Coronary heart disease (CHD) remains one of the leading causes of mortality in many industrialised countries. Several risk factors including hypertension, hypertriglyceridemia, hypercholesterolemia and high blood platelet aggregation have been identified as contributors to CHD. The major clinical manifestations of CHD include myocardial infarction, cardiac arrhythmias and sudden cardiac death, as well as disorders of atrial rhythm such as atrial fibrillation. Cardiac arrhythmias can occur during the early phase of 'ischaemia and, in certain situations, following the restoration of normal blood flow (reperfusion) to the ischaemic region of the myocardium. During ischaemia, the electrical properties of the heart are changed resulting in arrhythmias such as ventricular tachycardia and ventricular fibrillation (VF) which can lead to sudden cardiac death. Although arrhythmias can be of many types and vary in their aetiology, the inability of individual cardiomyocytes to function properly is fundamental to the generation of arrhythmias. Of particular concern in the present invention are ischaemic arrhythmias, reperfusion arrhythmias and atrial fibrillation.

Treatment of CHD may take many forms, including the use of antihypertensive and antiarrhythmic drugs. However, the range of such drugs currently available is quite limited.

Recent evidence suggests an inverse association between the consumption of various fish oils and mortality from CHD. The evidence supports the theory that the dietary intake of the n-3 (omega) polyunsaturated fatty acids (PUFAs) cis-5, 8, 11, 14, 17-eicosapentaenoic acid (EPA) and cis-4, 7, 10, 13, 16, 19-docosahexaenoic acid (DHA) which are present in fish-oil, reduces the incidence of fatal ventricular arrhythmias and contributes to the reduced cardiac disease mortality observed in human studies.

It has also been shown that consumption of a Mediterranean diet which is rich in the n-3 fatty acid a-linolenic acid (ALA) leads to a reduction in the rate of occurrence of cardiac events and overall mortality.

A number of theories exist as to how EPA and DHA influence the cardiovascular system.

They have been reported to lower the levels of very low density lipoproteins, decrease platelet aggregation, decrease blood viscosity and increase the fibrinolytic activity of the plasma. Billman et al. 1997 Lipids 32 1161-1168 have carried out a series of experiments on surgically-modified dogs which shows that the free fatty acid forms of EPA and DHA were antiarrhythmic in dogs when added in an intravenous emulsion form. In long-term feeding studies with rats, diets supplemented with fish oil enriched in DHA and EPA provided protection against ischaemia-and reperfusion-induced ventricular arrhythmias.

These results were later confirmed in a non-human primate model of cardiac arrhythmia.

In neonatal rat ventricular cardiomyocytes, EPA and DHA have been shown to block sodium and calcium channels, hyperpolarise the resting membrane potential, raise the threshold potential for the gating of the sodium channel and increase the duration of the refractory period. This altered electrophysiology of the cardiomyocytes induced by EPA and DHA has been suggested to play a role in preventing arrhythmias.

Animal studies have also suggested that dietary supplementation with fish oils high in EPA and DHA may alter the membrane phospholipid fatty acid composition of cells, including isolated heart cells (cardiomyocytes).

Breivik et al in US Patent 5, 698, 594 disclose a composition comprising at least 80% (w/w) of a mixture of n-3 PUFAs wherein at least 75% by weight of the fatty acids comprise a mixture of EPA and DHA. The composition is said to have a significant effect on risk factors for cardiovascular disease, including reduced coagulation factor VII phospholipid complex activity, lowering the level of serum triglycerides and improved diastolic and systolic blood pressure. However the disclosure does not demonstrate that this mixture has a direct effect on ischaemic arrhythmias, reperfusion arrhythmias, atrial fibrillation, and associated rhythm disorders.

Leaf et al in US Patent 5, 760, 081 disclose a composition comprising EPA, DHA or a mixture thereof for the prevention of ventricular fibrillation.

It has now been found that PUFAs of a particular class, namely the C18 PUFAs, can alleviate or reduce symptoms of coronary heart disease.

According to one aspect the present invention provides a method for the treatment or prevention of coronary heart disease including the step of administering a polyunsaturated fatty acid or derivative thereof, said polyunsaturated fatty acid including : a linear chain of 18 carbon atoms, and two or more double bonds in the linear chain, wherein said polyunsaturated fatty acid is an n-1, n-2, n-3, n-4 or n-5 fatty acid, but excluding 18:3n-3 (#9,12,15).

In another aspect the invention provides the use of a polyunsaturated fatty acid or a derivative thereof in the manufacture of a medicament for the treatment or prevention of coronary heart disease, said polyunsaturated fatty acid including : a linear chain of 18 carbon atoms, and two or more double bonds in the linear chain, wherein said polyunsaturated fatty acid is an n-1, n-2, n-3, n-4 or n-5 fatty acid, but excluding 18:3n-3 (#9,12,15).

It is to be understood that throughout specification and claims where a particular fatty acid is excluded, it is only excluded to the extent that one of the included fatty acids must be present in the defined composition or utilised in the method or process to the extent necessary to achieve the desired effect. Unless otherwise specified the exclusion of a

particular fatty acid is not intended to mean that the excluded fatty acid cannot be included in the composition as an additional component, or used as an additional agent in a method, such as in a combined therapy.

The fatty acids discussed herein are referred to using the convenient nomenclature of numerical designations based on the number of carbon atoms, the number of centres of unsaturation and the number of carbon atoms from the end of the chain to where the unsaturation begins. Thus DHA is designated 22 : 6n-3, indicating it is a 22 carbon chain with 6 double bonds and the first double bond is 3 carbon centres from the terminal methyl end of the fatty acid chain. Where it is necessary to specify the location of additional double bonds, this is done using the designation"A", where 9 refers to a double bond between C9 and C 10, counting from the carboxyl end.

The expression"treatment or prevention of coronary heart disease"is to be understood to include the treatment or prevention of one or more symptoms of coronary heart disease, such as myocardial infarction, cardiac arrhythmias, sudden cardiac death, and atrial rhythm disorders, such as atrial fibrillation and tachycardia. In preferred embodiments the PUFAs of the present invention are used to treat or prevent the various types of cardiac arrhythmias, such as those arising from fibrillation, the development of automaticity and/or re-entry circuits and/or abnormalities of repolarisation and the lilce.

It is believed that the C18 PUFAs of the present invention are capable of exhibiting an efficacy in the treatment or prevention of CHD which is at least comparable with EPA and DHA.

One method of determining the antiarrhythmic efficacy of a particular C18 fatty acid is by using isolated ventricular cardiomyocyte preparations. By using an isolated single cell (cardiomyocyte) bioassay system, it is possible to extrapolate the efficacy of a particular fatty acid from the cellular level to the effect it would lilcely have at the whole animal level or in the human clinical setting.

Some of the polyunsaturated fatty acids suitable for use in the present invention are novel and represent a further aspect of the invention.

According to this aspect the invention provides a substantially purified polyunsaturated fatty acid or a derivative thereof including a linear chain of 18 carbon atoms, and two or more double bonds in the linear chain wherein said polyunsaturated fatty acid is an n-1, n-2, n-3, n-4 or n-5 fatty acid, but excluding 18:3n-3 (#9,12,15), 18:4n-3 (#6,9,12,15) and 18:5n-3 (#3,6,9,12,15).

The fatty acid may be a derivative, such as an ester, amide, organic salt, inorganic salt, or a prodrug. These derivatives may not have activity in their own right, but may be converted to active forms in the body, or prior to administration.

The linear chain may be branched and may contain allcyl or halogen substituents. Examples of suitable substituents include, but are not limited to, hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, fluoromethyl, difluoromethyl, trifluoromethyl, fluoro, chloro, bromo and iodo.

The polyunsaturated fatty acids may have a variety of structures with between two and seven double bonds in cis or trans configuration. There can also be a mixture of cis and trans within the one molecule. The double bonds can be interrupted by either one or more methylene groups or they can be conjugated. Preferably the polyunsaturated fatty acids have three or more double bonds, more preferably four or more double bonds. Examples of some typical structural types include, but are not limited to, the following : Structural Type I

Where Rl-Rs are independently selected from the group consisting of hydrogen, allcyl, haloalkyl and halogen, specifically including but not limited to hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, fluoromethyl, difluoromethyl, trifluoromethyl, fluoro, chloro, bromo and iodo.

Structural Type II

Where RI-RIO are independently selected from the group consisting of hydrogen, allcyl, haloallcyl and halogen, specifically including but not limited to hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, fluoromethyl, difluoromethyl, trifluoromethyl, fluoro, chloro, bromo and iodo.

Structural Type III

Where R is selected from the group consisting of hydrogen, allcyl, haloalkyl, halogen, specifically including but not limited to hydrogen, methyl, ethyl, propyl, isopropyl, butyl,

isobutyl, fluoromethyl, difluoromethyl, trifluoromethyl, fluoro, chloro, bromo and iodo ; and n is an integer such that the linear chain, or baclcbone, is 18 carbons long.

Structural Type IV

Where R is selected from the group consisting of hydrogen, alkyl, haloallcyl and halogen, specifically including but not limited to hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, fluoromethyl, difluoromethyl, trifluoromethyl, fluoro, chloro, bromo and iodo ; each n is independently an integer from 0 to 3 ; and m is an integer such that the linear chain is 18 carbons long.

Other Structural Types

Where RI. 2 are independently selected from the group consisting of hydrogen, allcyl, haloalkyl, and halogen, specifically including but not limited to hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, fluoromethyl, difluoromethyl, trifluoromethyl, fluoro, chloro, bromo and iodo.

This corresponds to structural type 1 in which the A12 double bond is replaced with Ri and Ra, This type of substitution can be done with other double bonds of the structural types shown above.

This structure is an example of a fatty acid containing a mixture of cis and trans double bonds.

The fatty acids may be synthesised using any procedure known to the art for the synthesis of unsaturated fatty acids. Examples of suitable methods for synthesis are shown below in Schemes 1 and 2. 1. HI04. H20, EtOH 1. m-CPBA, DCM OH 2. HC (OEt) 3, Et Et I 2. Na2CO3, H20, 90 I pentane, CSA Et OE OH OH 0. 02M FeCI3, EtQ O acetone l ll EtO/H KOBS, DCM, 0°C + ~BrPh3P n xi X= (OEt) 2 0. 1M FeCI31 _ acetone, H2O X=O KOBut, DCM, 0°C or 6 > _< OR OR 0 R=Me LiOH, THF ERZ R=H SCHEME 1 Q BF3. Et20 PCC, NaOAc ft EtOH OR CM OR HO i-w t-I"" O OEt Et -BrPh3P OEt OR KOBut, DCM, 0°C 0 0. IMFeOs. HzO X= (OEt) z acetone, 40°C , X=O 'BrPh3P OEt KOBut, DCM, 0°C OR o 11 o 0. 1 M Fecal3, H20 X= (OEt) 2 acetone, 40°C X=O 'BrPh3P KOBut, DCM, 0°C OR o R=Et LiOH, THF R=H SCHEME 2 References J. Sandri & J. Viala, J. Org. Chem., 1995, 60, 6627-6630.

S. Michaud & J. Viala, Tetrahedron, 1999, 55, 3019-3024 M. Santelli & J. Viala, Synthesis, 1988, 395-397.

G. M. Ksander et al., J. Med. Chem., 1997, 40, 495-505.

The preparation of 18:5n-3 (#,3,6,9,12,15) is described by D. V. Kuklev, N. A. Aizdaicher, A.

B. Imbis, V. V. Bezuglov and N. A. Layshev, Phytochemistry, 31, pp2401-2403, 1992.

Some of the polyunsaturated fatty acids may also be obtained from natural sources such as algal, microalgal or other eukaryotic or prokaryotic sources, in which case the polyunsaturated fatty acid may be isolated and at least substantially fractionated from other compounds using standard techniques which may include standard chromatographic techniques, such as HPLC. Alternatively the fatty acids may be administered in the form of extracts, such as biomass extracts, or extracted oil mixtures. The biomass extract may be in the form of a total lipid extract containing sterols and other lipid soluble components. For nutraceutical applications the fatty acids may also be administered in the form of biomass.

It has also been surprisingly found that considerable quantities of some of these fatty acids, such as 18:4n-3 (#6,9,12,15) and 18:5n-3 (#3,6,9,12,15) can be obtained by culturing microalgae from the classes Dinophyceae (dinoflagellates), Cryptophyceae (cryptomonads), Bacillariophyceae (diatoms), Chlorophyceae, Prasinophyceae, Prymnesiophyceae or Raphidophyceae and subjecting the culture to extraction with an organic solvent and subsequent purification.

C18 PUFAs and their derivatives have been produced by logarithmic and stationary phase cultured microalgae of the classes Cryptophyceae e. g. unidentified cryptomonad (CS-412) : 18 : 4n-3 = 30. 7% of fatty acids (Renaud et al., 1999), Dinophyceae e. g. Scrippsiella sp. (CS- 295/c) : 18 : 4n-3 = 10. 6% of fatty acids, 18 : son-3 = 43. 1% of fatty acids (Mansour et al., 1999), Bacillariophyceae e. g. Thalassiosira pseudonana (CS-20) : 18 : 4n-3 = 12. 8% of fatty acids (Brown et al., 1996), Chlorophyceae e. g. Tetraselmis suecica : 18 : 4n-3 = 8. 4% of fatty acids (Volkman et al., 1989), Prasinophyceae e. g. Pyramimonas cordata (CS-140) : 18 : 4n-3 = 25. 6% of fatty acids, 18 : 5ni-3 3. 4% of fatty acids (Dunstan et al., 1992), Prymnesiophyceae e. g. Prymnesiophyte (CS-260) : 18 : 4n-3 = 19. 4% of fatty acids (Renaud et al., 1999) and Raphidophyceae e. g. Heterosigma akashiwo (CS-39) : 18 : 4n-3 = 18. 1% of fatty acids, 18 : 5n-3 = 4. 6% of fatty acids (Nichols et al., 1987).

All of these species are, to varying degrees, light-requiring (5-1000 umol photons PAR in-2 s-1) whether in the presence of an organic carbon source or not. Such microalgae may be cultured in temperatures ranging from 4°C to 32°C, between 12 : 12 hour light : dark (L : D) cycle and continuous light, with either no agitation (static) or some agitation by shaking and/or bubbling. The bubbling can be performed using air alone or using C02 (within a range of 0. 1 to 5%) mixed with air, at a rate generally between 20 and 500 mL min-'L-1.

To produce high biomass of microalgae, small cultures can provide inocula for the mass culturing of microalgae using a variety of different cultivation vessels and techniques.

Cultivation vessels can range from relatively low volume, high density open and closed (sterile) systems in a diversity of designs, such as photobioreactors (e. g. Tredici, 1999), through to high volume, low density open tanks and ponds (e. g. Jeffrey et al., 1989 ; Borowitzka, 1999). There is a wide range of cultivation techniques used in order to optimize growth and compound production ranging from the alteration of light intensity, photoperiod and nutrient source, through to shifts in temperature, salinity, aeration quality and rate, pH, dissolved oxygen and individual nutrients. In a scale up process, from 10 L vessels to 1000 L tanks, for example, a dilution factor between 50 : 1 (20 L inoculum) and 10 : 1 (100 L inoculum) is sufficient to obtain a relatively high biomass per volume in 1-3 weeks. Similarly, further scale up to open ponds is possible with the provision of sufficient inocula from smaller scale systems. At all scales of microalgal cultivation, cultures may be maintained in a productive state by semi-continuous and continuous dilution of culture with fresh sterile (autoclaved) media (e. g. Tredici et al., 1991). In such ways as those described, microalgae can be cultured to produce 18 : 4 (n-3) and 18 : 5 (n-3) PUFAs for extraction and purification at large and industrial scales.

Cultured microalgae are harvested in early logarithmic phase through to late stationary phase of growth by one of several available methods, according both to the species being harvested and the culture density and volume. Relatively fragile cells and/or cultures with low cell densities are filtered through glass fibre filters (Whatman 90 mm GF/C and GF/F) under low vacuum. More robust cells and/or cultures in high density and/or large culture volumes are either directly centrifuged at 1000-16000 x g for 10-30 min (e. g. Brown, 1991 ; Hodgson et al., 1991 ; Mansour et al., 1999) or concentrated (approximately 10-fold) by a

process of flocculation (Knuckey, 1998) followed by centrifugation to obtain a pellet for immediate extraction. If not extracted immediately, microalgal pellets are stored at-80°C or under liquid nitrogen.

According to another aspect of the invention there is provided a process for obtaining substantially purified C18 polyunsaturated fatty acids or derivatives thereof including : culturing microalgae of the class Dinophyceae, Cryptophyceae, Bacillariophyceae, Chlorophyceae, Prasinophyceae, Prymnesiophyceae or Raphidophyceae or other suitable microalga in a suitable culture medium under conditions amenable to microalgal growth, harvesting cultured microalgae, subjecting the harvested microalgae to solvent extraction and phase separation to obtain a fatty acid extract, transalkylating fatty acids with an alkanol and a suitable acid to form corresponding ; fatty acid esters, subjecting the fatty acid esters to a separation technique to obtain substantially pure fatty acid esters, optionally subjecting pure fatty acid esters to hydrolysis, transesterification or derivatisation.

