Evidence for numerous analogs of yessotoxin in Protoceratium reticulatum

Harmful Algae 4 (2005) 1075–1091 www.elsevier.com/locate/hal

Evidence for numerous analogs of yessotoxin in Protoceratium reticulatum Christopher O. Miles a,b,*, Ingunn A. Samdal a, John A.G. Aasen c, Dwayne J. Jensen d, Michael A. Quilliam e, Dirk Petersen f, Lyn M. Briggs b, Alistair L. Wilkins g, Frode Rise f, Janine M. Cooney d, A. Lincoln MacKenzie h a Veterinary Institute, PB 8156 Dep., 0033 Oslo, Norway AgResearch Ltd., Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand c Norwegian School of Veterinary Science, PB 8146 Dep., 0033 Oslo, Norway d HortResearch Ltd, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand e Institute of Marine Biosciences, National Research Council, 1411 Oxford Street, Halifax, Canada NS B3H 3Z1 f Chemistry Department, University of Oslo, P.O. Box 1033, Blindern, 0315 Oslo, Norway g Chemistry Department, The University of Waikato, Private Bag 3105, Hamilton, New Zealand h Cawthron Institute, Private Bag 2, Nelson, New Zealand b

Received 27 October 2004; received in revised form 31 March 2005; accepted 31 March 2005

Abstract A solid-phase extract from Protoceratium reticulatum was partitioned between water and butanol and the two fractions purified on an alumina column. Fractionation was monitored by ELISA and LC–MS. Results indicate that while almost all yessotoxin (1) was extracted into butanol, large amounts of yessotoxin analogs remained in the aqueous extract along with lesser amounts in the butanolic extract. NMR analysis of selected fractions from reverse-phase chromatography of the extracts confirmed the presence of yessotoxin analogs, although structure determinations were not possible due to the complexity of the mixtures. Analysis of fractions with LC–MS3 and neutral-loss LC–MS/MS indicated the presence of more than 90 yessotoxin analogs, although structures for most of these have not yet been determined. These analogs provide a mechanism to rationalise the discrepancy between ELISA and LC–MS analyses of algae and shellfish. # 2005 Elsevier B.V. All rights reserved. Keywords: Yessotoxin; Analogs; Protoceratium reticulatum; LC–MS; ELISA; NMR

1. Introduction * Corresponding author. Tel.: +47 2321 6201/+64 7 838 5041; fax: +47 2321 6201/+64 7 838 5189. E-mail addresses: [email protected], [email protected] (C.O. Miles).

Yessotoxins (YTXs) are a group of sulfated polyethers based on the structure of yessotoxin (1), first isolated by Murata et al. (1987). Yessotoxins are

1568-9883/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2005.03.005

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produced by the dinoflagellate algae Protoceratium reticulatum (Ciminiello et al., 2003; Samdal et al., 2004a; Satake et al., 1997a, 1999) and Lingulodinium polyedrum (Draisci et al., 1999; Paz et al., 2004). Filter-feeding shellfish accumulate and produce metabolites of YTXs, many of which are toxic by intraperitoneal injection (Ciminiello et al., 1998, 2000; Daiguji et al., 1998; Murata et al., 1987; Satake et al., 1996, 1997b; Terao et al., 1990) and therefore give rise to positive results in the traditional mouse bioassay for lipophilic biotoxins. Because of this, the maximum permissible level of YTX (1) and 45hydroxyYTX (2) and their 1a-homo congeners (4 and 45-hydroxy-4) in shellfish in the EU is 1000 mg/kg (European Union, 2002). Despite this, recent evidence indicates that 1 and several of its analogs may have no significant acute toxicity when administered orally to mice (Aune et al., 2002; Ogino et al., 1997; Tubaro et al., 2003) even at levels as high as 50000 mg/kg (R. Munday, unpubl. obs.), and may therefore be of limited public health significance. In addition to YTX (1), a number of YTXs have been identified in shellfish or algae. These include 45OHYTX (2) (Satake et al., 1997b) and carboxyYTX (3) (Ciminiello et al., 2000), produced by metabolism of 1 in shellfish. P. reticulatum also produces 1ahomoYTX (4) (Ciminiello et al., 2003), trinorYTX (5) (Satake et al., 1999), a methyl ketone analog (6) (Ciminiello et al., 2003) that gives rise to isomers 7 and 8 (Miles et al., 2004b), 41a-homoYTXs (9–12, 14, 15) (Finch et al., 2005; Miles et al., 2004a, 2005), 9methylYTXs (10, 12, 15) (Finch et al., 2005; Miles et al., 2004a, 2005), 44,55-diOHYTXs (13–15) (Finch et al., 2005; Samdal et al., 2004b), analogs in which the A-ring is absent (16–19) (Miles et al., 2004a), hydroxyamidoYTXs (11, 12) (Miles et al., 2005), as well 32-O-mono- (20) (Samdal et al., 2004b; Satake et al., 2002b; Souto et al., 2005) and di-glycosylYTXs (21) (Cooney et al., 2003; Satake et al., 2002b). Recently, Konishi et al. (2004) have also identified 32O-mono-, -di-, and -tri-arabinosides of 1a-homoYTX produced by a dinoflagellate closely resembling P. reticulatum. Both the 1H and 13C NMR resonances of the glycosyl moieties of 20 (Souto et al., 2005) and 21 (Miles et al., unpubl. obs.) are very similar to those of the 32-O-mono- and -di-arabinosides of Konishi et al. (2004), indicating that they have identical glycosyl moieties. Thus, evidence is accumulating that P.

