Shellfish are considered a delicacy by many consumers. In NZ, as in many overseas countries, there is a now thriv¬ing shellfish industry servicing both domestic and inter-national markets. Periodically shellfish accumulate harm¬ful levels of a variety of algal toxins, including domoic acid, yessotoxins, pectenotoxins and brevetoxins. When this occurs, regulatory authorities may impose harvesting closures which have a consequential economic impact on both farmers and staff employed to harvest and market shellfish products

Miles, Christopher O.

Wilkins, Alistair L.

English

Research Commons@Waikato

70Chemistry in New Zealand   October 2006Structure Determination of New Algal Toxins using NMR MethodsAlistair L. Wilkins*,a and Christopher O. Milesb,caChemistry Department, Waikato University (e-mail: a.wilkins@waikato.ac.nz)bAgResearch Limited, RuakuracNational Veterinary Institute, Oslo, NorwayIntroductionShellfish are considered a delicacy by many consumers. In NZ, as in many overseas countries, there is a now thriv-ing shellfish industry servicing both domestic and inter-national markets. Periodically shellfish accumulate harm-ful levels of a variety of algal toxins, including domoic acid, yessotoxins, pectenotoxins and brevetoxins. When this occurs, regulatory authorities may impose harvesting closures which have a consequential economic impact on both farmers and staff employed to harvest and market shellfish products.Quantification of algal toxins in sea water or shellfish tissue is dependent on the prior identification and struc-ture determination of the target toxin, and the subsequent availability of high purity reference material. Determina-tion of the structure of an algal toxin, prior to its toxic-ity being established and its level regulated, requires the consideration of spectral data derived from a variety of techniques. UV spectroscopy can facilitate the identifi-cation of chromophoric groups, while IR data assist in identifying the presence of functional groups. However, these techniques rarely define the precise location or the stereochemical orientation of groups such as hydroxyl, acetoxyl, or sulfate.Recent developments in mass spectrometry (MS), es-pecially improvements in the design and sensitivity of electrospray ion trap and time-of-flight (TOF) spectrom-eters and the interfacing of liquid chromatography (LC) columns to MS systems, have dramatically improved the ability of scientists to detect and monitor the level of po-tentially harmful algal toxins in shellfish. LC-MSn tech-niques can be used to define both the molecular weight of a target toxin, and characterize its fragmentation pattern. A particularly common approach is to determine the masses of a series of fragment ions generated by the progressive cracking of daughter ions generated by fragmentation of a parent ion. Ion trap and triple quad mass spectrometers are well suited to this approach, and while useful MSn, for n = 2, 3, 4, etc., can be generated and used to define similarities or differences in structures of related algal toxins, mass spectrometry alone is rarely able to define the stereochemistry of chiral carbons, or in many cases the specific site of attachment of a functional group.Thus, while a comparison of MSn data (Fig. 1) determined for yessotoxin (YTX) and a new homoyessotoxin ana-logue 2 (Chart 1) showed that an additional 14 units were associated with ring B–C region of the new analogue 2, it did not differentiate between, e.g. the replacement of a proton by a methyl group, the presence of a seven- rather than a six-membered ring, or replacement of a CH2 group by a C=O group.1Fig. 1. MS3 fragmentations observed for YTX and homoyes-sotoxin analogue 2 - see ref. 1.Notwithstanding the power of modern MS techniques, NMR spectroscopy remains the method of choice for