The degree to which the polyunsaturated fatty acid is purified is dependent somewhat on the intended use of the fatty acid. Thus for the purposes of structural identification or characterisation it may be preferable for the unsaturated fatty acid to be greater than 80% pure, whilst for the purposes of therapeutic use it may be preferred that the fatty acid is greater than 95% pure. This would be determined by the constituents and the nature of the remaining compounds in the mixture. The purity of the fatty acid, or a suitable derivative

thereof, may be determined using any of the standard techniques that are known in the art, which may include HPLC.

Preferably all of the double bonds in the polyunsaturated fatty acid are in the cis configuration, although the invention is not restricted thereto and polyunsaturated fatty acids having all trans or combinations of cis and trans double bonds are also encompassed.

The polyunsaturated fatty acids according to the present invention may also be used in combined therapies. For example, the fatty acids of the present invention can be administered with (or formulated with) other active agents used in the treatment of heart diseases, such as antiviral agents, antibiotics, anti-inflammatory agents, antihypertensive agents, anticonvulsants and the like.

If formulated as a fixed dose, such combination products employ the compounds of this invention in the dosage ranges described below and the other pharmaceutically active agent within its approved dosage range. The compounds of the invention may be used sequentially with known pharmaceutical agents when a combination formulation is inappropriate.

By an"effective amount"is meant a quantity of active compound which will upon single or multiple dose administration to the patient be effective in providing the desired effect.

In another aspect of the invention there is provided a composition including a polyunsaturated fatty acid including : a linear chain of 18 carbon atoms, and two or more double bonds in the linear chain, wherein said polyunsaturated fatty acid is a n-1, n-2, n-3, n-4 or n-5 fatty acid, but excluding 18:3n-3 (#9,12,15),

and a pharmaceutically or nutraceutically acceptable carrier.

Preferably the composition is a pharmaceutical composition or a nutraceutical (health supplement). The composition may contain the PUFAs in the form of pure or substantially pure compounds, or in the form of whole microalgal cells, partly purified microalgal cells or microalgal oil. Preferably the cells or microalgal oil are derived from autotrophic or substantially autotrophic microalgae (or at least mixotrophic), such as those previously described. It is possible that some heterotrophic microalgae may also provide sources of the PUFAs according to the invention.

Accordingly a preferred aspect of the invention provides a pharmaceutical or nutraceutical composition comprising wholly microalgal cells, partly purified microalgal cells or microalgal oil, wherein said cells or oil is derived from cultured autotrophic or substantially autotrophic microalgae.

When a compound of the invention is administered to a human subject the daily dosage can normally be determined by an attending physician with the dosage generally varying according to the age, weight, and response of the individual patient, as well as the severity of the patient's symptoms. The amount administered is preferably an effective amount to achieve the desired effect, whether that effect be prophylactic or therapeutic. In general a suitable dose of the compound of the invention will be in the range of 0. 1 to 50 mg per kilogram body weight of the recipient per day, preferably in the range of 0. 5 to 10 mg per kilogram body weight per day. The desired dose is preferably presented as two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, containing 1 to 1000 mg, preferably 10 to 500 mg of active ingredient per unit dosage form.

The compounds according to the invention, also referred to herein as the active ingredient, may be administered for therapy by any suitable route, including oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous and intradermal). Preferably, administration will be by the oral route, however it will be appreciated that the preferred route will vary with the condition and

age of the recipient, the nature of the invention and the chosen active ingredient. When administered by the oral route a prodrug of the active compound which is more efficiently absorbed than the unmodified compound is generally preferred.

The compositions of the present invention comprise the polyunsaturated fatty acid, optionally as a salt or other pharmaceutically acceptable derivative, together with one or more pharmaceutically acceptable carriers, diluents or excipients therefor, and optionally other therapeutic agents. Each carrier, diluent or excipient must be pharmaceutically "acceptable"in the sense of being compatible with the other ingredients of the composition and not injurious to the patient. Compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier, diluent or excipient which includes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient ; as a powder or granules ; as a solution or a suspension in an aqueous or non-aqueous liquid ; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.

The active ingredient may also be presented as a bolus, electuary or paste.

Tablets may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e. g. inert diluent, preservative disintegrant (e. g. sodium starch glycolate, cross- linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent). Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be

coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

Compositions suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured base, usually sucrose and acadia or tragacanth gum ; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acadia gum ; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Compositions for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter.

Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient ; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

Methods of suspending aliphatic alcohols, long-chain fatty acids and long-chain alkanes are well known in the art. One suitable method of making such a suspension is dilution of a nonionic detergent surfactant to 1 to 100 mg/ml in water or an aqueous solution such as a physiological saline solution and heating the solution (e. g., 37°C to 50°C). The active ingredient is then added to this surfactant solution to produce the desired final concentration of active ingredient and the combination is mixed (e. g., rotary or reciprocal mixing, stirring or sonicating), to produce a suspension of globular particles (about 0. 1, to 100//average size). Other acceptable carriers include emulsions (oil-in-water or water-in-oil), solutions,

creams, lotions, ointments, foams, gels and aerosols, all of which can be prepared using well-known methods.

The compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage compositions are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

The compounds according to the invention may also be presented for use in the form of veterinary compositions, which may be prepared, for example, by methods that are conventional in the art.

Preferably the pharmaceutical composition is used for treatment of coronary heart disease.

In particular the pharmaceutical composition may be used in the treatment of cardiac arrhythmias including ischaemic arrhythmias, reperfusion arrhythmias and atrial fibrillation and the like. The pharmaceutical composition may also include other agents useful in the treatment of coronary heart disease, including but not limited to therapeutically-used antiarrhythmic medications.

In one particularly preferred form of the invention the polyunsaturated fatty acid is 18 : 4n-3 (#6,9,12,15) or 18:5n-3 (#3,6,9,12,15).

The polyunsaturated fatty acid may be administered in any suitable form which gives rise to an elevation of the concentration of the unsaturated fatty acid in the cell membrane.

Treatment using the pharmaceutical composition of the present invention may be aimed at being preventative by reducing the risk of contracting the condition, or the pharmaceutical composition may be used to alleviate or obviate the condition.

Health supplements and nutraceuticals are becoming increasingly popular with those who remain unconvinced as to the nutritional benefit of the wide variety of processed foods available on supermarket shelves and from fast food outlets. Those wishing to live healthy lifestyles are also turning to such products. An example of a nutritional supplement rich in n-3 fatty acids is Ropufa by Roche Vitamins which has been incorporated as an oil or powder into various food products, such as breakfast cereals, margarine, spreads, bread and infant foods. The technology involved in preparing this supplement is said to eliminate the strong smell and taste of fish oil. DHA-Gold, by NutraSweetKelco is enriched with a microalgal ingredient and fed to chickens such that the resulting eggs are enriched with DHA. The PUFAs, microalgal biomass, extracts and oils of the present invention are similarly suitable for use as nutraceuticals.

Health supplements or nutraceuticals made from microalgae are already a rich source of nutritional benefit for humans. The majority of these are from the algal classes Cyanobacteria or blue-green algae and Chlorophyceae or green algae. They are supplements for protein, mixed carotenoids and other phytonutrients, B-vitamins, gammalinolenic acid and essential amino acids. Examples include Spirulina Pacifica TM from Cyanotech, natural antioxidants, such as the pigments astaxanthin (e. g. NaturRose from Cyanotech) and betacarotene. Cyanobacteria do not produce highly unsaturated fatty acids. Their major fatty acids are C 16 and C18 saturated and monounsaturated fatty acids (Romano et al. 2000).

Chlorophytes are abundant in the shorter chain C16 PUFA and also contain 18 : 3 PUFA but are almost always lacking in 18 : 4 and 18 : 5 PUFA. Algal oils commercially available include specific preparations produced from a heterotrophic dinoflagellate of the genus Crypthecodinium by Martek Biosciences. These contain various PUFA including EPA and DHA but the only C18 PUFA are 18 : 2 PUFA.

Microalgal nutraceuticals prepared from microalgae of the classes described above would be particularly useful in that they would include the C18 PUFAs according to the present invention, and depending on whether these PUFAs are used in pure form or as algal oils or whole biomass may include the benefits of additional components, such as C22 PUFAs and antioxidants. For example an oil extract of a Cryptomonad, such as Cryptomonad sp.

CRF101 CS-412, could provide a combination of 18 : 4 (n-3), DHA and EPA.

Nutraceuticals possible from use of this invention also include the use of total biomass of the microalga with the benefits ascribed above for Spirulina i. e. a source of protein but also including the benefits of the C18 and other unsaturated fatty acids. Other important constituents of microalgae are vitamins and natural antioxidants including ascorbic acid (vitamin C) and alpha-tocopherol (vitamin E) and pigments such as the carotenoids (Brown and Lavens, 2000, Brown et al. 1999 and Jeffrey and Vesk 1997). These would form part of the microalgal"health package"for human benefit, as well as acting as a stabilising agents by protecting the integrity of the unsaturated fatty acids. These nutraceuticals could take a variety of other forms including freeze-dried powders, compressed tablets and liquid extracts.

Microalgae offer a non-fish source of unsaturated fatty acids. This has benefits in being a non-animal source of these important fatty acids where consumption of animal derived material is not acceptable. There are several other advantages of Microalgae as an alternative source of n-3 PUFA over those derived from fish oils (e. g. Tuna oil).

Microalgae represent an enormous, renewable and relatively untapped biodiversity with a large range of biologically active compounds including long-chain polyunsaturated fatty acids such as DHA, EPA and AA and other novel PUFA such as 18 : 5 (n-3), 18 : 4 (n-3) and 28 : 8 (n-3).

Microalgae are a clean, green, high quality source for nutraceuticals. Their fatty acid profiles are usually much simpler compared with those of fish oils and specific to algal class. Hence certain algal species can be targeted for desirable fatty acid profiles (PUFA mixture in different ratios). They are one of the fastest growing plants on earth and they are amenable to mass culturing. In addition the synthesis of desirable PUFA and other bioactive constituents can be optimized by manipulation of culturing conditions.

In one embodiment the present invention provides use of a polyunsaturated fatty acid or a derivative thereof in the manufacture of a medicament for the treatment or prevention of coronary heart disease, said polyunsaturated fatty acid including :

a linear chain of 18 carbon atoms, and two or more double bonds in the linear chain, wherein said polyunsaturated fatty acid is an n-1, n-2, n-3, n-4 or n-5 fatty acid, but excluding 18:3n-3 (#9,12,15).

In one form of the invention the unsaturated fatty acid may be administered as part of a dietary regime. Thus the composition may be contained in a foodstuff or beverage, with consumption of the foodstuff or beverage thereby leading to an increase in levels of the unsaturated fatty acid. In such cases the foodstuff or beverage may constitute the nutraceutically acceptable carrier for the polyunsaturated fatty acid. A dietary regime may be particularly suited to a preventative treatment in a subject who is susceptible to the disease to be treated.

The nutraceuticals of the present invention could also include other dietary supplements, such as vitamins, carbohydrates, minerals, antioxidants, pigments and proteins.

With regard to dietary administration of the various C18 polyunsaturated fatty acids (PUFAs) and the likely effects they may have on protection of the heart from arrhythmias, it is important to note that, as opposed to acute administration, dietary administration would likely lead to metabolic conversions of these fatty acids to even longer chain, more polyunsaturated PUFAs. Thus dietary C18 PUFAs would be expected to be converted to C20 and C22 PUFAs, which have already been reported to exert potent antiarrhythmic effects on the heart. However, the extent to which these various C18 PUFAs would be converted to longer chain, more polyunsaturated fatty acids would be dependant on the initial degree of unsaturation of the C18 PUFA. Without wishing to be limited by theory, it would be most likely that 18 : 5n-3 would be more easily converted to the C20 and C22 PUFAs, than would 18 : 4n-3, which in turn would be greater than 18 : 3n-3. The reason for this lies in the number of rate-limiting desaturation steps required for the conversion of the C18 PUFAs to the longer chain PUFAs, in particular, 22 : 6n-3 (docosahexaenoic acid-DHA).

Dietary 18 : 3n-3 is further desaturated to 18 : 4n-3 by the action of A6 desaturase (step 1),

elongated to 20 : 4n-3 (step 2), desaturated to 20 : 5n-3 by the action of A5 desaturase (step 3), elongated to 22 : 5n-3 (step 4), elongated to 24 : 5 (step 5), further desaturated by A6 desaturase (step 6), and finally chain shortened by beta-oxidation to 22 : 6n-3 (step 7), ie three desaturation steps are involved. In the case of 18 : 4n-3 metabolism, step 1 is not required, and therefore only two desaturation reactions would occur in its conversion to 22 : 6n-3. Importantly is the fact that only one desaturation step is required (step 6) for the conversion of 18 : 5n-3 to 22 : 6n-3.

While the above metabolic pathways for the conversion of the C18 PUFAs to the longer chain C20 and C22 PUFAs have been well described, a number of points need to be considered. The situation in the rodent and man has been reported to vary markedly with respect to the efficiency at which these various reactions and conversions take place.

Competition also occurs between the differing classes of PUFAs (n-6 versus n-3) for desaturation by the various desaturating enzymes, and this will be dependant on the particular PUFA profile of the diet. However, notwithstanding the above, it would be reasonable to conclude that in terms of providing antiarrhythmic benefits either directly as the C 18 PUFA, or indirectly, by way of conversion to C20 or C22 PUFAs, dietary 18 : 5n-3 would be of greater benefit than 18 : 4n-3, which in turn would be greater than 18 : 3n-3.

With respect to the effect of acute 18 : 3n-3 on the extent of asynchronous contractile activity, it has been reported that this fatty acid is considerably less active than 22 : 6n-3 in a similar assay. Therefore, on the bases that C18 fatty acids of greater unsaturation (18 : 4n-3 and 18 : 5n-3) are shown to be as active as 22 : 6n-3 when added acutely, it would be reasonable to suggest that these fatty acids would also be more effective than 18 : 3n-3 in this assay.

It has also been found that the mechanism of action of PUFAs in the treatment of CHD is different to the mechanism of action of currently known therapeutic agents that are used in the treatment of coronary heart disease. It is therefore expected that there may be a synergistic effect between the PUFAs of the present invention and known antiarrhythmic therapies including sodium channel inhibitors, p-adrenergic receptor antagonists (hereinafter p-blockers) and calcium antagonists, or other antiarrhythmic therapies which work by a combination of sodium channel inhibition, (3-blocking or calcium antagonism and/or other

action. The synergistic effect may result in a reduced dose of the known antiarrhythmic agent being required to elicit an equivalent response. In particular it is proposed that the dosage of one or more of sodium channel inhibitors, p-blockers and calcium antagonists can be reduced when that agent is co-administered with a PUFA.

Accordingly a further aspect of the invention provides a composition for use in reducing the dose of a pharmaceutical agent used in the treatment of a coronary heart disease, the composition including a polyunsaturated fatty acid including a linear chain of 18 carbon atoms, and two or more double bonds in the linear chain, wherein said polyunsaturated fatty acid is an n-1, n-2, n-3, n-4 or n-5 fatty acid, and a pharmaceutically acceptable carrier.

The pharmaceutical agent may be one or more of the agents selected from the list including : sodium channel inhibitors, p-blockers and calcium antagonists, or other antiarrhythmic therapies which work by a combination of sodium channel inhibition, p-blocldng or calcium antagonism and/or other action.

The composition may be administered as a single dose containing the pharmaceutical agent.

Alternatively the composition may be administered prior to or during treatment with the pharmaceutical agent.

The polyunsaturated fatty acid may be any of the unsaturated fatty acids or derivatives described or exemplified herein.

Another aspect of the invention provides a method for reducing the dose of a pharmaceutical agent used in the treatment of a coronary heart disease, said method including the step of

administering a compound or pharmaceutical composition as described herein in conjunction with the pharmaceutical agent.

It is further noted that the C 18 PUFAs of the present invention are oxidatively more stable than the C22 PUFAs. In this regard it is usually necessary to formulate C22 PUFAs with antioxidants. The C18 PUFAs may likely require less stablisation by antioxidants. It has also been found that some algal oils obtained according to the present invention contain some natural antioxidants, thereby acting to stabilise C22 PUFAs present in the oil.

The invention will now be described with reference to the following examples and drawings which illustrate some preferred embodiments of the present invention. However it is to be understood that the following description is not to supersede the generality of the invention previously described.

Referring to the drawings : Figure 1 : is a graphical representation showing the effect of polyunsaturated fatty acids, at 20, uM final concentration, on the time course for development of spontaneous contractile activity (as percentage spontaneously contracting cells) in adult rat cardiomyocytes induced by 10, uM isoproterenol.

Figure 2 : is a histogram showing the effect of polyunsaturated fatty acids, 20, uM final concentration, on the percentage of adult rat cardiomyocytes displaying spontaneous contractile activity after 10 minutes of treatment with 10, uM isoproterenol.

Figure 3 : is a histogram showing the increase in stimulation voltage setting (Grass stimulator S4) required to electrically stimulate adult rat cardiomyocytes to synchronously contract after 5 minutes of treatment with various fatty acids at final concentration of 20, uM.