reticulatum is capable of producing a considerable number of yessotoxin analogs. That many of these analogs have been detected in P. reticulatum isolates or shellfish from Italy, New Zealand, Norway, Spain, Japan and Canada suggests that production of yessotoxin analogues is a normal in P. reticulatum. Recently, immunoassays have been developed against whole classes of algal toxins to facilitate rapid and cost-effective screening of shellfish for toxins (Garthwaite et al., 2001). As part of these efforts, an ELISA for YTXs has been developed (Briggs et al., 2002, 2004) that is so sensitive that it is possible to quantitate YTXs in single cells of P. reticulatum (Samdal et al., 2004a). This assay is also suitable for analysis of shellfish for YTXs (Aasen et al., 2004; Briggs et al., 2004; Samdal et al., 2005) and showed an excellent correlation with results obtained by LC–MS methods (Samdal et al., 2005). However, studies on algal (Briggs et al., 2002; Miles et al., 2004b; Samdal et al., 2004a) and shellfish (Aasen et al., 2004; Samdal et al., 2005) extracts indicate the presence of substantial quantities of unidentified YTXs. We have initiated studies to confirm the presence of large amounts of unknown YTXs in P. reticulatum and, where possible, to identify the most important components. Here we report application of ELISA, neutral-loss LC–MS/MS, LC–MS3 and NMR spectroscopic methods to demonstrate the presence of large numbers (in excess of 90) of what appear to be minor yessotoxin analogs in extracts from a single strain of P. reticulatum. We demonstrate that many of these YTXs partition preferentially into water rather than BuOH during the extraction procedure normally used for purification of 1, and also report m/z values and fragmentation patterns for many of these YTXs.

2. Materials and methods 2.1. Crude extract Crude YTX from 200 L of a batch culture of P. reticulatum (CAWD40) was obtained from the Cawthron Institute (Private Bag 2, Nelson, New Zealand). The crude toxin was produced by filtration of the culture under vacuum through glass fiber filters, passing the filtrate through a column of HP-20 resin

C.O. Miles et al. / Harmful Algae 4 (2005) 1075–1091

(Supelco, Bellefonte, PA, USA), rinsing the column with fresh water to remove salts, removing free water by suction, and eluting with MeOH. Evaporation of the solvent under vacuum produced a brown oil. 2.2. Fractionation of crude extract Half of the crude YTX was dissolved in water (200 mL), washed with EtOAc (200 mL), and extracted with 1-BuOH (3  50 mL). The butanolic extract was evaporated to dryness, and applied to a column of basic alumina (10 g) pre-equilibrated with 1:1 MeOH–CH2Cl2. The column was eluted with MeOH–CH2Cl2 (30 mL), MeOH (15 mL), and finally with NH4OH (1%)–MeOH (1:1, 40 mL). The ammoniacal fraction was immediately evaporated to dryness under vacuum to minimize the possibility of chemical modifications to base-sensitive analogs (Miles et al., 2004b). The aqueous residue was treated in an identical manner to the butanolic extract. The aqueous and butanolic ammonia-fractions from the alumina columns were each fractionated in identical manner on a reverse-phase flash column (4 cm  2 cm, LiChroprep1 RP-18, 40–63 mm; Merck, Darmstadt, Germany) and eluted with a step-wise gradient of MeOH in water (20, 40, 60, 80 and 100%, 20 mL per step) and fractions (10  10 mL) collected. All fractions were analyzed for YTXs by ELISA and for 1 by LC–MS. Fraction-6 (W6) (11.2 mg) from the water, and fraction-5 (B5) (4.5 mg) from the butanol, were evaporated to dryness and analyzed by NMR spectroscopy. Fractions 7 and 8 from the butanol (B7 and B8) were combined to afford a colorless solid (37.5 mg) that was also analyzed by NMR spectroscopy. The second half of the extract was then fractionated on an alumina column as described above. An aliquot of the ammonia fraction from the column was partitioned between 1-butanol and water, and the two fractions (hereafter referred to as butanol and water fractions) were analyzed by LC–MS/MS neutral loss scan. The remainder of the ammonia fraction was fractionated by reverse-phase flash column chromatography (80 mm  15 mm) on LiChroprep1 RP-18 (40– 63 mm; Merck, Darmstadt, Germany) using a stepwise gradient of 2:1 MeOH–MeCN (35, 40, 45, and 50%, 50 mL of each, then 30 mL of 100%) in water, with collection of 10 mL fractions. The fractions were analyzed for YTXs by LC–MS3 (Table 1).

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2.3. ELISA analyses The concentration of YTXs in each sample was determined by indirect competitive ELISA as described by Briggs et al. (2004), with only minor adjustments to plate coater and antibody concentrations to optimize the assay (Samdal et al., 2004a). All incubations were at room temperature. YTX standard in methanol (1 mg/mL) was diluted in phosphatebuffered saline–Tween 20 and sample-buffer, to give a methanol concentration of 10%. Serial three-fold dilutions of the YTX standard were performed with sample buffer, giving 10 standards from 0.0020 to 40 ng/mL. Serial dilutions of standards and samples were performed in duplicate. Absorbances were measured at 450 nm using a plate reader (Wallac 1420 Victor2 multilabel counter, Wallac, Turku, Finland). All ELISA results are reported as yessotoxin equivalents. 2.4. LC–MS analysis Four LC–MS systems with different attributes were used in this study. For clarity, these will be referred to as: LC–MS; LC–MS3; LC–MS/MS neutral loss scan; high resolution LC–MS (HR LC–MS). Preliminary experiments were monitored by LC–MS, based on the method of Quilliam et al. (2001), performed with an Agilent 1100 binary pump with a Perkin Elmer (PE) System 200 Autosampler, a PE System 200 column oven (30 8C), and a Sciex API 2000 (Applied Biosystems) mass spectrometer equipped with an electrospray interface (Turboionspray1). Eluents were: (A) ammonium formate (2 mM) + formic acid (50 mM) in water; (B) ammonium formate (2 mM) + formic acid (50 mM) in acetonitrile–methanol (2:1) containing 5% water. Chromatography was performed on an X-terra MS C18 column (Waters, 50 mm  2 mm, 3.5 mm) equipped with a 10 mm guard column, at 300 mL/min with a linear gradient elution from 40 to 100% B over 6 min followed by 14 min at 100% B. Injection volume was 5 mL, and selected ion monitoring was performed for mono-anions of YTXs. LC–MS3 analysis was conducted on an LCQ Deca ion trap mass spectrometer fitted with an ESI interface (ThermoQuest, Finnigan, San Jose, CA, USA) and coupled to a SurveyorTM HPLC and PDA detector.

