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Structure Determination of Carboxyhomoyessotoxin, a New Yessotoxin Analogue Isolated from Adriatic Mussels Patrizia Ciminiello,*,† Ernesto Fattorusso,*,† Martino Forino,† Roberto Poletti,‡ and Romano Viviani§,| Dipartimento di Chimica delle Sostanze Naturali, Universita` degli studi di Napoli “Federico II”, via D. Montesano 49, 80131 Napoli, Italy, Centro di Ricerche Marine, via A. Vespucci 2, 47042 Cesenatico (FO), Italy, and Dipartimento di Biochimica, Sezione di Biochimica Veterinaria, Universita` degli studi di Bologna, via Tolara di Sopra 30, 40064 Ozzano Emilia (Bologna), Italy Received March 1, 2000
The contamination of shellfish with marine biotoxins derived from microalgae represents a serious problem for shellfish industries and public health. This study investigated the composition of diarrhetic shellfish toxins in the digestive glands of mussels from the Northern Adriatic Sea. 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, carboxyhomoyessotoxin, whose structure was determined by mass spectrometry and 1H NMR spectroscopy.
Introduction Among the thousands of species of microscopic algae at the base of the marine food chain, there are a few dozen which produce potent toxins. One major category of impact occurs when toxic phytoplankton are filtered from the water as food by shellfish such as clams, mussels, oysters, or scallops, in which the algal toxins accumulate to levels that are potentially lethal to humans or other consumers (1, 2). Typically, the shellfish are only marginally affected, even though a single clam can sometimes contain sufficient toxin to kill a human. These poisoning syndromes have been named paralytic, diarrhetic, neurotoxic, and amnesic shellfish poisoning (PSP,1 DSP, NSP, and ASP, respectively). Except for ASP, all are caused by biotoxins synthesized by dinoflagellates. The toxins involved in DSP (diarrhetic shellfish poisoning) are the most important for their wide distribution over the world (3). The chemically defined DSP toxins fall into three structural classes, all being lipophilic compounds (4, 5): the first (acidic toxins) consisting of okadaic acid and related compounds, the second (neutral toxins) of pectenotoxins, and the third of yessotoxins. DSP episodes in Italy were first reported in 1989 in patients who have eaten mussels (Mytilus galloprovincialis) collected from the Northern Adriatic Sea (6). Unfortunately, DSP outbreaks, associated with blooms of harmful mi* Corresponding author. Phone: (39) 081 7486 503 or 507. Fax: (39) 081 7486 552. E-mail:
[email protected] or
[email protected]. † Universita ` degli studi di Napoli “Federico II”. ‡ Centro di Ricerche Marine. § Universita ` degli studi di Bologna. | Deceased June 1, 2000. 1 Abbreviations: 45-OHhomoYTX, 45-hydroxyhomoyessotoxin; ASP, Amnesic Shellfish Poisoning; ATX, Adriatoxin; CID, Collision Induced Dissociation; COOHhomoYTX, Carboxyhomoyessotoxin; COOHYTX, Carboxyyessotoxin; DSP, Diarrhetic Shellfish Poisoning; DTX-1, Dinophysistoxin-1; ESI, electron spray ionization; homoYTX, Homoyessotoxin; MALDI, Matrix Assisted Laser Desorption Ionization; NOE, Nuclear Overhauser Effect; NSP, Neurotoxic Shellfish Poisoning; OA, Okadaic Acid; PGME, Phenylglycine methyl ester; PSP, Paralytic Shellfish Poisoning; ROESY, Rotating-frame Overhauser Effect Spectroscopy; YTX, Yessotoxin.