de-fining both atom connectivities and the three-dimensional stereochemistry of a molecule. The quantity of material required for the successful structure determination of al-gal toxins with molecular weights in the range 750–1250 Daltons has progressively decreased from more than 5 mg a decade or two ago, to less than 100 µg, even when using only moderate-field (400–500 MHz) instruments fitted with ambient temperature gradient shielded 5 mm probes. The sensitivity of state-of-the-art 750–900 MHz instruments fitted with cryogenically cooled microprobes is even better.Raw power (highest possible field combined with the best hardware design) is always advantageous. However, care-ful attention to parameter setting, especially in two di-mensional experiments, particularly the number of incre-ments, repetition rate, number of scans per increment, and choice of transform conditions, can lead to the acquisition of data from a 400–500 MHz system, that approaches or sometimes even exceeds the quality and signal-to-noise of spectral data obtained from a less appropriately set up higher-field instrument, other than for factors related directly to field, e.g. spectral resolution. In our experi-ence, there is considerable merit in optimizing the sig-nal-to-noise ratio of spectral data that can be obtained in overnight or weekend experiments using known reference toxins, e.g. PTX-2 and yessotoxin, prior to embarking on Article71Chemistry in New Zealand   October 2006the more demanding task of defining the three dimension-al structure and deriving a complete assignment of the 1H and 13C NMR resonances of a new algal toxin analogue.Chart 1. Structures of yessotoxins shown in their sulfonic acid forms.Prior to a commitment being made in our laboratories in ca. 1996 to pursue the isolation and structural determi-nation of sub-milligram quantities of new algal toxins, potentially harmful substances isolated from extracts of NZ shellfish have, with the notable exception of the de-termination of the absolute stereochemistry of gymnodi-mine,2 been predominantly undertaken in Japanese or Ca-nadian laboratories. Working collaboratively with other NZ and overseas scientists, we have reported the isolation and structure elucidation of numerous new yessotoxins, pectenotoxins, and several other algal toxins.1,3-10 Re-cently, these studies have been aided greatly by access to a 600 MHz spectrometer equipped with microprobe and cryoprobe hardware, as part of a collaborative research agreement between the Chemistry Department of Oslo University, The National Veterinary Institute (NVI), Oslo, AgResearch at Ruakura (Miles) and a secondment agree-ment between the NVI and The University of Waikato (Wilkins).1H NMR SpectraDespite the wide range of NMR experiments now avail-able, a core group of 6–8 one- and two-dimensional NMR experiments frequently affords sufficient spectral data to define the structure of a new algal toxin. A standard ap-proach is to firstly determine the 1H NMR spectrum of the target compound. Invariably there is a substantial degree of overlap amongst methylene and methine proton sig-nals in algal toxins with molecular weights in the range 800–1300 Daltons, as is apparent in the 1H NMR spec-trum (Fig. 2) of 42,43,44,45,46,47,55-heptanor-39-en-41-oxoyessotoxin (1) which was recently isolated from extracts of cultured Protoceratium reticulatum.3 When solvent (or HOD) lines are excessive relative to target compound signals, they can be suppressed using single- or double-presaturation techniques that saturate the more slowly relaxing (long T1) solvent (or HOD) lines, while not attenuating more rapidly relaxing target compound signals. Alternatively, this can be achieved using the WA-TERGATE technique. Modern spectrometer software al-lows these and other advanced NMR experiments to be set up and recalled by less experienced users as essential-ly black box experiments, other than for frequency setting and, if required, saturation power level adjustment.Fig. 2. 