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Table 1 YTX analogs detected by LC–MS3 analysis of a fractionated extract of Protoceratium reticulatum Entry Mwt

Rt

[M

1 2

956c 984

3.2 3.3

955 983

3

986

2.2

985

4

992

5.2

991

5 6 7 8

992 992 1008 1010

6.1 991 7.1 991 3.2 1007 3.3 1009

9 10 11 12

1012d 1020e 1022f 1026g

2.7 4.4 4.1 2.8

1011 1019 1021 1025

13 14 15 16

1026 1038 1038 1040

3.1 3.3 4.0 5.4

1025 1037 1037 1039

17 18 19 20 21 22

1042 1048 1048 1048 1062 1062h

2.6 5.2 5.9 6.8 3.2 6.3

1041 1047 1047 1047 1061 1061

23 24 25

1082 1082 1086

3.0 1081 3.4 1081 8.8 1085

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

1090 7.5 1089 1118i 5.7 1117 1120 4.6 1119 1120j 5.3 1119 1134 5.8 1133 1138 4.8 1137 1142 7.5 1141 1142 8.0 1141 1142 9.0 1141 1142 9.4 1141 1142k 10.0 1141 1144 6.7 1143 1148 5.6 1147 1150l 4.7 1149 1156m 10.5 1155

41 42

1158n 1158n

7.2 1157 7.5 1157

H]

MS2

MS3 (see Fig. 5)

[M

875 903, 869, 653, 599 905, 815, 771 911

831, 795 –

477.3 –

++ –

– –

– –

797, 772, 645, 627, 583

439.0

+

–

–

868, 729, 868, 827 919, –

–

–

–

–

17

– – – –

– – – –

– – – –

– – – –

18 19

887, 851, 807, 696 939 – 927, 758

505.2 – – 512.0

+ – – +

– – – –

– – – –

927, 864 939, 877 929 927

– 518.3 – 519.5

– ++ – +

– – – –

– – – –

946, 943, 917, 915, 881 924, 907, 895, 855, 713, 671 924, 895, 855, 713, 671, 659 883 951 –

520.4 – – – – –

+ – – – – –

– – – – – –

– – – – – –

970, 927, 885, 855, 799, 713 983, 957, 927, 869, 855, 713 921, 868, 851, 822, 799, 773, 657 981, 967, 925, 855, 799, 671 924, 895, 855, 713 959, 895, 855, 799, 713, 687 1021, 941, 924, 895, 855, 713 967, 925, 855, 713 – 924, 896, 855, 757, 743, 713 924, 855, 839, 801, 785, 713 924, 879, 855, 799, 713, 659 993, 924, 907, 855, 801, 714 977, 924, 907, 879, 855, 801 1033, 924, 855, 801, 713 979, 868, 799, 657, 575, 448 – 967, 925, 896, 855

– – –

– – –

– – –

– – –

– – 559.4 559.4 – 568.3 – – – – – – 574.0 574.3 –

– – ++ + – +++ – – – – – – + +++ –

– – – – – 528 – – – – – – – 534 –

– – – – – 519, 499 – – – – – – – 525, 503, 229 –

– –

– –

– –

– –

911 911 927 929, 922, 850, 799 931 939, 799 980, 941, 925 945, 875, 847, 786 945 957 957 959, 929, 847, 598 961 967 967 967 981 981, 924, 855, 713 1001 1001 1005, 868 1009 1037 1039 1039 1053 – 1061 1061 1061, 939 1061 1061 1063 1067 – 1123, 1075, 1026 1077, 1059 1077

799, 757, 688, 575 657 912, 855, 759

924, 855, 713, 671 940, 925, 871, 815, 759, 729, 675

2H]2

Relative MS2 MS3 intensitya

Structureb

6 7 8

16

1

9

C.O. Miles et al. / Harmful Algae 4 (2005) 1075–1091

1079

Table 1 (Continued ) Entry Mwt

Rt

[M

H]

MS2

MS3 (see Fig. 5)

[M

967, 940, 895, 855 993, 967, 940, 925, 871, 855, 729 941, 924, 896, 855 1066, 1049, 1037, 924, 855, 840 1065, 924, 895, 855, 713, 587, 559, 501 – 981, 939, 869, 727 – 1053, 967, 924, 855, 799, 713 1053, 1037, 969, 925 1079, 1037, 924 – 1037, 924, 868, 855, 839, 785, 713 1079, 1037, 1012, 981, 924, 855 1079, 1032, 924, 855, 759, 713 981, 955, 940, 925 968, 925, 855, 785 967, 925, 855