croalgae, have occurred in the Adriatic Sea with alarming frequency since then, causing serious threat to human health and severe economic losses for shellfish industries, whose production areas have been forced to remain closed for some months. Therefore, in Italy a research program to prevent the risk of diarrhetic poisoning associated with the ingestion of contaminated shellfish was initiated in 1990. To obtain more detailed information about the DSP contamination in mussels of the Adriatic Sea, we have been analyzing toxic samples of shellfish collected along the Emilia Romagna coasts in correspondence to the highest level of toxicity since 1992. A constant providing of toxic shellfish instrumental analysis is indispensable because toxin profiles may change in both chemical structure and toxicological effects year after year. In fact, while okadaic acid (OA) was the major toxin in the toxic episode of 1989 (7), the toxin profile has completely changed in the last years, and yessotoxin (YTX, 1) and its analogues are now predominant. The first presence of yessotoxin in the Northern Adriatic Sea was discovered by our laboratory in 1995, when the analysis of mussels collected in June along the coasts of Emilia Romagna showed high levels of this DSP toxin in addition to trace amounts of okadaic acid (8). Successively, other analogues of YTX have been isolated from hepatopancreas of mussels of the Adriatic Sea, some of which, homoyessotoxin [homoYTX, 2 (9)], 45-hydroxyhomoyessotoxin [45-OHhomoYTX, 3 (9)], adriatoxin [ATX, 4 (10)], and carboxyyessotoxin [COOHYTX, 5 (11)], represent new additions to the class of yessotoxins, not reported until now in any other country (Figure 1). Recent etiological study revealed that the biogenetic origin of YTX is different from those of OA and dinophysistoxin-1 (DTX-1). YTX is produced by the dinoflagellate Protoceratium reticulatum (12), while OA and DTX1 are produced mainly by Dinophysis spp. (13). Toxicologically, YTX also differs from OA and DTX1 in causing neither intestinal fluid accumulation in infant mice by intubation nor inhibition of protein phosphatase 2A. Thus, a ques-
10.1021/tx000048q CCC: $19.00 © 2000 American Chemical Society Published on Web 07/26/2000
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Figure 1. Structures of yessotoxins.
tion arises as to whether YTX should remain in the DSP category or not. The mouse lethality of YTX by intraperitoneal injection (0.1 mg/kg) is the strongest among all DSP toxins, but its oral toxicity is the weakest as can be deduced by considering that the maximum oral dose of 1 mg/kg does not kill the mice (14). Therefore, it can be reasonably concluded that YTX is much less hazardous than OA or DTX1 to human health. Nevertheless, the cell detachment and the lethal effect on infant mice as observed in a toxicological study (14) should be borne in mind. Despite the wealth of data on OA, the molecular mechanism underlying toxicity of YTXs is unknown. Indeed, very limited data are available regarding the effects of this group of components on cellular systems. Histopathological analysis revealed that a target organ of YTX is the heart; marked intracytoplasmatic edema in cardiac muscle cells was observed in mice after ip injection of the toxin (15). An involvement of the nervous system in YTX toxicity can also be hypothesized on the basis of the chemical structure, since brevetoxins and ciguatoxins (5), both structurally strictly related to YTX, induce poisoning characterized by neurological and cardiovascular symptoms. As for the mechanism of action, by analogy with brevetoxins and ciguatoxins, YTX may act as a depolarizing agent, opening membrane channels of Na+-permeable excitable cells and leading to a Na+ influx (16). It remains to be established, however, to
which extent these toxins can be absorbed by the intestine, and then gain access to target organs. The scarcity of toxicological data on YTX and its analogues may be a quite dangerous lack, since these currently appear to be the major biotoxins of mollusks from the Adriatic Sea, which represents the main Italian producing area of edible shellfish covering about 90% of the total production of mussels. The key role of yessotoxins in the poisoning of Adriatic mussels is now confirmed by the isolation of a new YTX analogue, carboxyhomoyessotoxin (COOHhomoYTX, 6), which represents one of the major toxins in these mussels (Figure 1).
Experimental Procedures Isolation. COOHhomoYTX was isolated from the digestive glands (3.7 kg) of mussels M. galloprovincialis, collected in autumn 1998 from one sampling site located along the Emilia Romagna coasts of Italy. The hepatopancreas was found to be toxic by the official mouse bioassay method for DSP (17). Digestive glands were extracted with acetone twice. After evaporation of 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 COOHhomoYTX was carried out from the combined EtOAc extract. After removal of EtOAc, the extract was partitioned between 80% MeOH and hexane. The toxic residue
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Figure 3. ∆δ (S - R) values (in hertz) for PGME amide derivatives of compound 6.