1H NMR spectrum of 42,43,44,45,46,47,55-heptanor-39-ene-41-oxoyessotoxin (1).COSY and TOCSY SpectraThereafter, proton chemical shifts can be correlated in two dimensional correlation spectroscopy (COSY) and total correlation spectroscopy (TOCSY) experiments, or variants of these experiments, including double quan-tum filtered COSY, long range COSY, or COSY with solvent line presaturation. COSY data typically identify short range connectivities (2J and 3J couplings) whereas the spin-locked TOCSY experiment, also known as the HOHAHA experiment (Homonuclear Hartmann-Hahn Spectroscopy), can be optimized to detect short, medium, or long range correlations. Generally, in TOCSY experi-ments, a spin locked correlation (or mixing) time of the 72Chemistry in New Zealand   October 2006order 15 msec affords a COSY-like spectrum whereas a mixing time of 150 msec enables connectivities for pro-tons within 5–6 bonds of each other to be defined in ver-tical or horizontal columns appearing in the two dimen-sional spectra. The TOCSY spectrum of 1 is shown in Fig. 3 and detailed analyses of the multitude of correlations observed in the spectra of such medium to high molecular weight algal toxins is a time consuming, but rewarding task.Fig. 3. TOCSY spectrum of 1.1D SELTOCSYA powerful variant of the TOCSY experiment is the 1D-SELTOCSY (selective excitation TOCSY) experiment, a one dimensional selective excitation version of the more time-consuming two-dimensional experiment. Where knowledge of the correlations arising from only a limited number of protons is required, as is often the case for a substance known to differ from a known compound only in a single region of the structure, it may well be the method of choice. Advantages of the 1D-SELTOCSY experiment, compared to a conventional TOCSY experiment, include greater proton signal resolution (thereby allowing for coupling constant determination and the interpretation of vicinal couplings in six-membered rings using the Karplus equation), excitation only of a narrow region of the original 1H NMR (typically 5–30 Hz), and a reduction in spectrom-eter time where only limited spectral data are required to define the location and stereochemical disposition of a par-ticular group. Like TOCSY experiments, 1D-SELTOCSY experiments can be optimized for the detection of either short range (COSY-like) or long range correlations.Selective excitation effectively eliminates solvent and im-purity signals lines from these spectra. Despite ones best efforts at purification, detectable levels of phthalate and various tap grease components, surfactants and solvent sta-bilizers, are periodically encountered in precious samples that one is reluctant to subject to further cycles of clean-up and purification (particularly for samples that show a tendency to degrade during purification) pending initial establishment of the compound’s structure. Provided tar-get signals are not concealed by impurity signals, selective excitation techniques eliminate these impurity signals from consideration when setting up acquisition parameters such as receiver gain, and more importantly from plots of the resulting spectra.Proton-Carbon CorrelationsHMBC and HSQC SpectraHaving established short- and long-range proton connec-tivities, 1J and longer range 2J and 3J proton–carbon corre-lations can be defined in gradient-selected heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond connectivity (HMBC) experiments, re-spectively, or variants of these experiments. Using gradi-ent-selected inverse 1H detection techniques, HMBC and HSQC spectra can now be obtained more readily than is the case for a