– –

– –

– –

– –

– –

– –

– –

– –

–

–

–

–

583.4 – 586.2 –

+++ – + –

543 – – –

534, 511, 503 – 10 – – 13

588.3 588.0 588.3 588.4

+ + +++ +

– – 548 –

– – 526, 517, 496 –

588.6

+

–

–

–

–

–

–

– – – – –

– – – – –

– – – – –

– – – – –

–

–

–

–

–

–

–

–

598.3 – 601.3 – –

+++ – ++ – –

558 – 561 – –

549, 543, 519 – 530, 523, 498 – 15 –

–

–

–

–

608.2 – –

+++ – –

568 – –

497 – –

615.4 – 641.5 –

++ – +++ –

575 – 601 –

491, 385 – 20 593, 580, 561 –

645.3

++

605

529

648.4 651.9

+++ ++

608 612

599, 556, 248 536, 516 12

43 44

1158n 1158n

7.7 1157 8.5 1157

1077 1077

45 46

1160 1162

4.3 1159 4.5 1161

1079 1081

47

1164

6.6 1163

1083

48 49 50 51

1168 1170o 1174 1176p

4.1 10.1 5.3 6.1

1167 1169 1173 1175

– 1089 – 1095

52 53 54 55

1178 1178 1178 1178

3.8 4.2 4.4 4.6

1177 1177 1177 1177

1097 1097 – 1097

56

1178

5.0 1177

1097

57

1188

6.3 1187

1107

58 59 60 61 62

1188 1188 1188 1190 1190q

7.9 8.5 9.3 6.5 7.1

1187 1187 1187 1189 1189

1107 1107 1107 1109 1109

63

1192

3.5 1191

1111

64

1192

5.2 1191

1111

65 66 67 68 69

1198 1202 1204 1204 1212

3.7 8.3 5.2 6.0 6.6

– 1201 1203 1203 1211

– 1121 – 1123 1131

70

1216

8.2 1215

1135

71 72 73

1218 1222 1228

6.4 1217 9.9 1221 7.2 1227

– 1141 1147

74 75 76 77

1232 1274r 1284 1290

6.1 8.4 3.6 6.6

1231 1273 1283 1289

– 1193 – 1209

78

1291

5.8 1290

1210

79 80

1298 1305

4.4 – 5.5 1304

– 1224

1067, 924, 895, 855, 713, 643, 559, 489 1093, 1070, 1001, 940, 871, 843, 759 1091, 1053, 1028, 924, 895, 855, 743, 713 – 981, 939, 813, 727 – 1081, 981, 939, 869, 727 1116, 1053, 1038, 1009, 968, 924, 856 1093, 939, 924, 869, 757, 727 – 1109, 1077, 985, 855, 801 1132, 1069, 1037, 1009, 967, 924, 855, 713 – 1109, 1061, 1056, 987, 713 – 1125, 1077, 1072, 1055, 1003, 841 1077, 1049, 967, 925, 855, 713, 645 – 1091, 1063, 981, 939, 869, 727, 651

2H]2

Relative MS2 MS3 intensitya

Structureb

14

11

1080

C.O. Miles et al. / Harmful Algae 4 (2005) 1075–1091

Table 1 (Continued ) Entry Mwt

Rt

[M

H]

MS2

MS3 (see Fig. 5) – 1194, 1163, 1137, 1063, 1005 – 1269, 1178, 1049, 924, 855, 713 – 1241, 1193, 1188, 1061, 713 – – 1299, 1257, 1209, 1135, 1077, 939 1334, 1301, 1111, 927, 799, 713 – – –

81 82

1312 1350

4.2 1311 8.2 1349

– 1269

83 84

1354 1380

5.7 1353 3.9 1379

– 1299

85 86

1406 1406s

4.3 – 7.7 1405

– 1325

87 88 89

1418 1420 1422

4.1 – 4.1 1419 6.0 1421

– – 1341, 1077

90

1442

3.2 1441

1361

91 92 93

1472 1472 1538

2.9 – 3.1 1471 7.1 –

– – –

[M

2H]2

Relative MS2 MS3 intensitya

Structureb

1132,

655.2 –

+++ –

615 –

606, 585, 561 –

1018,

676.3 689.2

+++ ++

636 649

625, 568, 533 632, 617, 523

1119,

701.9 702.4

+++ ++

662 662

653, 638, 356 653, 642, 512 21

1204,

708.8 708.9 710.4

+++ +++ +

668 669 –

628, 623, 498 660, 653, 504 –

1063,

720.2

++

–

–

735.2 735.6 768.5

+++ ++ +++

695 695 728

635, 569, 464 667, 645, 569 719, 662, 653 22

For each entry is given the molecular weight, retention time, and m/z values for observed mono- and di-anions and of their daughter ions observed in the MS2 and MS3 spectra. MS2 spectra are of the parent [M H] ions, and MS3 spectra are of the most abundant daughter ion (which in all cases was [M H SO3] for monoanions or [M 2H SO3]2 for dianions) in the MS2 spectra. The most abundant daughter ion in each spectrum is denoted by bold values. a Relative intensity of the di-anion (compared to mono-anion). –: not observed; +: weak; ++: strong; +++: predominant. b Structure numbers refer to Fig. 1. c Apparent atomic composition from HR LC–MS, C41H64O21S2 (D = 0.9 ppm). d Apparent atomic composition from HR LC–MS, C44H68O22S2 (D = 2.0 ppm). e Apparent atomic composition from HR LC–MS, C43H72O23S2 (D = 3.0 ppm). f Apparent atomic composition from HR LC–MS, C46H70O21S2 (D = 0.5 ppm). g Apparent atomic composition from HR LC–MS, C45H70O22S2 (D = 10 ppm). h Apparent atomic composition from HR LC–MS, C45H74O24S2 (D = 5.0 ppm). i Apparent atomic composition from HR LC–MS, C49H82O24S2 (D = 2.3 ppm). j Apparent atomic composition from HR LC–MS, C44H80O28S2 (D = 2.7 ppm). k Atomic composition from HR LC–MS, C55H82O21S2 (D = 1.7 ppm). l Apparent atomic composition from HR LC–MS, C55H74O22S2 (D = 6.6 ppm). m Atomic composition from HR LC–MS, C56H84O21S2 (D = 6.4 ppm). n Apparent atomic composition from HR LC–MS (unresolved isomers), C55H82O22S2 (D = 0.6 ppm). o Atomic composition from HR LC–MS, C57H86O21S2 (D = 5.1 ppm). p Apparent atomic composition from HR LC–MS, C55H84O23S2 (D = 1.2 ppm). q Apparent atomic composition from HR LC–MS, C55H82O24S2 (D = 0.5 ppm). r Apparent atomic composition from HR LC–MS, C60H90O25S2 (D = 1.2 ppm). s Apparent atomic composition from HR LC–MS, C65H98O24S2 (D = 4.5 ppm).