Figure 2. HPLC chromatogram of yessotoxins isolated from Adriatic mussels. Table 1. Comparison of 1H NMR Chemical Shifts (δ) of CarboxyYTX and CarboxyhomoYTX in CD3OD carboxycarboxyposition carboxyYTX homoYTX position carboxyYTX homoYTX 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
4.21, 4.21 1.99, 2.21 1.31 4.24 1.75, 2.59 3.10 3.35 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 CH3-26 27 28 29 30 31 32 CH3-33 34 35 36 37 38 CH2-39 40 CH3-41 42 43 CH2-44 44 45 46 47
1.50, 1.75 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 3.89 1.38 5.61 5.77 2.92 2.25, 2.46 5.82 4.95, 5.09
1.50, 1.75 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 3.89 1.38 5.61 5.77 2.92 2.25, 2.46 5.82 4.95, 5.09
obtained in the 80% MeOH 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 a MeOH/ H2O mixture (8:2 and 9:1) and MeOH in this order. Toxins eluted in the last fraction were passed through a Toyopearl HW40 SF column with MeOH. The toxins were dissolved in a 6:4 MeOH/H2O mixture and further purified on a RP-8 column equilibrated with the same solvent. The column was then washed stepwise with a MeOH/H2O mixture (6:4 and 8:2) in this order. The presence of YTXs in the eluates was checked by TLC (silica gel 60, 30:10:1 CHCl3/MeOH/H2O) and by monitoring ultraviolet absorption at 230 nm. The final HPLC purification was carried out on a RP 18 column with a 1:1:2 CH3CN/MeOH/ H2O mixture as the eluent (Figure 2).
Figure 4. Structure of carboxyhomoYTX (6). Arrows indicate the significant NOEs confirming the ring conformations of 6. (a) Conformers and stereostructure around the last two rings of the eastern part of the molecule. CarboxyhomoYTX (6): [R]D25 +13.3 (c 0.0006, MeOH); ESIMS (negative ion mode) m/z 1209 (M - Na)-, 1187 (M - 2Na + H)-; HR-FABMS (negative mode) m/z 1209.4571 [calcd for C56H82O23S2Na (M - Na)- 1209.4586]. The 1H NMR data (CD3OD) are reported in Table 1. MS and NMR Spectral Measurements. The electron spray ionization (ESI) mass spectrum was measured with a LCQFinnigan (ion trap) spectrometer. High-resolution FABMS (negative ion mode) was performed at 70 eV on a Kratos MS50 mass spectrometer (CsI ions, glycerol matrix). Negative ion MALDI (matrix-assisted laser desorption ionization), CID (collision-induced dissociation), and MS/MS experiments were carried out on a AUTOSPECTOF mass spectrometer (DHB matrix). NMR spectra were measured on a Bruker AMX-500 spectrometer in CD3OD. Reaction of COOHhomoYTX with (S)- and (R)-PGME. To a solution of compound 6 (0.3 mg, 2.5 × 10-4 mmol) in DMF (200 µL) at 0 °C were added 0.07 mg (1.5 equiv) of (S)phenylglycine methyl ester (PGME) hydrochloride, 0.22 mg (1.5 equiv) of benzotriazolyloxytris(pyrrolidinyl)phosponium hexafluorophosphate (PyBoP), 0.06 mg (1.5 equiv) of 1-hydroxybenzotriazole (HOBT), and 1 µL (4 equiv) of N-methylmorpholine, and the mixture was stirred at room temperature for 3 h. After addition of brine, the reaction mixture was extracted three times with a 2:1 EtOAc/benzene mixture, and the obtained organic phase was washed in sequence with 5 mL of 1.2 N HCl, water, NaHCO3-saturated aqueous solution, and water. The obtained organic phase was dried over Na2SO4 and filtered, and the solvent was removed under reduced pressure, affording the (S)PGME amide 7 (0.33 mg, 95% yield). Using (R)-PGME hydro-
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Figure 5. Characteristic fragment ions observed in the tandem mass spectrum of carboxyhomoYTX (6). In the CID spectrum for the molecular ion at m/z 1209 as a precursor, all the fragmentations were accompanied by loss of sulfonate (SO3) which indicates that fragment ions linked one of the sulfate esters. chloride, the same procedure afforded the (R)-PGME amide 8 in the same yield. No racemization occurred during the reaction.