Figure 4 : is a dose response curve for the effect of polyunsaturated fatty acids, from 2. 5 to 20, uM final concentration, on the percentage of spontaneously-contracting adult rat cardiomyocytes after 10 minutes of treatment with 10, uM isoproterenol.

Figure 5 : is a dose response curve showing the effect of increasing concentrations of a PUFA mix from Cryptomonad sp. on the development of isoproterenol- induced asyachrony.

Figure 6 : is a dose response curve showing the effect of increasing concentrations of a PUFA mix from Scrippsiella sp. on the development of isoproterenol- induced asyachrony.

Figure 7 : is a histogram showing the affects of acute administration of 18 : 4 (n-3) against the percentage ventricular fibrillation incidence following artery ligation.

EXAMPLES Example 1-Extraction andpurification of the microalgal PUFAs 18 : 4n-3 and 18 : 5n-3 Culturing microalgae 18 : 4 (n-3) and 18 : 5 (n-3) were extracted from an unidentified cryptomonad designated CS- 412, and two strains of Scrippsiella sp designated CS-295/c and CS-297, held in the CSIRO Collection of Living Microalgae. Prior to extraction the microalgae were cultured and harvested as follows : The marine dinoflagellate Scrippsiella sp. CS-295/c was obtained from the CSIRO Collection of Living Microalgae, Tasmania, Australia, after isolation from ship ballast water. Cultures were grown in two 2 L Erlenmeyer flasks, each containing 100 mL of inoculum diluted to 1L with GSe medium (Blackburn et al., 2001),. a modification of the GP medium of Loeblich (1975. The culture was maintained at 18. 5 °C under 80 (miol photons PAR m-2s~l of cool white fluorescent light on a 12 : 12

h L : D cycle. The microalgae were harvested by centrifugation at 2500-4000 g for 10 min or filtered through GF/F glass-fibre filters. The collected microalgae pellet or filters were stored in polypropylene cryo-tubes (5 mL) under liquid nitrogen before extraction (Mansour et al., 1999).

An unidentified cryptomonad (CS-412) was obtained from the CSIRO Collection of Living Microalgae, Tasmania, Australia, after isolation from Fitzroy Island, Queensland. From 50 mL stock cultures, CS-412 was scaled up through 1. 5 L cultures in glass Erlenmeyer flasks to 10 L cultures in polycarbonate carboys.

Further 10 L batch cultures were subsequently inoculated with 1 L of culture in 9 L of growth medium. The culturing medium was GSe (Blackburn et al, 2001) with a growth temperature of 25+ 1 °C. Other culturing conditions included a light intensity of 100-150 llmol photons PAR m~2s~l, 12 : 12 hour L : D cycle, bubbling with 1% CO2 in air at a rate of 200 mL L min~1. The microalgae were harvested by centrifugation at 2500-4000 g for 10 min or filtered through GF/C (Whatman, 90 mm) glass-fibre filters. The collected microalgae pellet or filters were either extracted directly or stored in polypropylene cryo-tubes at-80°C before extraction.

Extraction of microalgal biomass The lipid soluble fraction of the algal pellet was extracted in chloroform-methanol-water (1 : 2 : 0. 8, v/v/v) by a modified version of Bligh and Dyer's (1959) method, using successive ultrasonication (15 min) and centrifugation (5-6 times). To initiate phase separation, chloroform and purified water (Milli-Q system, Millipore) were added to the combined extracts to give a final chloroform-methanol-water ratio of 1 : 1 : 0. 9 (v/v/v). The chloroform layer was rotary-evaporated and the yield of lipid determined gravimetrically. The extracts were then reconstituted in chloroform (1. 8 mL) and stored under nitrogen at- 20°C until analysis.

Derivatisation of fatty acids Fatty acid methyl esters (FAMEs) were formed by heating in MeOH : HCl (10 : 1) at 80° for 2h and extracted into hexane and stored under nitrogen at-20°C.

Clean-up offatty acid methylesters (FAMEs) The FAMEs were purified by elution through a short silica-alumina (0. 2g and 0. lg respectively) column with lOmL diethyl ether : hexane (3 : 97, v/v). The purified FAMEs were reconstituted in dichloromethane and stored at-20 °C until required for HPLC purification and GC, GC-MS analysis.

Analytical HPLC HPLC was performed using a preparative pump with a low pressure quaternary gradient controller and an autosampler and a reverse phase column : 4. 6 mm i. d. x 250 mm. with 5, um spherical particles, 100 A pore size, a 16 % carbon load of polymerically bonded C18 stationary phase and endcapped to remove free silanol groups (Alltima C18, Alltech Australia). The elution was isocratic using acetonitrile/water (97. 5 : 2. 5, v/v) and a flow rate of 1. 5 mL/min. Sample detection was by evaporative light scattering detection (see below).

Samples (100, uL) of a 0. 8 ug., uL-l solution of FAME in DCM were injected.

Preparative HPLC HPLC was performed using a reverse-phase column of i. d. 22 mm x 300 mm packed with the same type of particles as for the analytical column except they were 10, um in size. A binary solvent gradient starting with 100% acetonitrile and ending with 100% chloroform was used and samples (200, uL) of a 10, ug., uL-l solution. Dichloromethane was used as a replacement for chloroform in some instances.

Detection and collection Components eluting from the HPLC column were detected using an Evaporative Light- Scattering Detector (ELSD) (Mass Detector Model 750/14, Applied Chromatography Systems (ACS), ICI Australia Operations Pty Ltd, Scientific Instruments Division). The settings were as follows : attenuation range 16, time constant 5 sec, photomultiplier sensitivity 2 and evaporator set 40 °C and nitrogen cylinder outlet pressure of 300-400 lcPa.

Sample collection was achieved by use of a flow-splitter (Alltech Australia). Fractions were collected using an automatic fraction collector (Gilson model FC203B, Gilson Inc. WI USA).

Creation of PUFA mixtures PUFA FAME mixtures with a desirable PUFA composition were separated from unwanted fatty acids such as diunsaturated (e. g. 18 : 2n-6), monounsaturated (e. g. 18 : ln-9 or 16 : ln-7) and saturated fatty acids (e. g. 16 : 0 and 18 : 0) by preparative reverse-phase HPLC. In the example of PUFA mixtures obtained from the microalgal species, Scrippsiella sp. CS-295/c, the PUFA mixture contained the following fatty acids : 18 : 5n-3, 18 : 4n-3, 20 : 5n-3, 22 : 6 (n-3). These were separated by collection of the PUFA fraction between 12 and 16 minutes from a chromatographic injection of a total fatty acids (as FAME).

Preparation of purified PUFA mixtures Mixtures of suites of PUFA were prepared by preparative HPLC (see above), these included PUFA mixture containing : 18 : 4 (n-3) : 20 : 5 (n-3) : 22 : 6 (n-3) [2 : 1 : 1] from Cryptomonad sp. CRFI01 CS-412 18 : 5 (n-3) : 18 : 4 (n-3) : 20 : 5 (n-3) : 22 : 6 (n-3) [3. 5 : 1 : 0. 2 : 2] from Scrippsiella sp. CS-295/c.

The total fatty acid composition of Scrippsiella sp. CS-295/c and Cryptomonad CRFI01 CS- 412 are shown in Tables 1 and 2 below.

Table 1 Total fatty acid composition of CS-295/c Fatty acid % 16 : 1n-7 3. 5 16 : 0 13. 5 18 : 5n-3 33. 5 18 : 4n-3 12. 8 18 : 2n-3 2. 7 18 : 1n-9 3. 5 18 : 1n-7 0.9 18 : 0 1. 4 20 : 5n-3 2. 5 22 : 6n-3 23. 7 28 : 8n-3 2. 1 Total 100. 0 Table 2 Total fatty acid composition for CS-412 Fatty acid % 14 : 0 2. 4 15 : 0 0. 8 16 : 1 4. 7 16 : 0 12. 3 18 : 4 20. 6 18 : 2n-6 0. 6 18 : 3n-3 26. 4 18 : ln-9 0. 6 18 : 0 1. 8 22 : 4n-6 0. 0 20 : 5n-3 13. 8 22 : 5n-6 2. 1 22 : 6n-3 13. 6 22 : 5n-3/22 : 4n-6 0. 2 Total 100. 0

DMOXderivatization for determination of position of double bonds A low temperature derivatization method according to Christie (1998) was used to avoid degradation of PUFA and to minimise the formation of byproducts. Purified FAME mixtures or HPLC purified individual FAME were first hydrolyse to the free fatty acids by heating for 10 min at 80° with 10% KOH in 80% MeOH then acidified to pH 2 with Supra pur conc. HC1. The free acid (s) was converted to the acid chloride by reaction with oxalyl chloride (0. 5 mL) at ambient temperature overnight under nitrogen. Excess reagent was removed in a stream of nitrogen and the product reacted immediately with 0. 5 mL of 2- amino-2-methyl-1-propanol in dichloromethane (10 mg. mL-l ; stored over molecular sieve- type 5A (Alltech)) for 1 h at ambient temperature. The solvent was evaporated and trifluoroaceticanydride (1 mL) was added and the mixture heated at 40°C for 1 h. Excess reagent was then evaporated and the product taken up in hexane (5 mL) and washed with Milli-Q H20 water (2 x 2 mL). The solution was dried over anhydrous sodium sulphate, then evaporated and redissolved in hexane for GC and GC-MS analysis.

Gas chromatography GC was performed on an HP 5890 gas chromatograph fitted with an BPX-70 bonded phase capillary column (50 m ; 0. 32 mm i. d. ; 0. 25, um film thickness), a FID and an on-column injector. Samples were injected at 45°C ; after 2 min the oven temperature was raised at 30°C min''to 120 °C and then at 3°C min-'to 2400, where it was held for 20 min. FAME, hydrogenated FAME and DMOX derivatives were also analysed on an HP-1 non-polar column (50 m ; 0. 32 mm i. d. ; 0. 17 um film thickness) with the same conditions as above except the final temperature was 310°C.

Mass spectrometry EI GC-MS was performed on a Fisons MD-800 with an on-column injector set at 45°C. The sample was injected into a retention gap attached to an HP-5 Ultra 2 bonded phase column (50 m ; 0. 32 mm i. d. ; 0. 17 um film thickness). The initial temperature of 45°C was held for 1 min, followed by temperature programming at 30°C min-'to 1400C then at 3°C min~1 to 310°C, where it was held for 12 min. Helium was used as the carrier gas. Mass spectrometer operating conditions were : electron impact energy 70eV ; transfer line 310°C ; source temperature 250°C ; scan rate 0. 8 scans. s-1 and mass range 40-650 dalton. CI GC-MS

was achieved with methane and the following modifications to the operating conditions : electron impact energy 35eV, source temperature 200°C.

Preparation offree fatty acids The purified individual FAME, FAME mixture and total FAME were hydrolysed by heating at 80°C for 5 min. with 10% KOH in methanol. After acidification of the saponified fraction with HC1 to pH 2-3, the purified free fatty acids were extracted into hexane. The hexane was removed and the purified free fatty acid extracts were reconstituted in ethanol containing 0. 03% v/v butylatedhydroxytoluene and stored at-20°C until required for testing.

An equivalent stock solution of 22 : 6n-3 was also prepared.

Example 2-Effect of 18 : 4n-3 and 18 : 5n-3 on tlle time course of isoprotereliol-induced spontaneous contractile activity in rat cardiomyocytes Cellular Bioassay System The assay method used for testing the antiarrhythmic efficacy of polyunsaturated fatty acids derived from microalgae, uses heart cells (cardiomyocytes) isolated from 10 to 12-week'old Sprague-Dawley rats. These cells which are normally quiescent (i. e. do not beat without an external stimuli) are induced to beat by application of an electrical field at a frequency of one stimulus per second. Cells beat in synchron with the applied electrical field. Cells are then induced to beat in an asynchronous manner by a number of means one of which, as described in this invention, is by the addition of the ß-adrenergic receptor agonist isoproterenol. The antiarrhythmic (antiasynchronous) properties of the microalgal polyunsaturated fatty acids are determined by adding the test algal PUFAs to the rat cardiomyocytes prior to adding isoproterenol. Microalgal PUFAs are considered to be antiarrhythmic if they prevent the development of asynchronous contractile behaviour in such cardiomyocytes.

Perfusion Media Calcium-free Tyrode perfusion media, contained (in mN) 137. 7 NaCl, 4. 8 KC1, 1. 2 KH2P04, 1. 2 MgS04, 11 glucose, 10. 0 (N- [2-hydroxyethyl] piperazine-N'- [2-ethanesulfonic acid])

(HEPES), pH 7. 40. Tyrode solution was prepared using ultra-pure (Milli-Q) water and was filtered through a 0. 22, m Millipore filter prior to use and gassed with 100% 02.

Preparation of Adult Rat Ventricular Cardiomyocytes The heart of a 10 to 12 week old male Sprague-Dawley rat was excised and retrogradely perfused in a non-recirculating manner on a Langendorff apparatus with Tyrode solution containing 1. 5 mM Ca2+ for 4 min. The heart was then perfused with Ca+-free Tyrode solution (non-recirculating) for 2 mn. The buffers were maintained at 37°C and gassed with 100% 02. The heart was further perfused in a recirculating manner for 30 min with Tyrode solution supplemented with 20, M Ca2+, 500 U/ml collagenase (type 1A) and 0. 1% (w/v) dilipidated BSA (fraction V) at 37°C. After perfusion with collagenase, the ventricles were removed, minced with scissors, and agitated in Tyrode solution containing 20, uM Ca2+, 2% (w/v) BSA and 30 mM 2, 3-butane-dione monoxime at 25°C. The suspension was filtered through a 250, um nylon-mesh gauze. This method of preparation resulted in more than 70% of the cells being rod-shaped and displaying clear striations without cell membrane blebbing. The concentration of Ca2+ was gradually increased to 1 mM. Aliquots of the cardiomyocyte suspension (approximately 2 ml) were added to petri dishes containing 12 mm (diameter) round glass coverslips coated with laminin for 60 min. This procedure allowed >95% rod-shaped cardiomyocytes with clear cross striations without membrane blebs, to adhere to coverslips within 60 min at room temperature.

Measurement of Cardiomyocyte Contraction A CCD video camera (Sony) was mounted on an inverted Olympus microscope which transferred images to a TV monitor. The images were simultaneously transferred to a pentium computer connected between the camera and monitor housing a Neotech Image Grabber board (Neotech Ltd., Hampshire, UEC). Pixel resolution was 768 horizontal x 576 vertical. The G-programming language associated with the software LabVIEW (National Instruments, Victoria, Australia) and an add-on image analysis library, Concept Vi Level 2 (Graftek, Mirmande, France) were developed to allow on-line, real-time consecutive automated image analyses. The cell length in motion was measured with a time resolution of 40ms.

Spontaneous Contractile Activity of Cardiomyocytes Adult rat cardiomyocytes were superfused with Tyrodes solution containing test polyunsaturated fatty acids for 4 min at 37°C in the absence of electrical field stimulation.

Normally quiescent cardiomyocytes, in the absence of electrical-field stimulation can be induced to spontaneously contract over time by the addition of isoproterenol. 10, uM isoproterenol was added to cardiomyocytes in the absence or presence of polyunsaturated fatty acids, and the time course for the development of spontaneous contractile activity determined.

Electrical Field Stimulation of Cardiomyocytes Glass cover slips with adhering cells were gently removed from the petri dish and placed in a custom-designed superfusion chamber (20 mm diameter, 500, u1 volume). This chamber was mounted on the stage of an inverted Olympus microscope housed in a perspex chamber maintained at 37°C. The various agents tested were added to the superfusing solution.

Cardiomyocytes were stimulated to contract using two platinum wire electrodes fitted within the superfusion chamber 10 mm apart and positioned parallel to the flow of the superfusing medium. Biphasic electrical stimuli were applied at 1. 5x threshold voltage (unless otherwise stated) with a pulse duration of 5 ms at 1 Hz. To allow cells to adapt to the stimulating conditions, a conditioning procedure consisting of electrical stimulation of cardiomyocytes at a frequency of 1 Hz for 15 min with constant superfusing of Tyrode solution containing 1 mM Ca2+ was carried out prior to all experiments. Only those cardiomyocytes contracting at both ends of their longitudinal axis were measured.

Measurement of Spontaneous Asynchrony Asynchrony, usually observed as a series of rapid contractions, is defined here as a period of extra contractions, over and above those expected from the stimulation frequency. To ensure >95% cardiomyocytes were asynchronous, the superfusing medium was Tyrode solution containing 10, uM isoproterenol. Cardiomyocytes were electrically stimulated for 10 min. The test fatty acids were then added to the same superfusing solution and stimulation was allowed to proceed for a further 10 min. After this time, cardiomyocytes were observed for each treatment and counted to determine the percentage of asynchronously and synchronously contracting cardiomyocytes. To determine whether

microalgal polyunsaturated fatty acids prevent asynchronous and synchronous contractile activity cardiomyocytes were first superfused with Tyrode solution containing fatty acid.