˚ The column was a Prodigy 5 mm ODS(3) 100 A (Phenomenex, Torrance, CA, USA), 150 mm  2 mm. A 0.2 mm in-line filter (Alltech, Deerfield, IL, USA) was installed before the analytical column and the temperature of the column oven was maintained at 35 8C. Gradient elution was performed using methanol–0.1% aqueous ammonium formate (3:17) containing 0.1% formic acid (solvent A) and 100% methanol (solvent B). Linear gradients were

run from 70 to 100% B over 10 min, held for 3 min, then reset to the initial conditions. The flow rate was 200 mL/min, the injection volume 10 mL, and the PDA detector scanned from 200 to 600 nm. MS data was acquired in both positive and negative modes using a data-dependent LC–MSn method. The ESI voltage, capillary temperature, sheath gas pressure and auxiliary gas were set at 4 kV, 275 8C, 35 psi, and 0 psi, respectively.

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Fig. 1. Structures of selected yessotoxin analogs shown in their sulfonic acid forms. Entry numbers refer to Table 1. Values for m/z are for [M H] , and are rounded down. Compounds 1–6, 8–13, 16, and 20 have been unambiguously identified by NMR and mass spectrometry. Compound 20 was identified by Satake et al. (2002a) and comparison of its 13C NMR data (Miles et al., unpubl. obs.) with that of Konishi et al. (2004) indicates it to be the 32-O-diarabinoside of YTX. Structures of compounds 14–15, 17–19, and 22 are based solely on evidence from LC–MS studies.

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Fig. 1. (Continued ).

C.O. Miles et al. / Harmful Algae 4 (2005) 1075–1091

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2.5. LC–MS/MS neutral loss scan

2.7. NMR spectroscopy

Analyses were performed on an LC–MS/MS system consisting of an Agilent 1100 HPLC with binary pumping system and automated injector coupled to an API4000 mass spectrometer (PESCIEX, Concorde, ON) with TurboSpray interface. The column (150 mm  1 mm) was packed with 3 mm Hypersil-BDS-C8 (Thermo/Keystone, Bellefonte, PA) and maintained at 20 8C. The mobile phase consisted of water (A) and acetonitrile–water (95:5) (B), both containing 50 mM formic acid and 2 mM ammonium formate. The flow rate was 50 mL/min, with gradient elution from 10 to 100% B over 10 min with a hold at 100% for 15 min before returning to the initial conditions and re-equilibrating for 15 min, and the injection volume was 10 mL. The ESI voltage and nebulizer gas temperature were set at 4500 V and 100 8C, respectively. Neutral loss mass spectra were acquired in the negative ion mode by performing collision-induced dissociation of Q1-selected precursor ions with nitrogen in Q2 operated in radio frequency (rf)-only mode and synchronously scanning Q1 and Q3 from m/z 900 to 1400 (or 900 to 1600 for some runs) with an offset of 80 amu (SO3 loss). The collision energy was set at 65 V.

NMR spectra of butanol fraction 5 were obtained in CD3OD (+99.8 at.% D; Aldrich, USA) with a Bruker 400 MHz spectrometer. NMR spectra obtained were 1 H, 13C, DEPT135, 1D-TOCSY, COSY, TOCSY, gHSQC, g-HMBC, ROESY and NOESY. NMR spectra of water fraction 6 were obtained from CD3OD (+99.8 at.% D; Aldrich, USA) with a Bruker 500 MHz spectrometer. NMR spectra obtained were 1H, 13C, DEPT135, COSY, TOCSY, HSQC and HMBC spectra. Spectra were determined at 30 8C, and calibrated relative to internal CHD2OD (3.31 ppm) and CD3OD (49.0 ppm).

2.6. High resolution LC–MS High resolution LC–MS (HR LC–MS) analysis was performed on aqueous and butanolic extracts with a model 1100 liquid chromatograph (Agilent, Palo Alto, CA, USA) coupled to a QStar Pulsar mass spectrometer (Applied Biosystems/MDS Sciex, Concord, Ont., Canada) as described elsewhere (Miles et al., 2004b). HR LC–MS results are presented in Table 1. However, because the chromatography system used was different to that used for the LC–MS3 analyses, it was not possible to unambiguously assign accurate masses determined by HR LC–MS to peaks observed during the LC– MS3 analysis except where the compound had been isolated. The HR LC–MS measurements assigned to entries in Table 1 for compounds that have not yet been isolated must therefore be regarded as tentative, and are reported as the ‘‘apparent atomic composition’’.

3. Results and discussion Analyses of algal and especially of shellfish samples using an ELISA for YTXs gave much higher results than did analyses for 1 by LC–MS (Aasen et al., 2004; Briggs et al., 2002, 2004; Miles et al., 2004b, 2005; Samdal et al., 2004a, 2005). The possibility that these results were due to matrix effects on the ELISA was excluded by extensive use of dilution series. The conclusion was that the high results in the ELISA were probably due to the presence of compounds which bound to the anti-YTX antibodies but which were not detected in the LC–MS analyses (Aasen et al., 2004; Briggs et al., 2002; Samdal et al., 2004a, 2005). In shellfish samples, Samdal et al. (2005) have shown that much of the difference between ELISA and LC– MS results for shellfish samples is due to the presence of 45-hydroxyYTX (2) and carboxyYTX (3), but that there are still large amounts of YTXs that appear to be unaccounted for in the standard LC–MS analysis. P. reticulatum has been reported to produce not only 1, but also a mono- and a di-glycoside of 1 (Cooney et al., 2003; Samdal et al., 2004b; Satake et al., 2002b; Souto et al., 2005). When we analyzed extracts prepared from P. reticulatum strain CAWD40 by SPE, we found that LC–MS indicated the presence of 1 as the major component along with smaller peaks that appeared to be three heptanor-41-oxoyessotoxins (6–8) (Miles et al., 2004b). However, analysis of the same extracts by ELISA indicated that the total content of yessotoxins was about three times the level of 1 that had been measured by LC–MS. We dissolved