conventional 1H-decoupled 13C NMR spec-trum. Figs. 4 and 5 show the HSQC and HMBC spectra of 1.Fig. 4. HSQC spectrum of 1.Fig. 5. Partial HMBC spectrum (CH3 region) of 1.The resolution in the 1H NMR axis of slices taken from the phase-sensitive HSQC spectrum of 1 was such that axial or equatorial orientation of methylene and methine protons in 6-membered ring systems could be defined. Typically, large 3J axial-axial and 2J vicinal couplings are resolved, whereas smaller 3J axial-equatorial and 3J equa-torial-equatorial couplings are not resolved. Thus, the res-onances of the axially and equatorially oriented  C(37)H2- protons can be readily distinguished (Fig. 6).While 1H-detected HSQC and HMBC experiments indi-rectly identify 13C shifts with a precision of order 0.5–0.8 ppm, it is preferable to determine 13C shifts directly with a precision of 0.1 ppm, and to define carbon types (C, CH, CH2 or CH3) using the distortionless enhancement by polarization transfer using a 135 degree selection pulse 73Chemistry in New Zealand   October 2006(DEPT135) sequence. Frequently the acquisition of a 13C spectrum requires 2–3 times more spectrometer time than is required to obtain good quality 1H-detected HSQC and HMBC spectra.Fig. 6. HSQC slice showing the -C(37)H2- methylene proton signals of 1.NOESY or ROESY SpectraCareful consideration of a combination of 1H, 13C, DEPT135, COSY, TOCSY, HSQC and HMBC spectral data almost invariably enables a complete assignment of the 1H and 13C NMR assignments of new pectenotoxins, yessotoxins or other algal toxins to be derived, especially when considered alongside NMR data for known refer-ence compounds. These data do not, however, allow for definition of the stereochemical relationship between spe-cific functional groups. Data from nuclear Overhauser effect spectroscopy (NOESY) or the rotating frame vari-ant ROESY, or from 1D-SELNOESY or 1D-SELROESY variants of these experiments, or the classic NOE-DIF-FERENCE technique, can be utilized for this purpose. The spatial disposition of protons in ketone 1 was defined by correlations observed in its ROESY spectrum (Fig. 7).Fig. 7. ROESY spectrum of 1.We have used the NMR techniques described here to iden-tify a number of new algal toxins, including a series of new yessotoxins, pectenotoxins and some gymnodimine and okadaic acid analogues. Some of the recently iden-tified1,3,4 yessotoxins are depicted by 1-10 (Chart 1) and we have also reported evidence for numerous other minor yessotoxins.5 We anticipate that LC-NMR-MS3 data will facilitate the identification of some of the minor compo-nents when considered alongside NMR spectral data for known YTX analogues. Arrangements are in place for LC-NMR investigations of the multitude of minor yes-sotoxins to be conducted collaboratively with our Norwe-gian colleagues.Recently identified pectenotoxins include6 11-14 depicted in Chart 2 and a series of predominantly 37-O-acyl fatty acid esters of PTX-2 seco acid 15.7 Spectral data for PTX-11 (11) were determined collaboratively with Canadian workers while the structures of PTX-12 (12), and the lo-cation of the acyl group in a series of PTX-2 seco acids esters, were determined collaboratively with Norwegian associates. Complete assignments of the 1H and 13C NMR assignments of PTX-2 SA (15) and 7-epi-PTX-2 SA (16) have also been achieved,8 as has the structure elucidation of a number of other algal toxins including an okadaic acid cis-diol ester (17)9 and gymnodimines B (18) and C (19).10Chart 2. Structures of the pectenotoxins, okadaic acid deriva-tives, and gymnodimines.ConclusionThe structure determination of sub-milligram quanti-ties of newly isolation algal toxins using one- and two-dimensional NMR techniques, notably COSY, TOCSY, NOESY, ROESY, HSQC, HMBC, 1D-SELTOCSY, SEL-NOESY and SELROESY