To a natural-products chemist, the term "natural products" does not refer to all compounds from natural sources, as the name might imply. It is specifically used to refer to compounds known as secondary metabolites, structurally complex molecules, often of unknown function, with very limited biological distribution

Prinsep, Michèle R.

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Research Commons@Waikato

CHEM NZ 81, November 2000 37SLIMEANDSCUM:NATURAL PRODUCTSFROMLANDANDSEAMichèle Prinsep*Department of Chemistry, University of WaikatoJIVTRODUC7YON- WIfATARENATURAL PRODUCTS?To a natural-products chemist, the term "naturalproducts" does not refer to all compounds fromnatural sources, as the name might imply, It isspecifically used to refer to compounds known assecondary metabolites, structurally complexmolecules, often of unknown function, with verylimited biological distribution.The primary aim of our research group is to isolatebioactive natural products. A bioactive orbiologically active chemical has an effect on aliving system, which may be good or bad. Wescreen these compounds to find useful molecules,for example, ones that kill fungi or tumour cells.Such compounds are potential newpharmaceuticals and can be used as research toolsthat help us understand the complex processes thatoccur in living organisms. We are also interested incompounds with novel chemical structures.Ideally, a new secondary metabolite will have bothinteresting activity and a novel structure.Slime and scum are the types of organisms that we investigate to isolate secondmetabolites. Slime refers to terrestrial cyanobacteria and scum to marine bryozoans. Astudy these organisms in particular? Tests or screens on many different organisms overyears have shown that they contain many bioactive compounds, which seem to helporganisms in their everyday survival. Cyanobacteria, for example, live in a complex scontaining many other creatures, so need to compete for nutrients and space. Itadvantageous for them to produce compounds that may inhibit the growth of compet* Dr Michèle Prinsep is a senior lecturer in Chemistry at the University of Waikato, and winner of the ZontaScience Award 2000. The award is administered by the Zonta Club of Wellington and has been offeredbiennially since 1990. Its aims are to encourage women to pursue a scientific career, to actively promote scienceas a career for young women, to encourage others already in the scientific field and to acknowledge thevaluable contribution of women scientists. The prize consists of return travel to Britain, $5000.00 and a medal,which is presented to the recipient by the Governor General at a special reception held at Government House.38 CHEM NZ 81, November 2000bacteria or fungi, leaving more resources for themselves. Marine bryozoans could also usechemicals to help them in their lives: they are primitive animals that are anchored to theseabed and are therefore, vulnerable to predation, overgrowth or infection. With no immunesystem, the theory is that bryozoans produce chemicals to help in their defence.GENERAL APPROACH 7t RI'SEARCHHow do we isolate bioactive compounds? The same process applies for both cyanobacteriaand bryozoans. We extract the organism by homogenising it mashing it up in a blender witha suitable solvent. The extract is then filtered and the excess solvent is removed to leave acrude extract containing many different compounds, including the one that we are trying toisolate. Murphys law means that this substance is often present in trace amounts only, sogetting it out is a challenge. Fractionation techniques and various forms of chromatographyare used to separate and isolate the compounds of interest.Bioassays are tests that we use to monitor the isolation process and to determine in whichfractions biologically active compounds are located. The assays that we use routinely screenfor anti-tumour, anti-viral and anti-microbial activity, but we have access to some morespecialised assays if required. Active fractions are combined and the process of fractionationand assay is repeated until, hopefully, we isolate a pure, active compound.Once an interesting compound is isolated, the next step is to determine its structure. The mostuseful structural determination technique for the natural-products chemist is nuclear magneticresonance NMR spectroscopy. This technique allows us to exploit the magnetic properties ofcertain atomic nuclei to determine molecular structure. Not all elements are observable byNMR spectroscopy but, luckily for the organic chemist, hydrogen and carbon are, so we canusually obtain a lot of structural information about organic compounds. Other techniques,such as mass spectrometry and X-ray crystallography, are also used.SLLfEWhat are cyanobacteria? They are neither plants nor animals. They look just like slime andare similar to bacteria in that they are usually unicellular and lack a distinct nucleus.However, unlike most bacteria, they contain