Results and Discussion From the digestive glands (3.7 kg), 600 µg of COOHhomoYTX (6), 1 mg of homoYTX (2), and 400 µg of 45hydroxyhomoYTX (3) were obtained. In addition, we isolated 500 µg of a new toxin correlated with YTX, whose analysis is currently under investigation. The lethality of COOHhomoYTX to mice by intraperitoneal injection was approximately 0.5 mg/kg. Thus, COOHhomoYTX is less significant in lethal potency than YTX (0.1 mg/kg), but is comparable with both 45-hydroxyYTX and COOHYTX (0.5 mg/kg). The limited amount of available material has prevented until now both an accurate evaluation of their toxicology and the determination of their exact mouse lethality. ESI-MS gave an ion peak corresponding to [M - Na]of COOHhomoYTX at m/z 1209. Thus, COOHhomoYTX was indicated to be 14 mass unit larger than the recently reported carboxyYTX [COOHYTX, m/z 1195 (M - Na)-] (11). Most probably, COOHhomoYTX has an extra methylene or methyl compared to the structure of COOHYTX. Its molecular formula was determined to be
C56H82O23S2Na2 from the negative HRFABMS data on the pseudomolecular ion at m/z 1209 [(M - Na)-, m/z 1209.4571, calcd 1209.4586]. The structural elucidation was mainly carried out by comparing the 1H NMR data between COOHhomoYTX and COOHYTX. 1H NMR signal assignments were determined by 1H-1H COSY and HOHAHA measurements in CD3OD (Table 1). Proton connectivities from H-4 to H2-18, H-20 to H-22, H2-24 to H-32, H-34 to H-38, and H-42 to H2-47 in COOHhomoYTX were identical with those of COOHYTX. Chemical shifts and coupling constants of protons from H-7 to H2-18, H-20 to H-22, H2-24 to H-32, H-34 to H238, and H-42 to H2-47 and those of two exomethylenes (C-47 and C-53), as well as the chemical shifts of H-40 and of methyls except for Me-48, were virtually unchanged between COOHhomoYTX and COOHYTX (Table 1). Therefore, the structural difference between the two YTXs must arise from the western part of the molecule. 1 H-1H COSY allowed us to identify an extra methylene located in position 2a, whose protons resonate at δ 1.61 and 1.78, respectively. The 1H connectivities from H2-1 to H2-2a were easily determined with 1H-1H COSY and HOHAHA spectra and were identical with those of homoYTX (9).
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These results allowed us to determine the structure of COOHhomoYTX as shown in Figure 1. As in COOHYTX, the presence of a carboxylic group in the molecule was further proved by its transformation into a chiral amide by the method of Nagai and Kusumi (18), which allowed also to establish the absolute stereochemistry at C-44. The method is based on the reaction of a carboxylic acid and (S)- and (R)-PGME. The stereochemical determination was based on the chemical shift differences of the protons at C-42, C-43, C-45, and C-46 [(R)-PGME amide, H-42, δ 5.48; H-43, δ 5.58; H2-45, δ 2.03 and 2.11; H-46, δ 5.68; (S)-PGME amide, H-42, δ 5.44; H-43, δ 5.49; H2-45, δ 2.29 and 2.35; H-46, δ 5.76; ∆δ, H-42, -0.04 ppm; H-43, -0.09 ppm; H2-45, 0.26 and 0.24 ppm; H-46, 0.08 ppm]. The ∆δ (S R) values obtained by analysis of 1H NMR spectra of the PGME amides 7 and 8 are reported in Figure 3. As pointed out by Nagai and Kusumi in their original paper, the differences are not very marked; however, they are completely consistent and indicate the R configuration at C-44 of compound 6, as for COOHYTX. NOE (nuclear Overhauser effect) correlations observed on ROESY (rotating-frame Overhauser effect spectroscopy) spectra of 6 between angular protons, between angular protons and angular methyls (H-7 and Me-48, H-37 and Me-52), and between angular methyls (Me-49 and Me-50) (Figure 4) were also identical to those observed for COOHYTX, thus indicating that they have the same stereostructure. The structure of 6 was further tested for collisioninduced dissociation (CID) by negative ion MALDI MS/ MS experiments carried out on the [M - Na]- ion (m/z 1209) of COOHhomoYTX. Since 6 has sulfate esters at one terminus of the molecule, a negative charge was localized at this point, thereby allowing fragments arising from this part of the molecule. Fragment ions were generated by bond cleavage at the sites characteristic of ether rings, as established in previous experiments on yessotoxin (19) and maitotoxin (20). In the course of that study, it was demonstrated that the fused polycyclic ethers of yessotoxin and maitotoxin gave rise to characteristic fragmentations, from which sizes of ether rings and/or substituents on rings could be deduced directly (Figure 5). All prominent product ions in the MS/MS spectrum of 6 were 14 mass unit larger than the corresponding fragment ions observed in the MS/MS spectrum of COOHYTX, thus confirming, once again, that an extra methylene should be present on the sulfated side chain. An interesting aspect of the results of this study is that mussels harvested at the same location in two different years [1997 (8, 10, 11, 21) and 1998] contained different toxin compositions, with a predominance of homoYTXs in the 1998 cultivation, but similar, for the two years, with respect to the functionalization of the base skeleton. It would be quite interesting to determine if the slight differences in the toxin compositions of the two years (from the yessotoxin type to the homoyessotoxin type) arise from change in the plankton population. It still remains to be ascertained the real dinoflagellate species responsible for the poisoning that has now been recurring for years in the Adriatic Sea.