experiments, is now well es-74Chemistry in New Zealand   October 2006tablished. NMR analyses, in combination with MSn data, have proved to be the cornerstone techniques for defining the structures of new algal toxins. It is likely that this will continue to be the case for the foreseeable future, since no other spectroscopic technique with the exception of X-ray crystallography (suitable crystals are rarely available) of-fers the same degree of insight into structural and stereo-chemical issues as does NMR spectroscopy.AcknowledgementsWe thank our NZ collaborators at AgResearch Ruakura (Hamilton) and the Cawthron (Nelson), those at the Na-tional Veterinary Institute and the University in Oslo, and the Institute for Marine Biosciences, NRC, (Halifax, Canada), the Marine Institute (Galway, Ireland), and the Tohoku National Fisheries Research Institute (Japan). We also thank the NZFRS&T (contract number CAWX0301), the IIOF (contract number C10X0406), the Norwegian Research Council (grant 139593/140), and the BIOTOX project (part funded by the EC (6th Framework Programme contract 514074) for financial support.References1. Miles, C.O.; Wilkins, A.L.; Jensen, D.J.; Cooney, J.M.; Quilliam, M.A.; Aasen, J.; MacKenzie, A.L. Chem. Res. Toxicol. 2004, 17, 1414–1422.2. Stewart, M.; Blunt, J.W.; Munro, M.H.G.; Robinson, W.T.; Hannah, D.J. Tetrahedron Lett. 1997, 38, 4889–4890.3. Miles, C.O.; Wilkins, A.L.; Hawkes, A.D.; Selwood, A.; Jensen, D.J.; Aasen, J.; Munday, R.; Samdal, I.A.; Briggs, L.R.; Beuzenberg, V.; MacK-enzie, A.L. Toxicon 2004, 44, 325–336.4. Finch, S.C.; Wilkins, A.L.; Hawkes, A.D.; Jensen, D.J.; MacKenzie, A.L.; Beuzenberg, V.; Quilliam, M.A.; Olseng, C.D.; Samdal, I.A.; Aasen, J.; Selwood, A.I.; Cooney, J.M.; Sandvik, M.; Miles, C.O. Toxicon 2005, 46, 160–170. Miles, C.O.; Wilkins, A.L.; Hawkes, A.D.; Selwood, A.I.; Jensen, D.J.; Cooney, J.M.; Beuzenberg, V.; MacKenzie, A.L. Toxicon 2006, 47, 229–240. Miles, C.O.; Wilkins, A.L.; Hawkes, A.D.; Selwood, A.I.; Jen-sen, D.J.; Munday, R.; Cooney, J.M.; Beuzenberg, V. Toxicon 2005, 45, 61–71. Miles, C.O.; Wilkins, A.L.; Selwood, A.I.; Hawkes, A.D.; Jensen, D.J.; Cooney, J.M.; Beuzenberg, V.; MacKenzie, A.L. Toxicon 2006, 47, 510–516.5. Miles, C.O.; Samdal, I.A.; Aasen, J.A.G.; Jensen, D.J.; Quilliam, M.A.; Petersen, D.; Briggs, L.M.; Wilkins, A.L.; Rise, F.; Cooney, J.M.; MacKen-zie, A.L. Harmful Algae 2005, 4, 1075–1091.6. Miles, C.O.; Wilkins, A.L.; Hawkes, A.D.; Jensen, D.J.; Selwood, A.I.; Beuzenberg, V.; Mackenzie, A.L.; Cooney, J.M.; Holland, P.T. Toxicon 2006, 47, in press. 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.; Cooney, J.M. Chem. Res. Toxicol. 2004, 17, 1423–1433. Suzuki, T.; Walter, J.A.; LeBlanc, P.; MacKinnon, S.; Miles, C.O.; Wilkins, A.L.; Munday, R.; Beuzenberg, V.; MacKenzie, A.L.; Jen-sen, D.J.; Cooney, J.M.; Quilliam, M.A. Chem. Res. Toxicol. 2006, 19, 310–318.7. Wilkins, A.L.; Rehmann, N.; Torgersen, T.; Rundberget, T.; Keogh, M.; Petersen, D.; Hess, P.; Rise, F.; Miles, C.O. J. Ag. Food Chem. 2006, 54, in press.8. Miles, C.O.; Wilkins, A.L.; Munday, R.; Dines, M.H.; Hawkes, A.D.; Briggs, L.R.; Sandvik, M.; Jensen, D.J.; Cooney, J.M.; Holland, P.T.; Quil-liam, M.A.; MacKenzie, A.L.; Beuzenberg, V.; Towers, N.R. Toxicon 2004, 43, 1–9. Miles, C.O.; Wilkins, A.L.; Munday, J.S.; Munday, R.; Hawkes, A.D.; Jensen, D.J.; Cooney, J.M.; Beuzenberg, V. J. Ag. Food Chem. 2006, 54, 1530–1534.9. Miles, C.O.; Wilkins, A.L.; Hawkes, A.D.; Jensen, D.J.; Cooney, J.M.; Larsen, K.; Petersen, D.; Rise, F.; Beuzenberg, V.; MacKenzie, A.L. Toxicon 2006, 47, in press.10. Miles, C.O.; Wilkins, A.L.; Stirling, D.J.; MacKenzie, A.L. J. Ag. Food Chem. 2000, 48, 1373–1376, 2003, 51, 4838–4840.Article

Structure determination of new algal toxins using NMR methods

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