identical to that of YTX (15), as well as to that of all other YTXs analogues so far isolated, being observed the same key NOE correlations (4-9) (Figure 4). In addition, the NOE correlations between H-36 and Me-42 indicated the diaxial relationship between C-41 and H-36, thus defining the stereochemistry at C-40. The very limited amount of pure noroxohomoYTX did not allow evaluation of its toxicological properties; anyway, its toxicity seems to be comparable with that of 45OHYTX. In fact, both noroxohomoYTX and 45-OHYTX showed very similar mouse lethality (ca. 0.5 mg/kg). The finding of a new yessotoxin-like compound in Adriatic mussels highlights that, among shellfish toxins, YTXs are the ones which pose most serious problems to aquaculture in the Adriatic Sea because of their persistent occurrence in the past few years. Unfortunately, YTXs still remain unknown as to their mode of action. Therefore, a better insight into their toxicology and their effects at the molecular and cellular level is needed. Any sort of toxicity test and investigation onto the molecular basis of the effects in livings systems absolutely requires availability of toxins. Minute amounts of some YTX components characterized so far have been obtained only in a few laboratories, and quantities are not sufficient for widespread testing. A notable exception might be yessotoxin itself, which has been recently commercialized, even if the purchase of YTX on the market did not result, up to now, in a clear definition of its toxicology. Anyway, it is now evident that there is a variety of YTX analogues in some shellfish producing areas and toxicological investigations should be addressed to all the YTX-like compounds. Therefore, many efforts must be directed to the accumulation of these toxins to be addressed to further toxicological studies.
Acknowledgment. This work is a result of a research supported by MURST PRIN, Rome, Italy. NMR
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and FABMS spectra were performed at “Centro di Ricerca Interdipartimentale di Analisi strumentale”, Universita` degli studi di Napoli “Federico II”. The assistance of the staff is gratefully appreciated.
References (1) Anderson, D. M. (1989) Toxic algal bloom and red tides: A global perspective. In Red Tides: Biology, Enviromental Science and Toxicology (Okaichi, D. M., Anderson, D. M., and Nemoto, T., Eds.) pp 11-16, Elsevier Science Publishing Co, New York. (2) Smayda, T. J. (1990) Novel and nuisance phytoplankton blooms in the sea: Evidence for a global epidemic. In Toxic Marine Phytoplankton (Graneli, E., Sundstrom, B., Edler, L., and Anderson, D. M., Eds.) pp 29-40, Elsevier Science Publishing Co, New York. (3) Hallegraff, G. M. (1993) A review of harmful algal blooms and their apparent global increase. Phycology 32, 79-99. (4) Fattorusso, E., Ciminiello, P., Costantino, V., Magno, S., Mangoni, A., Milandri, A., Poletti, R., Pompei, M., and Viviani, R. (1992) Okadaic acid in mussels of Adriatic Sea. Mar. Poll. Bull. 24, 234237. (5) Ciminiello, P., Fattorusso, E., Forino, M., Magno, S., Poletti, R., Satake, M., Viviani, R., and Yasumoto, T. (1997) Yessotoxin in mussels of the northern Adriatic Sea. Toxicon 35, 177-183. (6) Satake, M., Tubaro, A., Lee, J. S., and Yasumoto, T. (1997) Two new analogues of yessotoxin, homoyessotoxin and 45-hydroxyhomoyessotoxin, isolated from mussels of the Adriatic Sea. Nat. Toxins 5, 107-110. (7) Ciminiello, P., Fattorusso, E., Forino, M., Magno, S., Poletti, R., and Viviani, R. (1998) Isolation of adriatoxin, a new analogue of yessotoxin from mussels of the Adriatic Sea. Tetrahedron Lett. 39, 8897-8900. (8) Ciminiello, P., Fattorusso, E., Forino, M., Poletti, R., and Viviani, R. (2000) A new analogue of yessotoxin, carboxyyessotoxin, isolated from Adriatic Sea mussels. Eur. J. Org. Chem. 2, 291295. (9) Ciminiello, P., Fattorusso, E., Forino, M., Poletti, R., and Viviani, R. (2000) Structure determination of carboxyhomoyessotoxin, a new yessotoxin analogue isolated from Adriatic mussels. Chem. Res. Toxicol. 13, 770-774. (10) Murata, M., and Yasumoto, T. (1993) Marine Toxins. Chem. Rev. 93, 1897-1909. (11) Gazzetta Ufficiale della Repubblica Italiana, September 10, 1990, no. 211; Decreti Ministeriali, August 1, 1990, nos. 256 and 257. (12) Naoki, H., Murata, M., and Yasumoto, T. (1993) Negative-ion fastatom bombardment tandem mass spectrometry for the structural study of polyether compounds: structural verification of yessotoxin. Rapid Commun. Mass Spectrom. 7, 179-182. (13) Murata, M., Naoki, H., Matsunaga, S., Satake, M., and Yasumoto, T. (1994) Structure and partial assignments for maitotoxin, the most toxic and largest natural nonbiopolymer. J. Am. Chem. Soc. 116, 7098-7107. (14) Morohashi, A., Satake, M., Murata, K., Naoki, H., Kaspar, H. F., and Yasumoto, T. (1995) Brevetoxin B3, a new Brevetoxin analogue isolated from the greenshell mussel Perna canaliculus involved in Neurotoxic Shellfish Poisoning in New Zealand. Tetrahedron Lett. 36, 8995-8998. (15) Satake, M., Terasawa, K., Kadowaki, Y., and Yasumoto, T. (1996) Relative configuration of yessotoxin and isolation of two new analogues from toxic scallops. Tetrahedron Lett. 37, 5955-5958.
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