After 10 min, isoproterenol (10, uM) was added to the same solution and superfused as above for a further 10 min. Following electrical stimulation at a suprathreshold voltage, the percentage of asynchronous and synchronous contracting cardiomyocytes was counted.

Addition of the test PUFAs to the superfusate medium was performed by dilution of the stock PUFA solution in Tyrodes buffer with the ethanol concentration not exceeding 0. 04% v/v. Ethanol alone at the above concentration did not effect the contractile properties of rat cardiomyocytes in any of the protocols used.

The effect of 18 : 4n-3 and 18 : 5n-3 on the time course of isoproterenol-induced spontaneous contractile activity in rat cardiomyocytes in comparison with the effect of no fatty acid addition, or the addition of the positive control PUFA, 22 : 6n-3, is shown in Figure 1. When tested at a final concentration of 20, uM, 18 : 4n-3 treatment completely suppressed the development of spontaneous contractile activity, while 18 : 5n-3 nd 22 : 6n-3 were of equal potency in suppressing the development of spontaneous contractile activity. It can also be seen from this figure that no addition of fatty acid results in the rapid development of spontaneous contractile activity over time as a result of the isoproterenol treatment.

When this data is analysed to show the extent of cardiomyocytes spontaneously contracting after 10 minutes, it can be seen from Figure 2 that 18 : 4n-3, 18 : 5n-3 and 22 : 6n-3 are nearly equivalent in their ability to reduce the development of spontaneous contractile activity, and far more effective than no addition of fatty acid. The ability of normally quiescent cardiomyocytes to undergo synchronous contractile activity in response to electrical field stimulation is related to the excitability of the cardiomyocyte and the ease at which it will undergo the depolarisation required for the generation of the excitation-contraction coupling cycle. Figure 3 shows that when no PUFA is added, the normal voltage required to stimulate cells (about 20-25 volts setting on the Grass Stimulator), does not need to be increased in order to elicit a contraction. However, treatment of cardiomyocytes with 20, uM PUFA requires between 10 volts and about 70 volts extra to elicit contractile activity

depending on the particular PUFA added. The greatest increase in voltage is required following treatment with 18 : 5n-3 (Figure 3).

A dose response curve of the spontaneous contractile activity of rat cardiomyocytes to various added fatty acids is shown in Figure 4. At 10, uM final concentration 18 : 4n-3, 18 : 5n- 3 and 22 : 6n-3 all appear equivalent in their efficacy in inhibiting the development of isoproterenol-induced spontaneous contractile activity after 10 minutes. At the lowest concentration tested (2. 5, uM), 22 : 6n-3 appears to be the most effective of the three fatty acids tested. These results are compared to no addition of PUFA in which there was a greater than 80% incidence of spontaneously contracting cardiomyocytes after 10 minutes of isoproterenol treatment.

Example 3-Effect of mixtures of micfoalgal n-3 fatty acids on electrically stimulated asynchrony in rat cardiomyocytes A. Measurement of asynchrony during electrical stimulation.

Adult rat cardiomyocytes on glass coverslips were placed in a custom prepared superfusion chamber and superfused with Tyrode buffer containing 1mM Ca2+ at,.

37°C (designated the zero time point) and then allowed to equilibrate for 2 minutes.

A video camera mounted on an inverted Olympus microscope housed in a perspex chamber, transferred images to a television screen where the cells were viewed and numbered. Contractility was induced by the presence of electrical field stimulation using a Grass S4 stimulator. After 2 minutes of acclimatisation, the cells were stimulated and voltage increased slowly until the majority of the cells being viewed on the TV monitor were beating synchronously. The electrical stimulation was turned off when at least twenty cells had been recorded. At the 4 minute time point, the cells were superfused for a further 4 minutes with 1 mM Ca2+ Tyrode buffer containing the particular test microalgal polyunsaturated fatty acid mix at the desired concentration (20, 10, 5 or 2. 5 uM). At the 8 minute time point, the electrical stimulation was re-applied, and retained for the remainder of the assay. Cells were then superfused with the desired concentration of microalgal PUFA mix in addition to 1 x 10-8 M of the ß-adrenergic receptor agonist, isoproterenol. The voltage was

increased if more than 50% of the cells were no longer beating. At the 9 minute time point, the number of asynchronous cells was recorded. At the 10 minute time point, the cells were superfused with the same fatty acid mix, however, the isoproterenol concentration was increased to 3. 12 x 10-8 M. Asynchronously contracting cells were recorded at the 11 minute time point. This procedure was repeated for concentrations of isoproterenol ranging from 1 x 10-7 M to 3. 12 x 10-6 M (half log concentrations) every 2 minutes (representing the 12, 14, 16 and 18 minute time points). Asynchronously contracting cells were recorded for each concentration of isoproterenol at 2 minute intervals (13, 15, 17, 19 and 20 minute time points). Each microalgal PUFA mix was tested at 20, 10, 5 and 2. 5 uM against a positive control, DHA, also tested at these four concentrations, and the negative control (no fatty acid).

Results Table 3 : Fatty Acid Compositions of Microalgal PUFA mixes.

Cryptomonad sp. 412 PUFA mix 18 : 4n-3 51. 0% 20 : 5n-3 24. 8% 22 : 6n-3 24. 2% Scrippsiella sp. PUFA mix 18 : 5n-3 52. 1% 22 : 6n-3 30. 7% 18 : 4n-3 14. 7% 20 : 5n-3 2. 5% Table 3 shows the fatty acid compositions of the two microalgal PUFA mixtures used to determine effects on isolated rat heart cardiomyocytes with respect to protection against isoproterenol-induced asynchronous contractile activity. In the cryptomonad sp. cs-412, 18 : 4n-3 represented 51% of the total fatty acid present in the extract. For the Scrippsiella sp.

18 : 5n-3 represented 52. 1% of the fatty acids present while 18 : 4n-3 represented 14. 7%.

B. Effect of acute administration of microalgal PUFA mixtures on the extent of asynchronous contractile activity in isolated rat cardiomyocytes.

Figure 5 shows the effect of increasing concentrations of the PUFA mix from the cryptomonad sp. on the development of isoproterenol-induced asynchronous contractile activity in rat cardiomyocytes tested as described in the methods. The cryptomonad sp. PUFA mix was as effective as DHA at the four concentrations tested in inhibiting the development of asynchronous contractions. For the cryptomonad sp. PUFA mix and for DHA maximum protection was achieved at a concentration of 20 uM for each. In contrast, the negative control (BHA-No fatty acid) was without effect.

The Scrippsiella sp, PUFA mix was also tested in the same system as described for cryptomonad sp. (Figure 6) however only concentrations of 10 and 20 IlM were tested. At these two concentrations the PUFA mix from Scrippsiella sp. appeared equal to, or slightly better than the equivalent concentration of DHA. Again the negative control was without effect.

Example 4-Effect of 18 : 4n-3 on ventricular fibrillation in the coronary artery ligated rat.

A. Coronary artery ligation in the whole animal : 12 weeks old male Sprague Dawley rats were maintained on a pro-arrhythmic diet- standard chow supplemented with 12% saturated sheep kidney fat-which has previously been shown to promote vulnerability to cardiac arrhythmia and ventricular fibrillation and allowed to age to 24 weeks.

The animals were fasted overnight and anaesthetised with a single injection of Nembutal (i. p.) and maintained at a suitable depth with subsequent supplementary doses of anaesthetic as required and prepared for the ligation of the left ascending coronary artery.

In brief, animals were intubated and artificially ventilated following the opening of the chest cavity. The right femoral artery and vein were cannulated. The arterial cannula was attached to a pressure transducer for continuous monitoring of blood pressure.

The chest cavity was opened exposing the heart. The pericardium was broken and the heart exteriorised allowing the insertion of the ligature around the left descending coronary artery. The heart was returned to the chest cavity and the animal allowed to stabilise for 15 minutes. The venous cannula was used for the administration of test compounds prior to the induction of occlusion. Fatty acids were administered at a dose of 20 mg/kg-bodyweight using rat serum, standardised to a protein concentration at 0. 05 mg/ml, as the vehicle. The test compounds were infused at a rate of 0. 5 ml/min using an automated syringe pump. Fifteen minutes after the infusion of fatty acids, the ligature was tightened to induce myocardial ischaemia and ECGs were recorded. An ischaemic period of 15 minutes was used before the release of the ligature and monitoring of the animal for'a further'10 minutes. At the conclusion of the experiment, any survivors were killed by exsanguination whilst still under anaesthesia.

From the ECGs the effectiveness of the treatments were assessed by calculating the incidence of'various parameters including ventricular tachycardia (VT) and. ventricular fibrillation (VF) and mortality.

B. Ventricular fibrillation The effects of acute administration of oleic acid (OA : l8 : ln-9), docosahexaenoic acid (DHA : 22 : 6n-3) and 18 : 4n-3 (obtained from Sigma Chemicals) against the % VF incidence following coronary artery ligation (N=11 rats per each fatty acid) as summarised in Figure 7. Oleic acid was used as a negative control while DHA acted as the positive control fatty acid. It is clear that compared to OA both DHA and 18 : 4n-3 were effective in reducing the % VF incidence.

Example 5 1 g of the oil extract from Table 1 in Example 1 is encased in a soft gelatin capsule to provide a pharmaceutical or nutraceutical composition.

Example 6-Synthesis of C18 PUFAs GENERAL EXPERIMENTAL 'H NMR and 13C NMR spectra were recorded on a Bruker AC 200. spectrometer at 200. 13 MHz and 50. 03 MHz respectively, in CDC13 solutions unless stated otherwise. Chemical shifts are given in ppm relative to the solvent (7. 26 ppm,'H ; 77. 0 ppm, 13C). THF was distilled over benzophenone/sodium. All other solvents were AR grade and were used without further purification. Reactions were monitored by TLC using Merck 60F-254 aluminium-backed silica gel plates. TLC plates were visualised by UV and by heating when. stained with 5% phosphomolybdic acid in EtOH. Column chromatography was performed using Merck silica gel, 230-400 mesh.

GENERAL PROCEDURES General Procedure for the LiAlH4Reduction of Esters The ester (10 mmol) in anhydrous Et2O (10 ml) was added dropwise to a suspension of LiAlH4 (10 mmol) in anhydrous Et20 (20 ml), maintained under N2. After the addition was complete, the mixture was allowed'to stir at room temperature for 30 min. The mixture was then carefully quenched with EtOAc and the reaction mixture partitioned between Et2O and H20 (10 ml). The phases were separated and the aqueous phase was extracted with Et20 (2 x 10 ml) and the combined organic extracts were washed with brine (10 ml), dried (MgS04), filtered and concentrated to afford the alcohol.

General Procedure for the Deprotection of Tetrahydropyranyl (THP) Ethers To a solution of the THP ether (10 mmol) in EtOH (15 ml) was added p-TsOH (2 mmol) and the mixture was stirred at room temperature for 2 h. After this time, the solvent was removed under reduced pressure and the residue was partitioned between Et2O (20 ml) and H20 (20 ml). The phases were separated and the aqueous phase was extracted with Et20 (2 x 20 ml). The combined organic extracts were washed with saturated aq. NaHCO3 (20 ml) and brine (20 ml), then dried (MgS04), filtered and concentrated to afford the alcohol.

General Procedure for the Conversion of Alcohols to Iodides To a solution of iodine (45 mmol) in CH2C12 (100 ml), under N2, was added at-15°C a solution of PPh3 (48 mmol) in CHUCK (100 ml). The yellow solution of Ph3PI2 was stirred at-15°C for 15 min and then a mixture of the alcohol (30 mmol) and pyridine (90 mmol) in CH2C12 (100 ml) were added dropwise. The mixture was allowed to warm to room temperature and was stirred overnight. After this time, the solvent was removed under ; reduced pressure and the residue was filtered through a silica plug (10% Et20/pet. spirits elution) [For large scale reactions, the reaction mixture was first filtered through a silica : plug with CH2C12 elution, followed by concentration of the filtrate and filtering the residue obtained through a second silica plug with 10% Et2O/pet. spirits elution.]. The filtrate was concentrated to afford the iodide.

General Procedure for the Conversion of Iodides to Phosphonium Salts A mixture of the iodide (20 mmol), PPh3 (36 mmol) and I (2CO3 (40 mmol) in CH3CN (90 ml) was refluxed for 4 h, maintained under N2. After this time, the solvent was removed and the residue was subjected to column chromatography (1% Et2O/pet. spirits to remove excess PPh3, then 5% MeOH/CH2Cl2). Concentration of the MeOH/CH2Cl2 fractions gave the phosphonium salt.

General Procedure for the Wittis Reaction with KOBut KOBut (10 ml of a 1 M solution in THF, 10 mmol) was added to a 0°C solution of the phosphonium salt (10 mmol) and the aldehyde (5 mmol) in CH2C12 (50 ml), maintained under N2. The mixture was allowed to warm to room temperature and was stirred for a further 1 h. After this time, the solvent was removed under reduced pressure and the residue was filtered through a silica plug (50% Et2O/pet. spirits elution). [For large scale reactions, the reaction mixture was first filtered through a silica plug with CH2Cl2 elution, followed by concentration of the filtrate and filtering the residue obtained through a second silica plug with 50% Et20/pet. spirits elution.] Concentration of the filtrate gave a yellow oil which was subjected to column chromatography (1% Et20/pet. spirits to 5% Et20/pet. spirits) to give the alkene.

General Procedure for the Wittig Reaction with NaN (SiMe » 2 To a suspension of the phosphonium salt (5. 51 mmol) in anhydrous THF (50 ml), at 0°C and under N2, was added sodium bis (trimethylsilylamide) (4. 90 ml of a 1 M solution in THF, 4. 90 mmol). The orange solution of ylide was stirred at room temperature for 1 h. The ylide solution was then cooled to-80°C and the aldehyde (5. 0 mmol) in anhydrous THF (20 ml) was added. The reaction mixture was left to warm to room temperature and was stirred overnight. Saturated aq. NH4C1 (20 ml) and Et20 were added and the phases separated. The aqueous phase was extracted with Et2O (2 x 20 ml) and the combined organic extracts were washed with water (20 ml), dried (MgS04), filtered and concentrated to afford the crude product.

General Procedure for the Hydrolysis of Esters with LiOH The ester (1 mmol) in THF (8 ml) and 0. 5 M'aqueous LiOH (4. 4 ml) was stirred at room temperature for 24 h. After this time, the reaction mixture was acidified with 2 M HC1 and Et2O (10 ml) and H20 (10 ml) were added. The phases were separated and the aqueous phase was extracted with Et2O (2 x 10 ml). The combined organic extracts were washed with brine (10 ml), dried (MgS04), filtered and concentrated to afford the acid.

to Aldehyde The alcohol (100 mmol) in CH2Cl2 (100 ml) was added to a suspension of PCC (54. 6 g, 150 mmol), NaOAc (50 mmol) and molecular sieves in CH2C12 (200 ml). The mixture was stirred at room temperature for 90 min. After this time, the mixture was filtered through a plug of celite and the filtrate was concentrated. The residue was filtered through a silica plug (Et20 elution) and the filtrate concentrated to afford the aldehyde.

A. Synthesis of Ethyl (6Z, 12Z)-6, 12-octadecenoate (i) trans-1, 2-Cyclohexanediol 0. 2 M Aqueous Na2C03 (204 ml) was added to cyclohexene oxide (20. 0 g, 20. 6 ml, 0. 204 mol) and the reaction was stirred at 92-94°C for 3 days. After this time, the mixture was cooled and most of the solvent was removed under reduced pressure. The residue was extracted with Et2O (4 x 50 ml) and then EtOAc (4 x 50 ml). The combined organic extracts were dried (MgSO4), filtered and concentrated to give a brown solid which was recrystallised (EtOH) to afford the diol (14. 4 g, 69%) as a pale-brown solid. 1H NMR 6 3. 34 (m, 2H), 3. 23 (s, 2H), 1. 97 (m, 2H), 1. 70 (m, 2H), 1. 28 (m, 4H).

(ii) 1, 1, 6, 6-Tetraethoxyhexane To a solution of trans-1, 2-cyclohexanediol (10. 0 g, 86. 1 mmol) in EtOH (340 ml), at-10°C, was poured solid HI04. H20 (21. 6 g, 94. 3 mmol). The mixture was stirred for 30 min at- 10°C, then Et2O/pet. spirits (1 : 1, 2 volumes) and water (0. 25 volume) were added. The phases were separated and the aqueous phase was extracted with 1 : 1 Et2O/pet. spirits (2 x 100 ml). The combined organic extracts were washed with H20 (50 ml), 1 M aq. Na2S203 (50 ml) and brine (50 ml) then dried (MgS04), filtered and concentrated. The crude material

was dissolved in pet. spirits (300 ml), then triethylorthoformate (24. 8 ml) and camphor sulfonic acid (20 mg) were added and the mixture was left to stir at room temperature overnight. After this time, solid KOH (290 mg) and H20 (50 ml) were added. The phases were separated and the organic phase was washed with H20 (50 ml) and brine (50 ml) and concentrated to afford a colourless oil. A by-product was distilled off the residue (b. p. 73- 80°C). The residue contained some aldehyde, so it was resubjected to the acetalization conditions to afford the bis-acetal (19. 6 g, 86%) as a colourless oil. 1H NMR 8 4. 48 (t, 2H, J 6 Hz), 3. 78-3. 21 (m, 8H), 1. 63 (m, 4H), 1. 37 (m, 4H), 1. 24 (m, 12H).