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the SPE extract in water and extracted successively with EtOAc and 1-BuOH, and analyzed both extracts as well as the residual water for 1 by LC–MS and for YTXs by ELISA. By LC–MS we found that there were only trace amounts of 1 in the EtOAc and water fractions; it had been essentially completely extracted into the BuOH. However, by ELISA we found approximately equal amounts of YTXs in both the water and in the BuOH, with only trace amounts present in the EtOAc. Only a small proportion of the YTXs in the water could be accounted for by the presence 6–8 (Miles et al., 2004b). The butanolic and aqueous extracts were treated by an alumina column cleanup procedure developed for the purification of YTXs and which removes most non-acidic components. However, this procedure did not significantly affect the concentrations of 1 (by LC–MS) or of total YTXs (by ELISA) in either the BuOH or water fractions. Our results suggested the presence of large amounts of unknown YTX analogs in the water, and to a lesser extent in the BuOH. To test this hypothesis, we conducted identical chromatographic fractionations of the BuOH and water fractions on reverse-phase flash column using a step-wise gradient. Fractions were analyzed by LC–MS and by ELISA and the results are shown in Fig. 2. These results clearly confirm that 1 is the dominant component of the butanolic extract, eluting in fractions 7–8. There was, nevertheless, a significant component of compounds in the butanolic fractions which were recognized as YTXs by the ELISA but in which LC–MS showed no detectable 1 (fractions 4–6). Results from the fractionation of the water extract were even more definitive. There were

only traces of YTX in fractions 7–8 from the water by LC–MS. However, ELISA indicated that even in these fractions, unknown YTX analogs appeared to predominate (Fig. 2). ELISA showed large amounts of unknown YTXs in fraction 6, but significant amounts were also present in fractions 3–9. The results of this experiment strongly suggest the presence of large quantities of unknown YTXs that are recognized by the anti-YTX antibodies used in the ELISA. These unknown YTXs behaved in a normal manner on reverse-phase chromatographic supports (Fig. 2), suggesting that they would be amenable to traditional methods of purification and chromatographic analysis. To confirm the presence of large amounts of YTXs in these extracts, we obtained NMR spectra (Fig. 3) for selected fractions from the above chromatographic steps. Butanol fractions 7–8 were combined and evaporated to dryness to give a white powder. NMR analysis showed this to be nearly pure 1, contaminated only with small amounts (about 1% each) of three new YTXs identified as 9, 10 and 16 (Miles et al., 2004a). Water fraction 6 contained no 1, but when evaporated to dryness gave a white powder. NMR analysis (Fig. 3) showed that most of the signals expected for 1 were also present in fraction W6, but that some of the resonances were either completely absent or partially suppressed. The two-dimensional COSY data for this fraction revealed the presence of all the connectivities normally observed for 1 from H-1 to ring I, after which there appeared to be further weaker connectivities associated with ca. 5 major and ca. 10 minor components. Particularly obvious in the 1H NMR spectrum of water fraction 6 was the almost complete

Fig. 2. Analyses of fractions from reverse-phase chromatography of (A) butanolic and (B) aqueous fractions after purification by alumina column chromatography. Analyses were performed by ELISA for YTXs (*) and LC–MS for YTX (1) (*).

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Fig. 3. 1H NMR spectra in CD3OD of: (top) butanol fraction 5; (center) yessotoxin (1); (bottom) water fraction 6.

absence of resonances associated with the C-40 side chain (H-40–H-47, H-54 and H-55) and the olefinic methylene (H-53) (Fig. 3). Furthermore, the intensity of the H-52 methyl resonance was reduced to about one-third that of the other methyl resonances, also suggesting that the majority of the YTXs present in this fraction are significantly modified in the ring-I–K region. No significant resonances were observed in any of the NMR spectra of this fraction that were not attributable to YTX analogs (e.g. Fig. 3). NMR analysis of butanol fraction 5 (Fig. 3) gave similar results and has in part been discussed elsewhere (Miles et al., 2004b). NMR analysis showed that about 20% of the total YTXs in butanol fraction 5 were due to 41ketoYTX (6). Again, the NMR spectra, including the COSY connectivities, showed that the analogs (with the exception of 6) were heavily modified in the region which normally would contain the three terminal (I–K) rings and C-40 side chain of 1. Again, no significant resonances attributable to non-YTXs were observable in the spectra (Fig. 3). Although these results demonstrated the presence of large amounts of unknown YTX analogs in P. reticulatum extracts, identification of individual

components requires use of LC–MS methods together with purification of the individual components for NMR analysis. We applied two LC–MS methods to obtain more information before proceeding with further purification steps. One was neutral loss (of m/z 80 = SO3 for singly charged ions) LC–MS/MS, a method that separates the components on an LC column and then detects all parent ions exhibiting loss of m/z 80 (SO3) during the MS fragmentation. Such compounds are almost certain in the present samples to be due to YTXs, because these disulfated compounds ionize predominantly as mono-anions carrying a single negative charge on one of the sulfate groups, and the fragmentation parameters were optimized for desulfation of the second (uncharged) sulfate group. Furthermore, the NMR studies had indicated that the extracts were composed almost exclusively of YTXs. Neutral loss LC–MS/MS was conducted on the butanolic and aqueous extracts after cleanup on the alumina column. The second method used was LC–MS3 performed with an ion trap MS detector. This method detects all the [M H] ions produced in the ionization source and then performs step-wise fragmentation to give first the desulfated