chlorophyll and, like plants, carry outphotosynthesis. A commonly used name for cyanobacteria is blue-green algae, even thoughthey are not really algae at all and only some of them have the distinctive blue-green cyancolouration.Why are we interested in cyanobacteria? They have existed for four billion years and are welladapted to their environment. They occur in a wide range of habitats such as fresh water, soiland salt water, from the cold of Antarctica to the heat of volcanic areas, so are very versatileorganisms.It is not feasible to collect sufficient quantities of cyanobacteria in the field for research into thesecondary metabolites that they contain. Even if there is an `algal bloom' in a water body, itCHEM NZ 81, November 2000 39will not be a clean, homogeneous culture. The solution to this problem is to collect a smallsample of material, isolate an individual species and culture it in the laboratory undercontrolled conditions to produce enough organisms to work with, The cyanobacteria arecultured on a medium of inorganic mineral salts and provided with constant light and a lowlevel of carbon dioxide as a carbon source, They are initially cultured in conical flasks, thentransferred to larger vessels. Cyanobacteria have proven to be an excellent source of novel,biologically active compounds as the following examples illustrate.Many different classes of compounds have been isolated from cyanobacteria, but the types ofcompounds most commonly isolated are cyclic peptides. Some of these, such as thetnicrocystins," have proven to be a problem in lakes and rivers, causing poisoning and evendeath of stock which drink there. These compounds are hepatotoxins, damaging the liver ofaffected animals.CO2H Ma 0Ma MsMa 0 CO2HOMicrocystin-LA, one of the family of microcystinsisolated from various species of cyanobacteria.Other cyclic peptides isolated from cyanobacteria have beneficial properties. Westiellanzide isa cyclic hexapeptide containing six amino acids that displays anti-tumour activity.3 It wasisolated from a terrestrial cyanobacteriuin from Hawaii. The same compound has been foundin a marine organism called a tunicate, but in this case it is thought to be produced by theprokaryotic symbionts located in the tun.icate cells.4Me Ma0Mt00 I "MaMa HNMeWestiellamideTolyporphin A was isolated from a cyanobacterium from Pohnpei in Micronesia.5 It displaysmulti-drug resistance MDR reversing activity. Patients on prolonged chemotherapy becomeresistant to a wide range of therapeutic drugs. The cancer cells may actively pump drugs out,causing the resistance. Tolyporphin A reverses that resistance by binding to the cellular pump40 C//EM NZ 81, November 2000and inhibiting it, so that drugs can get into cancer cells to kill them.6 We have subsequentlyisolated Tolyporphins B - K/ and conducted various studies on them, such as mode of actionand metal binding studies. Toyporphin A itself makes tumours more sensitive to a type ofcancer treatment called photodynaniic therapy and is currently under further investigation foruse for this purpose?ROJ_N uN__Tolyporphin ATolypodiol was isolated from the same cyanobacterium as tolyporphin A.'° It is also a novelcompound with excellent anti-inflammatory activity and has gone on to further investigationin the United States to assess its potential as an anti-arthritis compound.TolypodiolSCUMBryozoaris or `moss animals' are colonies of tiny filter-feeding individuals or zooids that arecommon fouling organisms on such things as ships, jetty piles and nets, hence the use of theterm scum to describe them. Bryozoans are somewhat understudied biochemically, but workto date has indicated that they are art excellent source of novel, biologically active compounds.The best example of this is the bryozoan species Bugula neritina, which is the source of acompound called bryostatin 1. This is currently in advanced clinical trials for a number ofcancers, including melanoma and leukaemia, and may well come on line as an anti-cancerdrug.2As stated above, bryozoans are understudied from a chemical point of view. There are severalreasons why, including accessibility problems. Many bryozoans live below the depths thatcan be reached by divers and require the use of a dredge or submersible for collection. Asubmersible is very expensive and a dredge is very non-selective.CIIEMNZ8I, November 2000c02Me H HOM,M011:0MQO''Y' Me'P10HLCO2MeBryostatin 1Obtaining sufficient quantities for research is another problem. Although some species aresoft and feathery, most are instead heavily calcified. Therefore, very large quantities ofmaterial have to be collected to obtain enough to work with. Still other species grow in thin,encrusting forms that are also difficult to collect.Accurate taxonomy is very important. You can get quite different colouration betweendifferent specimens of the same species, or you can have several samples that look identicalbut are in fact different species, which can only be distinguished by