(iii) 6, 6-Diethoxyhexanal To a 0. 2 M acetone solution (110 ml) of 1, 1, 6, 6-tetraethoxyhexane (5. 78 g ; 22. 0 mmol) was added at room temperature 0. 02 M aqueous solution of FeCl3 (2. 2 ml, 0. 044 mmol). The mixture was stirred at 40°C and the reaction was monitored by TLC (50% Et2O/pet. spirits as eluent). When the mono-aldehyde spot became darker than the bis-acetal spot, the reaction was cooled to-20°C and diluted with 3 volumes of pet. spirits. Filtration at-20°C of this mixture through a pad of silica gel and concentration of the filtrate gave a pale- yellow oil. Column chromatography (1% Et20/pet. spirits, then 5% Et2O/pet. spirits, then 50% Et2O/pet. spirits) gave the mono-aldehyde (1. 78 g, 43%) [unreacted bis-acetal was also recovered] as a colourless oil, which was used immediately in the next step.

(iv) (3Z, 6Z)-12, 12-Diethoxy-3, 6-dodecadiene KOBut (2. 31 g, 19. 0 mmol) in anhydrous THF (20 ml) was added to a 0°C solution of (Z)-3- hexenyl (triphenyl) phosphonium bromide (8. 08 g, 19. 0 mmol) and 6, 6-diethoxyhexanal (1. 78 g, 9. 5 mmol) in CH2CI2 (150 ml), maintained under N2, and the reaction proceeded as described in the general procedure for Wittig reactions with KOBut to give a colourless oil.

The oil was subjected to column chromatography (1% Et20/pet. spirits to remove impurities,

then 10% Et2O/pet. spirits to recover the product) to give the acetal (1. 38 g, 58%) as a colourless oil.'H NMR 8 5. 34 (m, 4H), 4. 44 (t, 1H, J 6 Hz), 3. 54 (m, 4H), 2. 74 (t, 2H, J 6 Hz), 2. 36 (t, 2H, J 6 Hz), 1. 99 (m, 2H), 1. 27 (m, 6H), 0. 83 (t, 3H, J 8 Hz) ; 13C NMR 8 131. 7, 129. 7, 128. 1, 127. 3, 102. 8, 60. 7, 33. 4, 29. 4, 27. 1, 25. 4, 24. 4, 20. 5, 15. 3, 14. 2.

(v) (6Z, 9Z)-6, 9-Dodecadienal H20 (0. 25 ml, 13. 9 mmol) and 0. 1 M Fecal3 in acetone (1. 39 ml, 0. 15 mmol) were added to a solution of (6Z, 9Z)-1-ethoxy-6, 9-dodecadienyl ethyl ether (700 mg, 2. 77 mmol) in acetone (14 ml) and the resulting mixture was stirred at room temperature and monitored by TLC.

After 40 min, the reaction had gone to completion and the reaction mixture was cooled to- 20°C and diluted with 3 volumes of pet. spirits. Filtration of this mixture through a pad of silica gel at-20°C and concentration of the filtrate gave the aldehyde (482 mg, 97%) as a colourless oil.'H NMR 8 9. 77 (t, 1H, J 2 Hz), 5. 34 (m, 4H), 2. 77 (t, 2H, J 6 Hz), 2. 45 (td, 2H, J 7 & 2 Hz), 2. 07 (m, 4H), 1. 64 (m, 2H), 1. 42 (m, 2H), 0. 97 (t, 3H, J7 Hz).

(vi) (6Z, 9Z)-6, 9-Dodecadien-1-ol A solution of (6Z, 9Z)-6, 9-dodecadienal (580 mg, 3. 22 mmol) in anhydrous THF (10 ml) was added dropwise, at-70°C, to a suspension of LiAlH4 (122 mg, 3. 22 mmol) in anhydrous THF (30 ml), maintained under N2. The mixture was allowed to warm to-20°C and was hydrolyse with H20 (1. 5 ml), then 2 M aq HC1 was added until a pH of 1. The mixture was saturated with solid NaCl, extracted with Et20 (3 x 20 ml), dried (MgSO4), filtered and concentrated to afford the alcohol (402 mg, 68%) as a colourless oil.'H NMR 8 5. 29 (m, 4H), 3. 57 (t, 2H, J7 Hz), 1. 99 (t, 4H, J 6 Hz), 1. 47 (m, 6H), 0. 90 (t, 3H, J7 Hz).

(vii) (3Z, 6Z)-12-Iodo-3, 6-dodecadiene To a solution of iodine (840 mg, 3. 31 mmol) in CH2C12 (10 ml), under N2 was added at -15°C a solution of PPh3 (924 mg, 3. 52 mmol) in CH2C12 (10 ml). The yellow solution of Ph3PI2 was stirred at-15°C for 15 min and then a mixture of (6Z, 9Z)-6, 9-dodecadien-1-ol (402 mg, 2. 21 mmol) and pyridine (0. 51 ml, 6. 62 mmol) in CH2C12 (10 ml) were added dropwise and the reaction proceeded as described in the general procedure for the preparation of iodides. Column chromatography (pet. spirits) gave the iodide (598 mg, 92%) as a colourless oil.'H NMR 8 5. 34 (m, 4H), 3. 18 (t, 2H, J 7 Hz), 2. 76 (m, 2H, J 6 Hz), 2. 06 (t, 4H, J7 Hz), 1. 84 (t, 2H, J7 Hz), 1. 42 (m, 4H), 0. 97 (t, 3H, J7 Hz).

(viii) (6Z, 9Z)-6, 9-Dodecadienyl (triphenyl) phosphonium iodide A mixture of (3Z, 6Z)-12-iodo-3, 6-dodecadiene (598 mg, 2. 04 mmol), PPh3 (960 mg, 3. 67 mmol) and K2CO3 (560 mg, 4. 07 mmol) in CH3CN (15 ml) was reacted as described in the general procedure for the preparation of phosphonium salts to give the phosphonium salt (1. 08 g, 95%) as a yellow gum. lH NMR 8 7. 79 (m, 15H), 5. 30 (m, 4H), 3. 78 (m, 2H), 2. 68 (t, 2H, J 6 Hz), 2. 03 (quint., 4H, J 7 Hz), 1. 65 (br s, 4H), 1. 40 (m, 2H), 0. 92 (t, 3H, J 7 Hz).

(ix) Methyl (Z)-6-dodecenoate KOBut (20. 2 ml of a 1 M solution in THF, 20. 2 mmol) was added to a 0°C solution of (6- methoxy-6-oxohexyl) (triphenyl) phosphonium iodide (10. 5 g, 20. 2 mmol) and hexanal (1. 0 g, 10. 1 mmol) in CH2C12 (150 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a colourless oil. The oil was subjected to column chromatography (1% Et2O/pet. spirits to 5% Et2O/pet. spirits) to give the ester (1. 56 g, 73%) as a colourless oil.'H NMR 8 5. 35 (m, 2H), 3. 66 (s, 3H), 2. 31 (t, 2H, J 8 Hz), 2. 02 (quint., 4H, J 7 Hz), 1. 63 (m, 2H), 1. 34 (m, 8H), 0. 88 (t, 3H,

J7 Hz), 13C NMR 8 174. 2, 130. 5, 129. 1, 51. 4, 34. 0, 31. 5, 29. 4, 29. 2, 27. 2, 26. 8, 24. 6, 22. 6, 14. 1.

(x) (Z)-6-Dodeceno-1-ol Methyl (Z)-6-dodecenoate (0. 75 g, 3. 53 mmol) in anhydrous EtaO (10 ml) was added dropwise to a suspension of LiAlH4 (134 mg, 3. 53 mmol) in anhydrous Et20 (30 ml), maintained under N2. After the addition was complete, the reaction proceeded as described in the general procedure for the reduction of esters to afford the alcohol (525 mg, 81%) as a colourless oil. 1H NMR 8 5. 29 (m, 2H), 3. 59 (t, 2H, J7 Hz), 1. 97 (t, 4H, J6 Hz), 1. 50 (t, 2H, J 7 Hz), 1. 26 (m, 10H), 0. 83 (t, 3H, J 6 Hz).

(xi) (Z)-6-Dodecenal (Z)-6-Dodeceno-1-ol (525 mg, 2. 85 mmol) in CH2C12 (40 ml) was added to a suspension of (; PCC (920 mg, 4. 28 mmol) and molecular sieves in CH2C12 (40 ml). The reaction proceeded as described in the general procedure for the PCC oxidation of alcohols to afford the aldehyde (250 mg, 48%) as a colourless oil.'H NMR 8'9. 69 (t, 1H, J 2 Hz), 5. 28 (m, 2H), 2. 37 (td, 2H, J7 & 2 Hz), 1. 99 (quint., 4H, J7 Hz), 1. 58 (m, 2H), 1. 26 (m, 8H), 0. 84 (t, 3H, J 7 Hz).

(xii) Ethyl 6-hydroxy hexanoate BF3. Et2O (1. 7 ml) was added to a solution ofs-caprolactone (20 g, 0. 175 mol) in EtOH (180 ml) and the resulting solution was stirred at room temperature for 3 days. After this time, the solvent was removed under reduced pressure and the residue was partitioned between Et2O (200 ml) and H20 (100 ml). The phases were separated and the aqueous phase was

extracted with Et20 (2 x 100 ml). The combined organic extracts were washed with brine (50 ml), dried (MgS04), filtered and concentrated to afford the hydroxy ester (27. 1 g, 97%) as a colourless oil. 1H NMR 8 4. 13 (q, 2H, J 7 Hz), 3. 65 (t, 2H, J 7 Hz), 2. 31 (t, 2H, J 7 Hz), 1. 70-1. 35 (m, 6H), 1. 26 (t, 3H, J7 Hz).

(xiii) Ethyl 6-iodo hexanoate To a solution of iodine (31. 7 g, 125 mmol) in CH2C12 (200 ml), under N2, was added at- 15°C a solution of PPh3 (34. 9 g, 133 mmol) in CHzClz (200 ml). The yellow solution of Ph3PI2 was stirred at-15°C for 15 min and then a mixture of ethyl 6-hydroxy hexanoate (14. 5 g, 83. 2 mmol) and pyridine (19. 1 ml, 250 mmol) in CH2C12 (200 ml) were added dropwise and the reaction was allowed to proceed as described in the general procedure for the preparation of iodides to afford the iodide (16. 7 g, 71%) as a yellow oil. IH NMR 8 4. 07 (q, 2H, J 7 Hz), 3. 14 (t, 2H, J 7 Hz), 2. 29 (t, 2H, J 7 Hz), 1. 79 (quint., 2H, J 7 Hz), 1. 60 (quint. 2H, J7 Hz), 1. 40 (m, 2H), 1. 21 (t, 3H, J7 Hz).

(xiv) (6-Ethoxy-6-oxohexyl) (triphenyl) phosphonium iodide A mixture of ethyl 6-iodo hexanoate (16. 7 g, 58. 8 mmol), PPh3 (27. 7 g, 106 mmol) and I (2CO3 (16. 2 g, 117 mmol) in CH3CN (250 ml) was reacted as described in the general procedure for the preparation of phosphonium salts to give the phosphonium salt (24. 4 g, 81 %) as a yellow oil which later solidified. tH NMR 8 7. 77 (m, 15H), 4. 09 (q, 2H, J 7 Hz), 3. 79 (m, 2H), 2. 31 (t, 2H, J7 Hz), 1. 71 (m, 6H), 1. 25 (t, 2H, J7 Hz).

(xv) Ethyl (6Z, 12Z)-6, 12-octadecenoate KOBut (2. 7 ml of a 1 M solution in THF, 2. 7 mmol) was added to a 0°C solution of (6- ethoxy-6-oxohexyl) (triphenyl) phosphonium iodide (1. 46 g, 2. 74 mmol) and (Z)-6-dodecenal

(250 mg, 1. 37 mmol) in CH2Cl2 (30 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a colourless oil. The oil was subjected to column chromatography (1% Et2O/pet. spirits to 5% Et2O/pet. spirits) to give the ester (253 mg, 60%) as a colourless oil. 1H NMR 8 5. 53 (m, 4H), 4. 13 q, 2H, J7 Hz), 2. 30 (t, 2H, J 7 Hz), 2. 03 (m, 8H), 1. 63 (m, 2H), 1. 33 (m, 15H), 0. 89 (t, 3H, J7 Hz) ; 13C NMR 8 173. 7, 130. 2, 130. 0, 129. 6, 129. 2, 60. 1, 34. 2, 31. 5, 29. 4, 29. 3, 29. 2, 27. 2, 27. 0 (8), 27. 0 (3), 26. 8, 24. 6, 22. 6, 14. 2, 14. 1 (one peak superimposed or missing).

B. Synthesis of (6Z, 12Z)-6, 12-Octadecenoic acid Ethyl (6Z, 12Z)-6, 12-octadecenoate (84 mg, 0. 272 mmol) in THF (4 ml) and 0. 5 M aqueous LiOH (1. 1 ml) was stirred at room temperature for 24 h. After this time, the reaction mixture was worked-up as described in the general procedure for the hydrolysis of esters to afford a white solid. The solid was chromatographed (10% Et2O/pet. spirits then 10% MeOH/CHaCIa) and the appropriate fractions concentrated to afford the acid (56 mg, 73%)-. as a white solid NMR 8 5. 36 (m, 4H), 2. 35 (t, 2H, J 7 Hz), 2. 01 (m, 8H), 1. 65 (quint.,.

2H, J7 Hz), 1. 35 (m, 12H), 0. 92 (t, 3H, J7 Hz) ; 13C NMR 8 189. 0, 130. 4, 130. 0, 129. 6, 129. 1, 31. 5, 29. 4 (3), 29. 3 (6), 29. 3 (0), 29. 1, 27. 2, 27. 1, 26. 8, 24. 3, 22. 6, 14. 1 (two peaks superimposed or missing).

C. Synthesis of Ethyl (6Z, 12Z, 15Z)-6, 12, 15-octadecatrienoate KOBu' (600 mg, 5. 35 mmol) in anhydrous THF (10 ml) was added to a 0"C solution of (6- ethoxy-6-oxohexyl) (triphenyl) phosphonium bromide (2. 85 g, 5. 35 mmol) and (6Z, 9Z)-6, 9- dodecadienal (482 mg, 2. 67 mmol) in CH2C12 (50 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a colourless oil. The oil was subjected to column chromatography (1% Et2O/pet. spirits to 5% Et2O/pet. spirits) to give the ester (620 mg, 76%) as a colourless oil.'H NMR 8 5. 35 (m, 6H), 4. 13 (q, 2H, J7 Hz), 2. 78 (t, 2H, J6 Hz), 2. 30 (t, 2H, J7 Hz), 2. 09 (m, 8H), 1. 62 (m, 2H), 1. 39 (m, 4H), 1. 26 (t, 3H, J7 Hz), 0. 97 (t, 3H, J 7 Hz).

D. Synthesis of (6Z, 12Z, 15Z)-6, 12, 15-Octadecatrienoic acid Ethyl (6Z, 12Z, 15Z)-6, 12, 15-octadecatrienoate (200 mg, 0. 653 mmol) in THF (5 ml) and 0. 5 M aqueous LiOH (2. 9 ml) was stirred at room temperature for 24 h. After this time, the reaction mixture was worked-up as described in the general procedure for the hydrolysis of esters to afford a colourless oil. The oil was chromatographed (10% Et2O/pet. spirits to remove high Rf impurities and then 10% MeOH/CH2Cl2) to afford the acid (135 mg, 74%) as a colourless oil. 1H NMR 8 5. 36 (m, 6H), 2. 78 (t, 2H, J 6 Hz), 2. 36 (t, 2H, J 7 Hz), 2. 03 (m, 8H), 1. 66 (m, 2H), 1. 40 (m, 6H), 0. 97 (t, 3H, J8 Hz) ; 13C NMR 8 180. 3, 131. 7, 130. 3, 129. 9, 129. 1, 128. 1, 127. 3, 34. 0, 29. 3, 29. 0, 27. 1, 26. 8, 25. 5, 24. 2, 20. 5, 14. 3 (two peaks superimposed).

E. Synthesis of Ethyl (6Z, 9Z, 12Z, 15Z) 6, 9, 12, 15-octadecatetraenoate (i) (3, 3-Diethoxypropyl) (triphenyl) phosphonium bromide To a vigorously stirred mixture of triphenyl phosphine (65. 5 g, 0. 25 mol), HBr (48%, 35. 1 ml, 0. 3 mol), CH2C12 (50 ml) and 2-propanol (50 ml), was slowly added, at-10°C, a solution of acrolein (18. 4 ml, 0. 28 mol) in CH2C12 (300 ml). After completion of the reaction at room temperature (checked by TLC), excess water was removed by decantation.