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[M H SO3] mono-anions, with subsequent fragmentation giving structurally-informative cleavages across the YTX ring system (Ciminiello et al., 2002a; Fernandez Amandi et al., 2002; Miles et al., 2004a, 2004b, 2005). The LC–MS3 method also detected [M 2H]2 ions for certain compounds, and fragmentation of these yielded [M SO3 2H]2 ions indicating that the second charge was not carried on the 1- or 4-sulfate groups (see below). Neutral loss LC–MS/MS of the butanolic fraction revealed the presence of numerous signals correspond-

ing to parent ions that lost m/z 80 in the MS/MS step. The results are best summarized by a contour plot as shown in Fig. 4A. It can be presumed that all the spots in the contour plot correspond to known or, in most cases, unknown, YTXs because the pre-fractionation procedure and LC–MS conditions used were highly specific towards this class of disulfated polyethers, and because the NMR spectra showed the presence of high levels of modified YTXs in all fractions examined. The dominant signal in the neutral loss plot was due to 1 (m/z 1141), but around 40 other spots were observed

Fig. 4. Neutral loss (m/z 80) LC–MS/MS contour plots of the butanol (A) and water (B) extracts of P. reticulatum, with the corresponding chromatogram shown above each contour plot. Compound numbers (bold, in parentheses) refer to structures in Fig. 1.

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Fig. 4. (Continued ).

(Fig. 4A). Several of these signals corresponded to known YTXs, including peaks at m/z 991 (17), 1047 (6–8), 1085 (16), 1155 (9), 1169 (10), 1175 (13), 1273 (20), 1290 (11) and 1304 (12), suggesting that compounds detected by this method were indeed YTXs. Many of the other signals in the neutral loss plot for the butanol extract (Fig. 4A) also corresponded to compounds that displayed fragmentations characteristic of YTXs during LC–MS3 analysis (Table 1). Neutral loss LC–MS/MS of the aqueous fraction also revealed a complex mixture of YTXs in which 1 (m/z 1141) was only a minor component (Fig. 4B).

The dominant signals in the contour plot were at m/z 955, 1009, 1011, 1021, 1025, 1047 (6–8), 1061, 1081, 1117, 1119, 1135, 1141 (1), 1161, 1175 (13), 1177, 1290 (11), and 1304 (12), but in total about 60 YTXs could be observed in the water extract. Again, many of the spots corresponded to compounds in the LC–MS3 analysis (Table 1) that displayed fragmentations characteristic of YTXs. A number of the compounds detected in the neutral loss LC–MS/MS plots of the two extracts were common to both. However, in total, the neutral loss LC–MS/MS analysis indicated that there were about 80 YTXs detectable in extracts of P. reticulatum

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CAWD40. These conclusions are consistent with results obtained by LC–MS3 analyses of an extract of the same alga that had been fractionated on a reversephase flash column instead of being partitioned between BuOH and water (Table 1). LC–MS3 analysis revealed the presence of at least 90 putative YTXs in the extract (Table 1). Peaks were considered to be attributable to a YTX analog when either the parent mono-anion [M H] lost m/z 80 to give a desulfated daughter mono-anion [M H SO3] , or when the parent di-anion [M 2H]2 lost m/z 40 to give a desulfated daughter di-anion [M 2H SO3]2 , during the first fragmentation (MS2) step. The [M H SO3] daughter ions tended to give rise to information-rich cleavages across the polyether skeleton (Fig. 5) in the MS3 spectra, whereas the [M 2H SO3]2 daughter ions did not (although loss of H2O (m/z 9) was often observed). In several cases we obtained MS3 spectra for both mono- and di-anions of the same compound (see entries 78 (11), 80 (12), 84 and 86, Table 1),

confirming that this behavior in di-anions should be considered evidence that they too originate from YTXs. These di-anions are not to be confused with those that can be observed when compounds such as 1 are analyzed by LC–MS using neutral (rather than weakly acidic) eluents (e.g. Can˜a´s et al. (2004)), where both sulfate groups are ionized and facile neutral loss of SO3 is not expected from such species. Entry 93 (Table 1), which corresponds to the 32-Otriglycoside of YTX (22), displayed a dianion during LC–MS3 analysis. Cooney et al. (2003) have shown that under these conditions 32-O-glycosides of YTX show increased di-anion intensity (with a corresponding decrease in mono-anion intensity) as the number of added furanose units is increased from 0 to 2 (also see entries 36, 75, and 86 in Table 1), so the presence of a relatively strong [M 2H]2 ion for 22 is not unexpected. In addition to the dianion at m/z 768, 22 also displayed prominent loss of SO3 (80 amu) in the MS2 step to give a daughter di-anion at m/z 728.

Fig. 5. Typical fragmentations observed for [M H SO3] ions (MS3 in Table 1) of some common yessotoxin skeletons during LC–MS3 analysis (Cooney et al., 2003; Miles et al., 2004a). Ions characteristic of these or modified yessotoxin skeletons were observed for many of the entries in Table 1.