examination under themicroscope. We are very fortunate to have expert assistance in bryozoan taxonomy fromscientists at NIWA Oceanographic in Wellington.Despite the problems associated with bryozoan research, New Zealand is quite a good placein which to study them. Obviously, we are surrounded by water and have a long coastlineand we also have a very good species diversity of bryozoans with a high proportion ofendemic species.Our research strategy is to collect as wide a species diversity as possible, extract them andscreen for biological activity,Investigation of a New Zealand bryozoan, Cribricellina cribraria resulted in the isolation of aseries of fl'.carboline alkaloids of which two novel ones are shown below. These exhibit arange of biological activities including anti-tumour and anti-fungal activities.13 Other bryozoansin the same family contain these alkaloids, but composition and quantities vary quite widely.'4O=s=OOH CH2CH3Novel alkaloids isolated from Cribricellina cribrariaSome recent examples of our work include an investigation of the bryozoan Ama thia wilsoni,which contains a series of alkaloids, the amathaspiramides.'5 These compounds arestructurally very similar to a series of compounds called the amathamides, which were42 CHEMNZ81, November 2000isolated from Australian collections of Amathia wilsoni." Interestingly, although bothAustralian and New Zealand organisms have been identified as the same speciestaxonomically, the Australian samples only seem to produce amathamides, while NewZealand colonies only produce the amathaspiramides.BrMaoLBrr,,LoH1 MenoA:RMe,R2=H B:RM EC:RH,R2=H D:R=HG: R1 MaR2 = AcAmathaspiramides A-COMeB A2 a Amathamide A:2Br OMeOMOAmathamide DAniathamides A-GThe species Euthyroides episcopalis contains the euthyroideones, which represent examples of anew ring structure.'9 These compounds contain an unusual combination of functional groupsand are very interesting from a structural point of view.FAmathamida F: R BrA B CEuthyroidenes A-CCHEM NZ 81, November 2000 43The bryozoan Watersipora subtorquata contains some compounds described by their basic ringstructure as anthraquinones. This ring structure was already known, but our group was ableto carry out research into their biological activity and determine how altering variousfunctional groups in the structures affected the biological activity.20OHO Oil OHO OHiiót::OAnthraquinones isolated from Watersipora subtorquataThis ongoing research demonstrates the utility of natural products research as a source ofcompounds that may be useful as pharmaceuticals and/or are of structural interest to achemist. As we search for potential new pharmaceutical compounds in both terrestrialcyanobacteria and marine bryozoans, we are constantly amazed at nature's ability for organicsynthesis.RE4ENCL5'1. RE. Moore, Pure &AppI. Chein., 54, 1919-1934 1982.2. MR. Prinsep, FR. Caplan, R.E. Moore, G.M.L. Patterson, RE, Honkanen and AL, Boynton,Phytochem., 31, 1247-1248 1992.3. M.R. Prinsep, RE. Moore, IA. Levine and G.M.L. Patterson, J. Nat. Prod., 55, 140-142 1992.4. T.W. Hambley, C.J. Hawkins, M.F. Lavin, AL. van den Brenk, D.J. Watters, Tetrahedron, 48, 341-348 1992.5. MR. Prinsep, P.R. Caplan, RE. Moore, G.M.L. Patterson and CD. Smith, J. Am. Chem. Soc., 114,385-387 1992.6. CD. Smith, M.R. Prinsep, P.R. Caplan, RE. Moore and G.M.L. Patterson, Oncol. Res., 6, 211-2181994.7. MR. Prinsep, G.M.L. Patterson, L.K. Larsen and CD. Smith, Tetrahedron , 51, 10523-10530 1995.8. MR. Prinsep, G.M.L. Patterson, L.K. Larsen and C.D. Smith, J. Nat. Prod., 61, 1133-11361998.9, P. Morlière, J-C Mazière, R Santus, C.D. Smith, MR. Prinsep, CC. Stobbe, M.C. Fenning, J.L.Goldberg and J.D Chapman, Cancer Res., 58, 3571-3578 1998.10. M.R. Prinsep, R.A. Thomson, M.L. West and B.L. Wylie, J. Nat. Prod., 59, 786-788 1996.11. G.R. Pettit, C.L. Herald, D.L. Doubek and DL, Herald, J. Am. Chem. Soc., 104, 6846-6848 1982.12. D. Newman, Bryostatin-From Bryozoan to Cancer Drug, in"Bryozoans in Space and Time-Proceedings of the Tenth International Bryozoology Association Conference", 1995 pp. 9-17.13. MR. Prinsep, J.W. Blunt and M.H.G. Munro, J. Nat. Prod., 54, 1068-1076 1991.14. MR. Prinsep and B.D. Morris, Secondary Metabolites from New Zealand Marine Bryozoans in"Bryozoans in Space and Time. Proceedings of the 10th International Bryozoolog-y AssociationConference", 1995 pp. 199-205.15. B.D. Morris and MR. Prinsep, J. Nat. Prod., 62, 688-693 1999.16. A.J. Blackman and D.J. Mathews, Heterocycles, 2& 2829-2833 1985.17. A.J. Blackman and R.D. Green, Aust. J. Chem., 40, 1655-1662 1987.44 CHEM NZ 81, November 200018. A.J. Blackman, T.P.D. Eldershaw and SM. Garland, Aust. J. Chem., 46, 401-405 1993.19, B,D. Morris and M.R. Prinsep, J. Org. Chem., 63, 9545-9547 1998.20. B.D. Morris, Ph.D. thesis, Studies of Natural Productsfrom New Zealand MarineBr,yozoans, University of Waikato, 1998.

Slim and scum: Natural products from land and sea

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