Triethyl orthoformate (167 ml, 1 mol) was then added at 0°C. When acetalization was complete (ca. 30 min), excess acid was neutralized by the addition of Et3N (13. 5 ml, 0. 1 mol). The mixture was concentrated under reduced pressure and then pet. spirits (300 ml) was added. The mixture was stirred at room temperature for 30 min and then the pet. spirits layer was decanted off the residue. The sticky residue was transferred into a beaker was and

dried under vacuum in the presence of P205 for 48 h to afford the phosphonium salt (91. 7 g, 77%) as a white solid NMR 8 7. 67 (m, 15H), 4. 91 (t, 1H, J 5 Hz), 3. 61 (m, 6H), 1. 87 (m, 2H), 1. 09 (t, 6H, J 7 Hz).

(ii) Ethyl 6-oxohexanoate

Ethyl 6-hydroxy hexanoate (27. 1 g,. 169 mol) in CH2C12 (200 ml) was added to a suspension of PCC (54. 6 g, 0. 261 mol), NaOAc (6. 66 g, 81 mmol) and molecular sieves in CH2C12 (400 ml) and the reaction proceeded as described in the general procedure for the PCC oxidation of alcohols to afford the aldehyde (21. 4 g, 80%) as a yellow oil.'H NMR 8 9. 76 (t, 1H, J2 Hz), 4. 13 (q, 2H, J7 Hz), 2. 39 (m, 4H), 1. 66 (m, 4H), 1. 25 (t, 3H, J7 Hz).

(iii) Ethyl (Z)-9, 9-diethoxy-6-nonenoate

KOBut (14. 1 g, 126 mmol) in anhydrous THF (130 ml) was added to a 0°C solution of (3, 3- diethoxypropyl) (triphenyl) phosphonium bromide (59. 7 g, 126 mmol) and the ethyl 6- oxohexanoate (9. 98 g, 63. 1 mmol) in CHaCIa (300 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a pale-yellow oil which was subjected to column chromatography (1% Et20/pet. spirits to 10% Et2O/pet. spirits) to give the acetal (6. 86 g, 40%) as a colourless oil. 1H NMR 8 5. 43 (m, 2H), 4. 48 (t, 1H, J6 Hz), 4. 13 (q, 3H, J7 Hz), 3. 58 (m, 4H), 2. 38 (t, 2H, J6 Hz), 2. 30 (t, 2H, J 7 Hz), 2. 07 (br q, 2H, J 7 Hz), 1. 62 (m, 2H), 1. 40 (m, 2H), 1. 25 (t, 3H, J 7 Hz), 1. 21 (t, 3H, J 7 Hz) ; 13C NMR 8 173. 3, 131. 4, 124. 1, 102. 4, 61. 0, 59. 9, 34. 0, 31. 9, 28. 8, 26. 9, 24. 4, 15. 1, 14. 1.

(iv) Ethyl (6Z, 12Z)-12, 12-diethoxy-6, 9-dodecadienoate

To a 0. 2 M acetone solution (78 ml) of ethyl (Z)-9, 9-diethoxy-6-nonenoate (4. 21 g, 15. 5 mmol) was added at room temperature H20 (1. 4 ml, 77 mmol) and a 0. 1 M solution of FeCl3 in acetone (7. 7 ml, 0. 85 mmol). The mixture was stirred at 40°C and the reaction monitored by TLC until completion (about 20 min). Then, the mixture was cooled to-20°C and then diluted with 3 volumes of pet. spirits. Filtration at-20°C of this mixture over a pad of silica gel and concentration of the filtrate gave the aldehyde as a colourless oil which was used immediately in the next step.

KOBut (31 ml of a 1 M solution in THF, 31 mmol) was added to a 0°C solution of the phosphonium salt (14. 7 g, 31 mmol) and the aldehyde (15. 5 mmol) in CHzCLz (180 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a yellow oil. The oil was subjected to column chromatography (1% Et20/pet. spirits to 5% Et20/pet. spirits) to give the acetal (892 mg, 29%) as a colourless oil. 1H NMR b 5. 40 (m, 4H), 4. 50 (t, 1H, J 6 Hz), 4. 14 (q, 2H, J 7 Hz), 3. 61 (m, 4H), 2. 79 (t, 2H, J 6 Hz), 2. 41 (t, 2H, J 6 Hz), 2. 30 (t, 2H, J 7 Hz), 2. 08 (m, 2H), 1. 65 (m, 2H), 1. 41 (m, 2H), 1. 26 (t, 3H, J 7 Hz), 1. 21 (t, 6H, J 7 Hz). 13C NMR 8 173. 3, 130. 1, 129. 5, 127. 9, 124. 0, 102. 3, 61. 0, 59. 9, 34. 0, 31. 9, 28. 9, 26. 7, 25. 7, 24. 4, 15. 1, 14. 1.

(v) (Z)-3-Hexenyl (triphenyl) phosphonium bromide To a solution of (Z)-3-hexenol (4. 58 g, 45 mmol) and pyridine (5. 8 ml, 59 mmol) in CH3CN (90 ml) was added, at 0°C in several portions, Ph3PBr2 (25. 0 g, 59 mmol). The reaction was checked by TLC (disappearance of alcohol). The reaction mixture was added to 50% Et20/pentane (400 ml), and then filtered through a short pad of silica gel. Slow distillation of solvents gave the crude 1-bromo-3-hexene, which was diluted with CH3CN (60 ml).

Ph3P (12. 2 g, 90 mmol) was added and the mixture was refluxed under N2 for 24 h. After concentration, the crude material was chromatographed (50% Et20/pet. spirits, Et2O, 5%

MeOH/CH2Cl2, Rf=0. 2 in 5% MeOH/CH2Cl2) to give the phosphonium salt (15. 5 g, 81%) as a sticky solid which later solidified. 1H NMR 8 7. 74 (m, 15H), 5. 42 (m, 2H), 3. 82 (m, 2H), 2. 43 (m, 2H), 1. 78 (br quint., 2H, J 8 Hz), 0. 83 (t, 3H, J 8 Hz).

(vi) Ethyl (6Z, 9Z, 12Z, 15Z) 6, 9, 12, 15-octadecatetraenoate To a 0. 2 M acetone solution (14 ml) of ethyl (6Z, 12Z)-12, 12-diethoxy-6, 9-dodecadienoate (892 mg, 2. 85 mmol) was added at room temperature H20 (0. 26 ml, 14. 3 mmol) and a 0. 1 M solution of FeCl3 in acetone (1. 43 ml, 0. 16 mmol). The mixture was stirred at 40°C and the reaction monitored by TLC until completion (about 20 min). Then the mixture was cooled to-20°C and then diluted with 3 volumes of pet. spirits. Filtration at-20°C of this mixture over a pad of silica gel and concentration of the filtrate gave the aldehyde as a colourless oil which was used immediately in the next step.

KOBut (5. 7 ml of a 1 M solution in THF, 5. 7 mmol) was added to a 0°C solution of (Z)-3- hexenyl (triphenyl) phosphonium bromide (2. 69 g, 5. 7 mmol) and the aldehyde (2. 85 mmol) in CH2C12 (50 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOB* to give the ester (333 mg, 38%) as a pale yellow oil. 1H NMR 8 5. 39 (m, 8H), 4. 14 (q, 2H, J7 Hz), 2. 82 (br t, 6H, J 5 Hz), 2. 31 (t, 2H, J7 Hz), 2. 09 (m, 4H), 1. 65 (m, 2H), 1. 41 (m, 2H), 1. 26 (t, 3H, J7 Hz), 0. 98 (t, 3H, J 7 Hz) ; 13C NMR 8 173. 5, 129. 6, 128. 4, 128. 2, 128. 0, 127. 8, 127. 0, 60. 0, 34. 1, 30. 3, 28. 0, 26. 8, 25. 6, 24. 5, 20. 5, 14. 2 (four peaks superimposed).

F. Synthesis of (6Z, 9Z, 12Z, 15Z) 6, 9, 12, 15-Octadecatetraenoic acid Ethyl (6Z, 9Z, 12Z, 15Z) 6, 9, 12, i5-octadecatetraenoate (1. 0 g, 3. 28 mmol) in THF (20 ml) and 0. 5 M aqueous LiOH (14. 4 ml) was stirred for 24 h at room temperature. After this time, the reaction was worked-up as described in the general procedure for the hydrolysis of esters to afford a colourless oil. The oil was chromatographed (10% Et2O/pet. spirits to

remove high Rf impurities and then 10% MeOH/CH2Cl2) to afford the acid (605 mg, 67%) as a colourless oil. 1H NMR 8 5. 38 (m, 8H), 2. 82 (br t, 6H, J 5 Hz), 2. 37 (t, 2H, J 7 Hz), 2. 09 (m, 2H), 1. 67 (m, 2H), 1. 45 (m, 2H), 0. 94 (t, 3H, J7 Hz) ; 13C NMR 8 180. 5, 131. 9, 129. 5, 128. 6, 128. 5, 128. 2, 128. 1, 127. 9, 127. 0, 34. 0, 28. 9, 26. 8, 25. 6, 25. 5, 24. 4, 24. 2, 20. 5, 14. 2 (one peak superimposed or missing).

G. Synthesis of Ethyl (6Z, 9Z, 12Z, 15Z)-17-methyl-6, 9, 12, 15-octadecatetraenoate (i) (Z)-1, 1-Diethoxy-5-methyl-3-hexene KOBut (86. 3 ml of a 1 M solution in THF, 86. 3 mmol) was added to a 0°C solution of (3, 3- diethoxypropyl) (triphenyl) phosphonium bromide (40. 84 g, 86. 3 mmol) and isobutyraldehyde (3. 11 g, 3. 92 ml, 43. 1 mmol) in CH2C12 (300 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a colourless oil which was distilled (Kugelrohr, 300°C @ atm. pressure) to afford the ether (7. 85 g, 98%) as a colourless oil.'H NMR 8 5. 28 (m, 2H), 4. 48 (t, 1H, J 6 Hz), 3. 61 (m, 4H), 2. 60 (m, 1H), 2. 39 (t, 2H, J 6 Hz), 1. 21 (t, 6 H, J7 Hz), 0. 95 (d, 6H, J7 Hz).

(ii) (Z)-5-Methyl-3-hexenal To a 0. 2 M acetone solution (237 ml) of (Z)-l, 1-diethoxy-5-methyl-3-hexene (8. 74 g, 46. 9 mmol) was added at room temperature H20 (4. 2 ml, 231 mmol) and a 0. 1 M solution of FeCl3 in acetone (23. 5 ml, 2. 58 mmol). The mixture was stirred at 40°C and the reaction monitored by TLC until completion (about 40 min). Then the mixture was cooled to-20°C and then diluted with 3 volumes of pet. spirits. Filtration at-20°C of this mixture over a pad of silica gel and concentration of the filtrate gave the aldehyde (4. 95 g, 94%) as a colourless oil which was used immediately in the next step.

(iii) (Z)-1-Iodo-5-methyl-3-hexene (Z)-5-Methyl-3-hexenal (4. 95 g, 44. 1 mmol) in EtOH (80 ml) was added to a-70°C solution of NaBH4 (1. 68 g, 44. 1 mmol) in EtOH (80 ml). The mixture was warmed to room temperature and stirred for 1 h. After this time, 2 M HC1 was added to neutralise the excess NaBH4. Silica gel was added to the mixture and concentration under reduced pressure gave a white powder. The supported substrate was introduced onto the top of a silica gel plug and eluted with Et2O. Concentration of the filtrate gave the alcohol (3. 45 g, 69%) as a colourless oil which was used immediately in the next step.

To a solution of iodine (11. 5 g, 45. 4 mmol) in CH2Cl2 (100 ml), under N2, was added at- 15°C a solution of PPh3 (12. 7 g, 48. 3 mmol) in CH2C12 (100 ml). The yellow solution of Ph3PI2 was stirred at-15°C for 15 min and then a mixture of the alcohol (3. 45 g, 30. 2 mmol) and pyridine (6. 9 ml, 90. 6 mmol) in CH2C12 (100 ml) were added dropwise and the reaction proceeded as described in the general procedure for the preparation of iodides to afford the iodide (3. 96 g, 59%) as a colourless oil. 1H NMR 8 5. 29 (m, 2H), 3. 14 (t, 2H, J 7 Hz), 2. 60 (m, 3H), 0. 97 (d, 6H, J7 Hz).

(iv) [ (Z)-5-Methyl-3-hexenyl] (triphenyl) phosphonium iodide A mixture of (Z)-1-iodo-5-methyl-3-hexene (3. 96 g, 17. 7 mmol), PPh3 (8. 32 g, 31. 8 mmol) and K2CO3 (4. 87 g, 35. 3 mmol) in CH3CN (80 ml) was reacted as described in the general procedure for the preparation of phosphonium salts to give the phosphonium salt (4. 37 g, 51 %) as a brown oil which later solidified.'H NMR 8 7. 74 (m, 15H), 5. 25 (m, 2H), 3. 60 (m, 2H), 2. 39 (m, 2H), 2. 15 (m, 1H), 0. 77 (d, 6H, J 7 Hz).

(v) Ethyl (6Z, 9Z, 12Z, 15Z)-17-methyl-6, 9, 12, 15-octadecatetraenoate To a 0. 2 M acetone solution (15 ml) of ethyl (6Z, 12Z)-12, 12-diethoxy-6, 9-dodecadienoate (938 mg, 3. 00 mmol) was added at room temperature H20 (0. 27 ml, 15. 0 mmol) and a 0. 1 M solution of FeCl3 in acetone (1. 50 ml, 0. 17 mmol). The mixture was stirred at 40°C and the reaction monitored by TLC until completion (about 20 min). The mixture was then cooled to -20°C and then diluted with 3 volumes of pet. spirits. Filtration. at-20°C of this mixture over a pad of silica gel and concentration of the filtrate gave the aldehyde as a colourless oil which was used immediately in the next step.

KOBut (6. 0 ml of a 1 M solution in THF, 6. 0 mmol) was added to a 0°C solution of [ (Z)-5- methyl-3-hexenyl] (triphenyl) phosphonium iodide (2. 92 g, 6. 00 mmol) and the aldehyde (3. 00 mmol) in CHzClz (50 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a yellow oil.

The oil was subjected to column chromatography (1% Et2O/pet. spirits to 5% Et20/pet. spirits) to give the ester (264 mg, 28%) as a pale yellow oil.'H NMR 8 5. 40 (m, 6H), 5. 23 (m, 2H), 4. 14 (q, 2H, J 7 Hz), 2. 82 (m, 6H), 2. 62 (m, 1H), 2. 31 (t, 2H, J7 Hz), 2. 11 (m, 2H), 1. 64 (m, 2H), 1. 44 (m, 2H), 1. 26 (t, 3H, J 7 Hz), 0. 97 (d, 6H, J 7 Hz) ; 13C NMR 8 173. 6, 137. 9, 129. 7, 128. 6, 128. 3, 128. 0, 127. 8, 125. 2, 60. 1, 34. 2, 29. 1, 26. 8, 26. 5, 25. 6, 24. 6, 23. 1, 14. 2 (four peaks superimposed or missing).

H. Synthesis of Ethyl (6Z, 9Z, 12Z, 15Z)-17-methyl-6, 9, 12, 15-octadecatetraenoate Ethyl (6Z, 9Z, 12Z, 15Z)-17-methyl-6, 9, 12, 15-octadecatetraenoate (42 mg, 0. 132 mmol) in THF (4 ml) and 0. 5 M aqueous LiOH (0. 58 ml) was reacted as described in the general procedure for the hydrolysis of esters to afford a pale-yellow oil. The oil was chromatographed (10% Et20/pet. spirits to remove high Rf impurities and then 50% Et2O/pet. spirits) to afford the acid (26 mg, 68%) as a pale-yellow oil. 1H NMR 8 5. 38 (m,

6H), 5. 23 (m, 2H), 2. 84 (m, 6H), 2. 64 (m, 1H), 2. 37 (t, 2H, J7 Hz), 2. 10 (m, 2H), 1. 65 (m, 2H), 1. 44 (m, 2H), 0. 96 (d, 6H, J7 Hz).

I. Synthesis of Methyl (4Z, 8Z, 12Z, 16Z)-4, 8, 12, 16-octadecatetraenoate (i) Methyl (Z)-4-hexenoate KOBut (72 ml of a 1 M solution in THF, 72 mmol) was added to a 0°C solution of (4- methoxy-4-oxobutyl) (triphenyl) phosphonium bromide (31. 95 g, 72. 1 mmol) and acetaldehyde (1. 59 g, 2. 0 ml, 36. 0 mmol) in CH2C12 (300 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig raction with KOBut to give a colourless oil, which was distilled (Kugelrohr, 200°C @ ca. 20 mm Hg) to give the ester (2. 38 g, 52%) as a colourless oil. 1H NMR 8 5. 44 (m, 2H), 3. 68 (s, 3H), 2. 37 (m, 4H), 1. 64 (d, 3H, J7 Hz).

(ii) (Z)-4-Hexen-1-ol Methyl (Z)-4-hexenoate (2. 38 g, 18. 6 mmol) in anhydrous Et20 (10 ml) was added dropwise to a suspension of LiAlH4 (705 mg, 18. 6 mmol) in anhydrous Et2O (60 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the reduction of esters to afford the alcohol (1. 49 g, 80%) as a colourless oil.