Acknowledgment. This work is a result of research supported by MURST PRIN “Chimica dei Composti Organici di Interesse Biologico”, Rome, Italy. NMR and FABMS spectra were recorded at “Centro di Ricerca
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Interdipartimentale di Analisi strumentale”, Universita` degli studi di Napoli “Federico II”. The assistance of the staff is gratefully appreciated.
References (1) Shumway, S. E. (1990) A review of the effects of algal blooms on shellfish and Aquaculture. J. World Aquacult. Soc. 21, 65-104. (2) Ahmed, F. E., Ed. (1991) Seafood Safety, pp 432, National Academy Press, Washington, DC. (3) Hallegraff, G. M. (1993) A review of harmful algal blooms and their apparent global increase. Phycology 32, 79-99. (4) Yasumoto, T. (1990) Marine microorganism toxins. An overview. In Toxic Marine Phytoplankton (Graneli, E., Sundstron, B., Edler, L., and Anderson, D. M., Eds.) pp 3-8, Elsevier, Amsterdam. (5) Yasumoto, T., and Murata, M. (1993) Marine Toxins. Chem. Rev. 93, 1897-1909. (6) Boni, L., Mancini, L., Milandri, A., Poletti, R., Pompei, M., and Viviani, R. (1990) First cases of DSP in the Northern Adriatic Sea. International Conference Regione Emilia Romagna, Bologna, Italy, March 21-24. (7) 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. (8) 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. (9) Satake, M., Tubaro, A., Lee, J. S., and Yasumoto, T. (1997) Two new analogs of yessotoxin, homoyessotoxin and 45-hydroxyhomoyessotoxin, isolated from mussels of the Adriatic Sea. Nat. Toxins 5, 107-110. (10) 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. (11) 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. (12) Satake, M., MacKenzie, L., and Yasumoto, T. (1997) Identification of Protoceratium reticulatum as the biogenetic origin of yessotoxin. Nat. Toxins 5, 164-167. (13) Lee, J. S., Igarashi, T., Fraga, S., Darhl, E. P., Hovgaard, T., and Yasumoto, T. (1989) Determination of diarrhetic shellfish toxins in various dinoflagellate species. J. Appl. Phycol. 1, 147-152. (14) Ogino, H., Kumagai, M., and Yasumoto, T. (1997) Toxicological evaluation of yessotoxin. Nat. Toxins 5, 255-259. (15) Terao, K. (1990) Histopathological studies on experimental marine toxin poisoning-5. The effect in mice of yessotoxin isolated from Patinopecten yessoensis and of a desulfated derivative. Toxicon 28, 1095-1104. (16) Gawley, R. E., Rein, K. S., Kinoshita, M., and Baden, D. G. (1992) Binding of brevetoxins and ciguatoxin to the voltage-sensitive sodium channel and conformation analysis of brevetoxin-B. Toxicon 30, 780-785. (17) Gazzetta Ufficiale della Repubblica Italiana 10 settembre 1990, Vol. 211; Decreti Ministeriali 1 agosto 1990, Vol. 256 and 257. (18) Nagai, Y., and Kusumi, T. (1995) New chiral anisotropic reagents for determining the absolute configuration of carboxylic acids. Tetrahedron Lett. 36, 1853-1856. (19) 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. (20) Murata, M., Naoki, H., Matsunaga, S., Satake, M., and Yasumoto, T. (1994) Structure and partial assignments for maitotoxin, the most toxic and largest natural non-biopolymer. J. Am. Chem. Soc. 116, 7098-7107. (21) Ciminiello, P., Fattorusso, E., Forino, M., Magno, S., Poletti, R., and Viviani, R. (1999) Isolation of 45-hydroxyyessotoxin from mussels of adriatic sea. Toxicon 37, 689-693.
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