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During the MS3 step, the daughter ion at m/z 728 displayed prominent neutral losses of water (18 amu, to give m/z 719), furanose (132 amu, to give m/z 662), and furanose + water (150 amu, to give m/z 653). Entry 89 (Table 1) has similarly been identified as an analog of 32-O-digycosyl-YTX that has been hydroxylated in rings A–E (Cooney et al., 2003). The LC– MS3 fragmentations for entry 82 (Table 1) are consistent with those expected for the 32-O-diglycoside of nor-ring-A-yessotoxin (16). Many of the entries in Table 1 also displayed MS3 fragments consistent with yessotoxin (m/z 924/925, 855, 713), 1a-homo- or 9-methylyessotoxin (m/z 939, 869, 727), nor-ring-A-yessotoxin (m/z 868, 799, 657), or ring-oxidized-yessotoxin (m/z 940, 871, 729) skeletons. In some cases (entries 1, 9–12, 22, 27, 29, 36, 39–44, 49, 51, 62, 75, and 86, Table 1), HR LC–MS data (for [M + NH4]+ or [M H + 2NH4]2+ ions) was also available which was consistent with disulfated polyethers of the yessotoxin type. Although few of these compounds can be definitively identified from the LC–MS analysis alone, tentative partial structure characterization is in some cases possible. For example, entry 1 appears to be a carboxylic acid derivative of YTX containing only the A–I rings. Like entry 1, entry 9 also showed a prominent loss of 44 amu from the [M H SO3] ion. All the ions observed for entry 1 had corresponding ions with an additional 56 amu in entry 9, suggesting that the two may differ by the presence of a six-membered ether ring (C3H4O, 56 amu) in a similar manner to 1 and 16. Entry 50 may correspond to 3, although supporting fragmentations were not observed due to the weakness of the signal. Entries 21 and 22 had the same mass as 1-desulfoyessotoxin (m/z 1061), but these compounds eluted much earlier and their fragmentation patterns (which included neutral loss of SO3) were consistent with their being disulfated. A series of overlapping peaks was observed at m/z 1157, corresponding to the mass of hydroxyyessotoxin, eluting before 1. One of these may be due to the known analog 45-hydroxyyessotoxin (2), but it is clear from the fragmentation patterns that oxygenation can occur at more than one location in both the C-40-side-chain (entries 41, 42, and 44) and also (entries 42–44) in the ring system. Entries 51–56 all appear to be analogs of 1 dihydroxylated in the C-41–C-47 side chain, and entries 27–28 could be oxidized derivatives of 5.

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Thus, the results of ELISA, neutral-loss LC–MS/ MS, LC–MS3, high resolution LC–MS, and NMR evidence presented here indicate that P. reticulatum strain CAWD40 extracts contain a complex array of more than 90 YTX analogs, of which approximately one-third is YTX (1) itself. The complexity of the array of YTX analogs in P. reticulatum appears to be unprecedented among algae producing polyether toxins of concern to the shellfish industry. This complexity appears to arise from the ability of the yessotoxin biosynthetic pathway to accommodate a range of polyketide chain lengths, chain termini, methylene insertions, methylations, oxygen insertions, amidations, and glycosylations. Some of the analogs observed in this study may also arise from abiotic conversions of YTXs, and this possibility is currently under investigation in our laboratories. Many of the YTX analogs appear to be much more hydrophilic than 1, because they were poorly extracted by butanol and they eluted earlier than 1 during reverse-phase LC. Some of the more abundant minor analogs from P. reticulatum have already been identified using NMR and LC–MS techniques (6–21) (Ciminiello et al., 2002b; Finch et al., 2005; Miles et al., 2004a, 2004b, 2005; Satake et al., 2002b). However, the majority are of unknown structure and the limited NMR and MS evidence presented here suggests that they contain major alterations to rings I–K. Such analogs could reasonably account for the higher values obtained when analyzing algal extracts by ELISA when compared to LC–MS methods in this and previous (Briggs et al., 2002; Miles et al., 2004b; Samdal et al., 2004a) studies. Little information is available regarding the toxicity of yessotoxin analogues, although 8 and 12 were of low toxicity to mice by intraperitoneal injection (Miles et al., 2004b, 2005) while 6 might be expected to be toxic based on results for 1a-homo-6 (Ciminiello et al., 2001). Metabolism by shellfish of such a complex mixture of YTXs from P. reticulatum would be expected to produce an even more complex array of YTX analogs, and could be the reason for the discrepancy in quantitation of mussels for YTXs by ELISA (for YTXs) and by LC–MS (for 1–3) (Aasen et al., 2004; Briggs et al., 2002; Samdal et al., 2005). Thus, ELISA appears to be an effective method for analyzing the total content of YTXs in algal and shellfish samples, and when combined with LC–MS is a powerful tool for identifying analogs of YTX.

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Further work will be required to test whether the new analogs detected by LC–MS and ELISA in this study, or their metabolites, are toxic. To date, all YTXs and 1a-homoYTXs containing an intact A–K ring system, including 1–5, 1a-homo-6, 20, and 21, appear to be toxic by intraperitoneal injection in the mouse bioassay (Ciminiello et al., 2000, 2001; Murata et al., 1987; Satake et al., 1997b, 2002b). It is therefore likely that some of the new analogs will be toxic by intraperitoneal injection. However, it should be noted that every YTX analog so far tested has displayed very low oral toxicity (Aune et al., 2002; Ogino et al., 1997; Tubaro et al., 2003), even though the analogs were highly toxic to mice by the intraperitoneal route. Many of the analogs observed in our study, as well as some of the more polar metabolites such as carboxy- and hydroxycarboxy-YTXs identified in other studies, are unlikely to be efficiently extracted into organic solvents. Such analogs would therefore not be expected to be detected effectively in the traditional mouse bioassay for lipophilic marine biotoxins, which involves partitioning of shellfish extracts between water and an organic solvent followed by mouse bioassay of the residue from the organic phase. These observations suggest that the range of YTX analogs regulated, their regulatory levels, and the suitability of the current mouse bioassay for monitoring them, may need to be reassessed.

Acknowledgements We thank Trine Torgersen (Norwegian School for Veterinary Science, Oslo, Norway) for assistance with LC–MS analyses, and Bill Hardstaff (NRC IMB, Halifax, Canada) for assistance with LC–MS/MS analyses. This study was supported by Norwegian Research Council grant 139593/140, and New Zealand Foundation for Research, Science and Technology Contract Nos. C10X0220, CAWX0301.[SS]

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