(iii) (Z)-4-Hexenal (Z)-4-Hexen-1-ol (1. 49 g, 14. 9 mmol) in CH2CI2 (20 ml) was added in one portion to a suspension of PCC (4. 81 g, 22. 3 mmol), NaOAc (610 mg, 7. 45 mmol) and molecular sieves in CH2C12 (80 ml). The resulting mixture was stirred at room temperature for 1 h. After this time, the mixture was filtered through a silica plug (CH2Cl2 elution). The filtrate was carefully concentrated until the volume of solvent in the flask was ca. 200 ml. The aldehyde in ca. 200 ml of CH2C12 was used immediately in the next reaction.

(iv) Methyl (4Z, 8Z)-4, 8-decadienoate KOBut (23. 8 ml of a 1 M solution in THF, 23. 8 mmol) was added to a 0°C solution of (4- methoxy-4-oxobutyl) (triphenyl) phosphonium bromide (10. 6 g, 23. 8 mmol) and (Z)-4- hexenal (11. 9 mmol-based on an estimated 80% yield in the previous reaction) in CH2C12 (200 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a yellow oil which was subjected to column chromatography (1% Et20/pet. spirits to 5% Et20/pet. spirits) to give the ester (984 mg, 36%) as a colourless oil. 1H NMR 8 5. 40 (m, 4H), 3. 67 (s, 3H), 2. 36 (m, 4H), 2. 10 (m, 4H), 1. 61 (d, 3H, J5 Hz), 13C NMR 6 173. 4, 130. 7, 129. 8, 127. 7, 124. 1, 51. 3, 34. 0, 27. 0, 26. 7, 22. 7, 12. 7.

(v) (4Z, 8Z)-4, 8-Decadien-1-ol Methyl (4Z, 8Z)-4, 8-decadienoate (984 mg, 5. 40 mmol) in anhydrous Et2O (10 ml) was added dropwise to a suspension of LiAlH4 (205 mg, 5. 40 mmol) in anhydrous Et2O (20 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the reduction of esters to afford the alcohol (590 mg, 71%) as a colourless oil. 1H NMR 8 5. 41 (m, 4H), 3. 66 (t, 2H, J7 Hz), 2. 14 (m, 6H), 1. 68 (t, 2H, J7 Hz), 1. 62 (d, 3H, J5 Hz).

(vi) (2Z, 6Z)-10-Iodo-2, 6-decadiene To a solution of iodine (1. 46 g, 3. 82 mmol) in CH2C12 (15 ml), under N2, was added at -15°C a solution of PPh3 (1. 60 g, 6. 12 mmol) in CH2C12 (15 ml). The yellow solution of Ph3PI2 was stirred at-15°C for 15 min and then a mixture of (4Z, 8Z)-4, 8-decadien-1-ol (590 mg, 3. 82 mmol) and pyridine (0. 88 ml, 11. 5 mmol) in CH2C12 (15 ml) were added dropwise and the reaction proceeded as described in the general procedure for the preparation of

iodides to afford the iodide (1. 16 g, quant.) as a colourless oil. 1H NMR 8 5. 41 (m, 4H), 3. 20 (t, 3H, J7 Hz), 2. 15 (m, 8H), 1. 62 (d, 3H, J6 Hz).

(vii) (4Z, 8Z)-4, 8-Decadienyl (triphenyl) phosphonium iodide A mixture of (2Z, 6Z)-10-iodo-2, 6-decadiene (1. 16 g, 3. 82 mmol), PPh3 (1. 80 g, 6. 87 mmol) and K2CO3 (1. 05 g, 7. 63 mmol) in CH3CN (20 ml) was reacted as described in the general procedure for the preparation of phosphonium salts to give the phosphonium salt (1. 74 g, 87%) as a colourless oil which later solidified. lH NMR 8 7. 75 (m, 15H), 5. 37 (m, 4H), 3. 70 (m, 2H), 2. 42 (m, 2H), 2. 04 (m, 6H), 1. 56 (d, 3H, J5 Hz).

(viii) 4-(Benzyloxy)-1-butanol 1, 4-Butanediol (90. 2 g, 1. 0 mol) and KOH (85%, 26. 4 g, 0. 4 mol) were stirred at room temperature overnight. The mixture was then heated to 130°C and vacuum applied to remove water. The mixture was then cooled to 90°C and benzyl chloride (46. 5 ml, 0. 40 mol) was added dropwise. The mixture was stirred at 90°C for 1 h, then heated to 130°C and stirred for 1 h. The mixture was allowed to cool to room temperature overnight.

CH2C12 (200 ml) and H20 (200 ml) were added and the phases separated. The aqueous phase was extracted with CH2C12 (2 x 50 ml) and the combined organic extracts were washed with water (50 ml), dried (MgS04), filtered and concentrated to afford a yellow oil, which was distilled (b. p. 160°C @ 20 mbar) to give the mono-protected diol (46. 1 g, 64%) as a colourless oil.'H NMR 8 7. 34 (m, 5H), 4. 53 (s, 2H), 3. 65 (t, 2H, J 6 Hz), 3. 53 (t, 3H, J 6 Hz), 2. 26 (s, 1H), 1. 71 (m, 4H). 13C NMR 8 138. 2, 128. 3, 127. 61, 127. 55, 72. 8, 70. 2, 62. 1, 29. 6, 26. 3.

(ix) 4- Benzyloxy) butanal 4- (Benzyloxy)-l-butanol (10. 0 g, 55. 5 mmol) in CH2Cl2 (30 ml) was added in one portion to a suspension of PCC (17. 9 g, 83. 2 mmol) and molecular sieves in CH2Cl2 (120 ml) and the reaction proceeded as described in the general procedure for the PCC oxidation of alcohols to give the aldehyde (8. 78 g, 89%) as a yellow oil.'H NMR 8 9. 97 (t, 1H, J 2 Hz), 7. 33 (m, 5H), 4. 49 (s, 2H), 3. 52 (t, 2H, J 6 Hz), 2. 56 (td, 2H, J7 & 2 Hz), 1. 96 (quint, 2H, J 6 Hz).

(x) tert-Butyl (Z)-8- (benzyloxy)-4-octenoate KOBut (24. 1 ml of a 1 M solution in THF, 24. 1 mmol) was added to a 0°C solution (4- methoxy-4-oxobutyl) (triphenyl) phosphonium bromide (10. 7 g, 24. 1 mmol) and 4- (benzyloxy) butanal (2. 15 g, 12. 1 mmol) in CH2C12 (100 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a yellow oil. The oil was subjected to column chromatography (1% Et2O/pet. spirits <BR> <BR> <BR> to 10% Et2O/pet. spirits) to give the tert-Bu ester (1. 39 g, 38%) as a colourless oil. 1H NMR 8 7. 33 (m, 5H), 5. 36 (m, 2H), 4. 50 (s, 2H), 3. 48 (t, 2H, J 7 Hz), 2. 23 (m, 6H), 1. 68 (quint, 2H, J7 Hz), 1. 44 (s, 9H) ; 13C NMR 8 172. 5, 138. 6, 130. 3, 128. 3, 127. 6, 127. 5, 80. 0, 72. 9, 69. 7, 35. 5, 29. 6, 28. 1, 23. 8, 22. 9 (two peaks superimposed or missing).

(xi) (Z)-8-Hydroxy-4-octenoic acid BC13 (23. 0 ml of a 1 M solution in CH2C12, 23. 0 mmol) was added to a-78°C solution of tert-butyl (Z)-8- (benzyloxy)-4-octenoate (1. 40 g, 3. 84 mmol) in CH2C12 (70 ml), maintained under a N2 atmosphere. The mixture was stirred at-78°C for 90 min. The reaction mixture was then allowed to warm to room temperature and was stirred for a further 30 min. The mixture was then cooled to-78°C and quenched with MeOH. The mixture was concentrated, and MeOH added and evaporated several times to remove the trimethyl

borate. The mixture was then filtered through a silica plug (Et2O elution). The filtrate was concentrated to afford a colourless oil which was chromatographed (50% Et2O/pet. spirits) to afford the hydroxy acid (760 mg, quant.) as a colourless oil.'H NMR 8 5. 40 (m, 2H), 3. 65 (t, 2H, J6 Hz), 2. 37 (m, 4H), 2. 17 (m, 2H), 1. 61 (m, 2H).

(xii) Methyl (Z)-8-hydroxy-4-octenoate Concentrated H2S04 (0. 5 ml) was added to a solution of (Z)-8-hydroxy-4-octenoic acid (3. 84 mmol) in MeOH (30 ml) the resulting mixture was refluxed overnight. After this time, the mixture was cooled and the solvent was removed under reduced pressure. The residue was partitioned between Et20 (20 ml) and H20 (20 ml) and the phases separated. The aqueous phase was extracted with Et2O (2 x 20 ml) and the combined organic extracts were washed with water (20 ml), dried (MgSO4), filtered and concentrated to afford the methyl ester (420 mg, 64%) as a colourless oil. 1H NMR 8 5. 41 (m, 2H), 3. 67 (s, 3H), 3. 65 (t, 2H, J 6 Hz), 2. 36 (m, 4H), 2. 17 (m, 2H), 1. 64 (m, 2H).

(xiii) Methyl (Z)-8-oxo-4-octenoate Methyl (Z)-8-hydroxy-4-octenoate (420 mg, 2. 43 mmol) in CHsClz (10 ml) was added in one portion to a suspension of PCC (790 mg, 3. 66 mmol), NaOAc (100 mg, 1. 22 mmol) and molecular sieves in CH2C12 (20 ml) and the reaction proceeded as described in the general procedure for the PCC oxidation of alcohols to afford the aldehyde (240 mg, 58%) as a pale yellow oil. 1H NMR 8 9. 78 (br t, 1H), 5. 40 (m, 2H), 3. 68 (s, 3H), 2. 46 (m, 8H).

(xiv) Methyl (4Z, 8Z, 12Z, 16Z)-4, 8, 12, 16-octadecatetraenoate KOBut (2. 61 ml of a 1 M solution in THF, 2. 61 mmol) was added to a 0°C solution of (4Z, 8Z)-4, 8-decadienyl (triphenyl) phosphonium iodide (1. 37 g, 2. 61 mmol) and methyl (Z)- 8-oxo-4-octenoate (240 mg, 1. 30 mmol) in CHUCK (40 ml), maintained under N2, and the

reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a yellow oil. The oil was subjected to column chromatography (1% Et2O/pet. spirits to 10% Et20/pet. spirits) to give the ester (20 mg, 5%) as a colourless oil. IH NMR 8 5. 42 (m, 8H), 3. 68 (s, 3H), 2. 37 (m, 6H), 221 (m, 10H), 1. 61 (d, 3H, J 6 Hz).

J. Synthesis of (4Z, 8Z, 12Z, 16Z)-4, 8, 12, 16-Octadecatetraenoic acid Methyl (4Z, 8Z, 12Z, 16Z)-4, 8, 12, 16-octadecatetraenoate (16 mg, 0. 053 mmol) in THF (4 ml) and 0. 5 M aqueous LiOH (0. 23 ml) was reacted as described in the general procedure for the hydrolysis of esters to afford a pale-yellow oil. The oil was chromatographed (10% Et2O/pet. spirits to remove high Rf impurities and then 50% Et20/pet. spirits) to afford the acid (7 mg, 47%) as a pale-yellow oil. 1H NMR 8 5. 38 (m, 8H), 2. 40 (m, 4H), 2. 09 (m, 12H), 1. 61 (d, 3H, J5 Hz).

K. Ethyl (6z, 12, Z, 15Z)-9-methyl-6, 12, 15-octadecatrienoate (i) 5-Methyl-2-oxepanone A solution of mCPBA (117. 2 g, 0. 476 mol) and 4-methyl cyclohexanone (35. 8 g, 39. 2 ml, 0. 319 mol) in CH2C12 (800 ml) was stored under N2 in the dark at room temperature for 90 h. The precipitated 3-CIPhCOZH was filtered and the filtrate was transferred to a large beaker and saturated aq. Na2C03 was added [CAUTION : vigorous reaction]. The mixture was then transferred to a separating funnel and the phases separated. The organic phase was washed with saturated aq. NaHCO3 and brine, then dried (MgS04), filtered and concentrated to afford the lactone as a colourless oil (49 g, quant.). 1H NMR 8 4. 22 (m, 2H), 2. 62 (m, 2H), 1. 84 (m, 3H), 1. 43 (m, 2H), 0. 99 (d, 3H, J6 Hz).

(ii) Ethyl 6-hydroxy-3-methylhexanoate BF3. Et2O (4. 1 ml) was added to a solution of 5-methyl-2-oxepanone (49 g, 0. 43 mol) in EtOH (450 ml) and the resulting solution was stirred at room temperature for 48 h. After this time, the solvent was removed under reduced pressure and the residue was partitioned between Et20 (200 ml) and H20 (100 ml). The phases were separated and the aqueous phase was extracted with Et20 (2 x 100 ml). The combined organic extracts were washed with brine (50 ml), dried (MgS04), filtered and concentrated to afford the hydroxy ester (28. 9 g, 39%) as a colourless oil.'H NMR 8 4. 12 (q, 2H, J 7 Hz), 3. 70 (m, 2H), 2. 34 (m, 2H), 1. 57 (m, 5H), 1. 25 (t, 3H, J7 Hz), 0. 92 (d, 3H, J 6 Hz) ; 13C NMR 8 174. 1, 60. 4, 60. 3, 39. 3, 31. 9, 31. 6, 29. 0, 19. 2, 14. 1.

(iii) Ethyl 3-methyl-6-oxohexanoate Ethyl 6-hydroxy-3-methylhexanoate (5. 0 g, 29. 0 mmol) in CH2C12 (20 ml) was added in one portion to a suspension of PCC (9. 39 g, 43. 5 mmol), NaOAc (1. 19 g, 14. 5 mmol) and molecular sieves in CH2C12 (130 ml) and the reaction proceeded as described in the general procedure for the PCC oxidation of alcohols to afford the aldehyde (4. 18 g, 84%) as a colourless oil. 1H NMR 8 9. 76 (t, 1H, J2 Hz), 4. 13 (q, 2H, J7 Hz), 2. 35 (m, 2H), 2. 10 (m, 1H), 1. 61 (m, 2H), 1. 25 (t, 3H, J 7 Hz), 0. 98 (d, 3H, J 7 Hz).

(iv) Ethyl (6Z, 9Z)-3-methyl-6, 9-dodecadienoate KOBu' (23. 2 ml of a 1 M solution in THF, 23. 2 mmol) was added to a 0°C solution of (Z)-3- hexenyl (triphenyl) phosphonium iodide (11. 0 g, 23. 2 mmol) and ethyl 3-methyl-6- oxohexanoate (2. 0 g, 11. 6 mmol) in CH2C12 (120 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a yellow oil. The oil was subjected to column chromatography (1% Et2O/pet. spirits to 5%

Et2O/pet. spirits) to give the ester (1. 82 g, 66%) as a pale-yellow oil.'H NMR 8 5. 37 (m, 4H), 4. 12 (q, 2H, J 7 Hz), 2. 76 (t, 2H, J 6 Hz), 2. 32 (m, 2H), 2. 04 (m, 2H), 1. 68 (m, 2H), 1. 48 (m, 2H), 1. 25 (t, 3H, J 7 Hz), 0. 97 (t, 3H, J 7 Hz), 0. 90 (d, 3H, J 6 Hz) ; 13C NMR 8 173. 9, 131. 8, 129. 2, 127. 9, 127. 1, 60. 1, 34. 1, 33. 0, 32. 2, 31. 5, 25. 6, 20. 5, 19. 1, 14. 0 (one peak superimposed or missing).

(v) (6Z, 9Z)-3-methyl-6, 9-dodecadieno-1-ol Ethyl (6Z, 9Z)-3-methyl-6, 9-dodecadienoate (1. 82 g, 7. 67 mmol) in anhydrous Et20 (20 ml) was added dropwise to a suspension of LiAlH4 (290 mg, 7. 64 mmol) in anhydrous Et2O (40 ml) under N2 and the reaction proceeded as described in the general procedure for the reduction of esters to afford the alcohol (1. 07 g, 71%) as a pale-yellow oil. 1H NMR 8 5. 37 (m, 4H), 3. 64 (t, 2H, J 7 Hz), 2. 04 (m, 4H), 1. 51 (m, 2H), 1. 20 (m, IH), 0. 97 (t, 3H, J 7 Hz), 0. 90 (d, 3H, J 7 Hz) ; 13C NMR 8 131. 8, 128. 9, 128. 4, 127. 2, 63. 2, 34. 4, 33. 2, 32. 6, 30. 4, 25. 6, 20. 5, 19. 5, 14. 3.

(vi) [(6Z, 9Z)-3-Methyl-6, 9-dodecadienyll (triphenyl) phosphonium iodide To a solution of iodine (2. 08 g, 8. 19 mmol) in CH2C12 (30 ml), under N2, was added at -15°C a solution of PPh3 (2. 28 g, 8. 72 mmol) in CH2C12 (30 ml). The yellow solution of Ph3PI2 was stirred at-15°C for 15 min and then a mixture of (6Z, 9Z)-3-methyl-6, 9- dodecadieno-1-ol (1. 07 g, 5. 45 mmol) and pyridine (1. 25 ml, 16. 3 mmol) in CH2C12 (30 ml) were added dropwise and the reaction proceeded as described in the general procedure for the preparation of iodides to afford the iodide as a yellow oil, which was used immediately in the next reaction.

A mixture of iodide (5. 45 mmol), PPh3 (2. 56 g, 9. 80 mmol) and K2CO3 (1. 50 g, 10. 9 mmol) in CH3CN (25 ml) was reacted as described in the general procedure for the preparation of phosphonium salts to give the phosphonium salt (2. 78 g, 90%) as a yellow oil. 1H NMR 8 7. 78 (m, 15H), 5. 28 (m, 4H), 3. 70 (m, 2H), 2. 67 (t, 2H, J 6 Hz), 1. 96 (m, 3H), 1. 70 (m, 4H), 0. 94 (t, 3H, J 7 Hz), 0. 79 (d, 3H, J 6 Hz).

(vii) Ethyl (6Z, 12Z, 15Z)-9-methyl-6, 12, 15-octadecatrienoate KOBut (4. 9 ml of a 1 M solution in THF, 4. 9 mmol) was added to a 0°C solution of [ (6Z, 9Z)-3-methyl-6, 9-dodecadienyl] (triphenyl) phosphonium iodide (2. 78 g, 4. 89 mmol) and ethyl 6-oxohexanoate (387 mg, 2. 44 mmol) in CHzCIs (100 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a yellow oil. The oil was subjected to column chromatography (1% Et20/pet. spirits to 5% Et20/pet. spirits) to give the ester (424 mg, 54%) as a pale-yellow oil. 1H NMR 8 5. 34 (m, 6H), 4. 12 (q, 3H, J 7 Hz), 2. 77 (t, 2H, J 5 Hz), 2. 30 (t, 2H, J 7 Hz), 2. 01 (m, 8H), 1. 64 (m, 2H), 1. 42 (m, 5H), 1. 25 (t, 3H, J7 Hz), 0. 97 (t, 3H, J8 Hz), 0. 89 (d, 3H, J 7 Hz) ; 13C NMR 8 173. 7, 131. 7, 130. 3, 129. 0, 128. 8, 128. 5, 127. 3, 60. 1, 36. 6, 34. 4, 34. 2, 32. 9, 29. 2, 26. 8, 25. 6, 24. 8, 24. 6, 20. 5, 19. 4, 14. 2 (one peak superimposed or missing).

L. Synthesis of (6Z, 12Z, 15Z)-9-methyl-6, 12, 15-octadecatrienoic acid Ethyl (6Z, 12Z, 15Z)-9-metl1yl-6, 12, 15-octadecatrienoate (102 mg, 0. 318 mmol) in THF (10 ml) and 0. 5 M aqueous LiOH (1. 3 ml) was reacted as described in the general procedure for the hydrolysis of esters to afford the acid (43 mg, 46%) as a pale-yellow oil. 1H NMR 8 5. 37 (m, 6H), 2. 77 (t, 2H, J 5 Hz), 2. 36 (t, 2H, J 7 Hz), 2. 03 (m, 8H), 1. 64 (m, 2H), 1. 40 (m, 5H), 0. 97 (t, 3H, J 7 Hz), 0. 89 (d, 3H, J 7 Hz) ; 13C NMR 8 179. 9, 131. 8, 130. 5, 128. 8, 128. 5, 127. 3, 36. 6, 34. 4, 33. 9, 33. 0, 29. 1, 26. 8, 25. 6, 24. 9, 24. 3, 20. 5, 19. 4, 14. 3 (one peak superimposed or missing).

M. Synthesis of Ethyl (6Z, 9Z, 15Z)-6, 9, 15-octadecatrienoate (i) 3-(Tetrahydro-2H-pyran-2-yloxy)-1-propanol 3, 4-Dihydro-2H-pyran (60 g, 65 ml, 0. 71 mol) was added dropwise to a chilled (ca. 5°C) solution of 1, 3-propane diol (50 ml, 0. 71 mol) and p-TsOH (300 mg) in CH2C12 (300 ml) such that the temperature did not rise above 5°C (ca. 30 min). The mixture was left to stir at 5°C for 30 min and was then allowed to warm to room temperature. The reaction mixture was stirred at room temperature for 4 h. After this time, the reaction mixture was diluted with Et2O (1 1) and washed with saturated aq. NaHCO3 (200 ml), H20 (200 ml) and brine (200 ml), then dried (MgS04), filtered and concentrated to give a colourless oil. The oil was chromatographed (20% Et2O/pet. spirits then 50% Et20/pet. spirits) to afford the mono- protected alcohol (24 g, 21%) as a colourless oil. 1H NMR 5 4. 59 (m, 1H), 3. 88 (m, 4H), 3. 54 (m, 2H), 1. 94-1. 50 (m, 8H). 13C NMR 8 98. 9, 65. 2, 62. 3, 60. 1, 32. 2, 30. 5, 25. 2, 19. 5.

(ii) 3-Iodopropyl tetrahydro-2H-pyran-2-yl ether To a solution of iodine (50. 9 g, 201 mmol) in CH2C12 (300 ml), under N2, was added at -15°C a solution of PPh3 (56. 0 g, 213 mmol) in CH2C12 (300 ml). The yellow solution of Ph3PI2 was stirred at-15°C for 15 min and then a mixture of 3- (tetrahydro-2H-pyran-2- yloxy)-1-propanol (21. 4 g, 134 mmol) and pyridine (30. 7 ml) in CH2C12 (300 ml) were added dropwise. The reaction proceeded as described in the general procedure for the preparation of iodides to afford the iodide (27. 2 g, 75%) as a colourless oil.

(iii) Triphenyl [3-(tetrahydro-2H-pyran-2-yloxy) propyl] phosphonium iodide A mixture of 3-iodopropyl tetrahydro-2H-pyran-2-yl ether (27. 2 g, 101 mmol), PPh3 (47. 7 g, 181 mmol) and K2CO3 (27. 7 g, 201 mmol) in CH3CN (300 ml) was reacted as described in the general procedure for the preparation of phosphonium salts to give the phosphonium salt

(46. 3 g, 86%) as a white solid.'H NMR 8 7. 73 (m, 15H), 4. 56 (m, 1H), 3. 83 (m, 3H), 3. 47 (m, 1H), 1. 87 (m, 4H), 1. 51 (m, 4H).

(iv) Ethyl (Z)-6-nonenoate KOBut (155 ml of a 1 M solution in THF, 155 mmol) was added to a 0°C solution propyl (triphenyl) phosphonium bromide (59. 6 g, 155 mmol) and ethyl 6-oxohexanoate (11 g, 77. 4 mmol) in CH2C12 (300 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBu to give a yellow oil. The oil was subjected to column chromatography (1% Et2O/pet. spirits to 5% Et20/pet. spirits) to give the ester (3. 03 g, 21%) as a colourless oil. 1H NMR 8 5. 34 (m, 2H), 4. 12 (q, 2H, J 7 Hz), 2. 30 (t, 2H, J 7 Hz), 2. 03 (quint., 4H, J 7 Hz), 1. 64 (m, 2H), 1. 42 (m, 2H), 1. 25 (t, 3H, J7 Hz), 0. 95 (t, 3H, J7 Hz).

(v) (Z)-6-Noneno-1-ol Ethyl (Z)-6-nonenoate (3. 03 g, 16. 4 mmol) in anhydrous Et2O (20 ml) was added dropwise to a suspension of LiAlH4 (624 mg, 16. 4 mmol) in anhydrous Et20 (100 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the reduction of esters to afford the alcohol (2. 26 g, 97%) as a colourless oil. 1H NMR 8 5. 35 (m, 2H), 3. 64 (t, 2H, J 7 Hz), 2. 03 (m, 4H), 1. 56 (m, 2H), 1. 37 (m, 4H), 0. 95 (t, 3H, J 7 Hz).

(vi) (Z)-6-Nonenal 6-Noneno-l-ol (2. 26 g, 15. 9 mmol) in H2C12 (20 mi) was added in one portion to a suspension of PCC (5. 13 g, 23. 8 mmol), NaOAc (652 g, 7. 95 mmol) and molecular sieves in CH2C12 (100 ml) and the reaction proceeded as described in the general procedure for the PCC oxidation of alcohols to afford the aldehyde (1. 46 g, 65%) as a pale-yellow oil. 1H NMR 8 9. 76 (t, 1H, J2 Hz), 5. 35 (m, 2H), 2. 44 (td, 2H, J7 & 2 Hz), 2. 04 (br quint., 4H, J7 Hz), 1. 65 (m, 2H), 1. 39 (m, 2H), 0. 95 (t, 3H, J7 Hz).

(vii) (3Z, 9Z)-3, 9-Dodecadienyl tetrahydro-2H-pyran-2-yl ether KOBut (20. 8 ml of a 1 M solution in THF, 20. 8 mmol) was added to a 0°C solution of triphenyl [3-(tetrahydro-2H-pyran-2-yloxy) propyl] phosphonium iodide (10. 3 g, 20. 8 mmol) and (Z)-6-nonenal (1. 46 g, 10. 4 mmol) in CH2C12 (200 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a yellow oil. The oil was subjected to column chromatography (1% Et2O/pet. spirits to 5% Et2O/pet. spirits) to give the ester (1. 55 g, 56%) as a colourless oil. 1H NMR 8 5. 38 (m, 4H), 3. 78 (m, 2H), 3. 45 (m, 2H), 2. 35 (br q, 2H, J 7 Hz), 2. 04 (br t, 6H, J 6 Hz), 1. 82- 1. 35 (m, 10H), 0. 94 (t, 3H, J7 Hz) ; 13C NMR 8 131. 8, 131. 6, 129. 1, 125. 5, 98. 7, 67. 1, 62. 3, 30. 7, 29. 3, 29. 2, 27. 9, 27. 2, 27. 0, 25. 4, 20. 5, 19. 6, 14. 4.

(viii) (3Z, 9Z)-3, 9-Dodecadien-1-ol To a solution of (3Z, 9Z)-3, 9-dodecadienyl tetrahydro-2H-pyran-2-yl ether (1. 55 g, 5. 82 mmol) in EtOH (10 ml) was added p-TsOH (330 mg, 1. 64 mmol) and the reaction : proceeded as described in the general procedure for the deprotection of THP ethers to afford the alcohol (870 mg, 82%) as a colourless oil. 1H NMR 8 5. 55 (m, 1H), 5. 34 (m, 3H), 3. 64 (t, 2H, J 6 Hz), 2. 33 (br q, 2H, J 7 Hz), 2. 02 (m, 6H), 1. 33 (m, 4H), 0. 95 (t, 3H, J 7 Hz).

(ix) (3Z, 9Z)-3, 9-Dodecadienyl (triphenyl) phosphonium iodide To a solution of iodine (1. 82 g, 7. 17 mmol) in CH2C12 (20 ml), under N2, was added at -15°C a solution of PPh3 (2. 0 g, 7. 64 mmol) in CH2C12 (20 ml). The yellow solution of Ph3PI2 was stirred at-15°C for 15 min and then a mixture of (3Z, 9Z)-3, 9-dodecadien-1-ol (870 mg, 4. 77 mmol) and pyridine (1. 1 ml, 14. 3 mmol) in CH2C12 (20 ml) were added dropwise and the reaction proceeded as described in the general procedure for the preparation of iodides to afford the iodide as a colourless oil, which was used immediately in the next reaction.

A mixture of the iodide (4. 77 mmol), PPh3 (2. 24 g, 8. 58 mmol) and K2CO3 (1. 31 g, 9. 53 mmol) in CH3CN (20 ml) was reacted as described in the general procedure for the preparation of phosphonium salts to give the phosphonium salt (1. 58 g, 60%) as a sticky orange oil.'H NMR 8 7. 75 (m, 15H), 5. 51 (m, 1H), 5. 33 (m, 3H), 3. 75 (m, 2H), 2. 45 (m, 2H), 1. 98 (m, 6H), 1. 24 (m, 4H), 0. 92 (t, 3H, J 7 Hz).

(x) Ethyl (6Z, 9Z, 15Z)-6, 9, 15-octadecatrienoate KOBut (2. 85 ml of a 1 M solution in THF, 2. 85 mmol) was added to a 0°C solution of (3Z, 9Z)-3, 9-dodecadienyl (triphenyl) phosphonium iodide (1. 58 g, 2. 85 mmol) and ethyl 6- oxohexanoate (320 mg, 2. 02 mmol) in CH2C12 (50 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a yellow oil. The oil was subjected to column chromatography (1% Et2O/pet. spirits to 5% Et20/pet. spirits) to give the ester (381 mg, 61%) as a colourless oil. IH NMR S 5. 36 (m, 6H), 4. 13 (t, 2H, J 7Hz), 2. 77 (br t, 2H, J 5 Hz), 2. 30 (t, 2H, J7 Hz), 2. 05 (m, 8H), 1. 64 (m, 2H), 1. 40 (m, 6H), 1. 22 (t, 3H, J7 Hz), 0. 95 (t, 3H, J7 Hz) ; 13C NMR S 173. 6, 131. 6, 130. 0, 129. 4, 129. 0, 128. 4, 127. 8, 60. 1, 34. 2, 29. 3, 29. 2, 29. 1, 27. 1, 26. 9, 26. 8, 25. 6, 24. 6, 20. 5, 14. 3, 14. 2.

N. Synthese of (6Z, 9Z, 15Z)-6, 9, 15-Octadecatrienoic acid Ethyl (6Z, 9Z, 15Z)-6, 9, 15-octadecatrienoate (107 mg, 0. 349 mmol) in THF (10 ml) and 0. 5 M aqueous LiOH (1. 4 ml) was reacted as described in the general procedure for the hydrolysis of esters to afford the acid (55 mg, 57%) as a pale-yellow oil. 1H NMR 8 5. 35 (m, 6H), 2. 77 (br t, 2H, J 5 Hz), 2. 36 (t, 2H, J 7 Hz), 2. 05 (m, 8H), 1. 65 (m, 2H), 1. 41 (m, 6H), 0. 95 (t, 3H, J 7Hz) ; 13C NMR 8 180. 1, 131. 7, 130. 2, 129. 3, 129. 1, 128. 6, 127. 9, 34. 0, 29. 4, 29. 3, 29. 0, 27. 1, 27. 0, 26. 8, 25. 7, 24. 3, 20. 6, 14. 4. O. Synthesis of Ethyl (lOZ, 13Z, 16Z)-10, 13, 16-octadecatrienoate (i) 3-(Tetrahydro-2H-pyran-2yloxy) propanal 3-(Tetrahydro-2H-pyran-2-yloxy)-1-propanol (12. 0 g, 74. 9 mmol) in CH2C12 (50 ml) was added to a suspension of PCC (24. 2 g, 112 mmol), NaOAc (3. 07 g, 37. 5 mmol) and molecular sieves in CH2C12 (150 ml) and the reaction proceeded as described in the general procedure for the PCC oxidation of alcohols to afford the aldehyde (7. 75 g, 65%) as a colourless oil. 1H NMR 8 9. 82 (t, 1H, J 2 Hz), 4. 63 (m, 1H), 4. 10 (m, 1H), 3. 60 (m, 2H), 3. 52 (m, 1H), 2. 70 (td, 2H, J 6 & 2 Hz), 2. 09-1. 51 (m, 8H) ; 13C NMR 8 201. 0, 98. 6, 61. 8, 60. 9, 43. 5, 30. 2, 25. 1, 19. 0.

(ii) (Z)-3-Penten-1-ol A solution of sodium borohydride (530 mg, 14. 0 mmol) in EtOH (5 ml), followed by. ethylene diamine (1. 6 ml) and 3-pentyn-1-ol (2. 0 g, 23. 8 mmol) was added successively under a balloon of hydrogen to a stirred solution of nickel acetate tetrahydrate (2. 96 g, 11. 9 mmol) in EtOH (50 ml). The mixture was left to stir for 4 h. After this time, the mixture was filtered through celite, and subsequently the filtrate was filtered through a 5 cm silica plug (Et2O elution). H20 (100 ml) was added to the filtrate and the phases separated. The aqueous phase was extracted with Et2O (5 x 50 ml). The combined organic extracts were washed with aq. 2 M HC1 (20 ml) and H20 (20 ml), then dried (MgS04), filtered and carefully concentrated to afford the alkene (1. 78 g, 87 %) as a pale-yellow oil. 1H NMR 8 5. 63 (m, 1H), 5. 39 (m, 1H), 3. 65 (t, 2H, J 7 Hz), 2. 34 (br q, 2H, J 7 Hz), 1. 65 (d, 3H, J 8 Hz) ; 13C NMR 8 126. 5, 126. 1, 61. 9, 30. 2, 12. 7.

(iii) (Z)-3-Pentenyl (triphenyl) phosphonium iodide To a solution of iodine (10. 9 g, 42. 9 mmol) in CH2C12 (100 ml), under N2, was added at- 15°C a solution of PPh3 (12. 0 g, 45. 7 mmol) in CH2C12 (100 ml). The yellow solution of Ph3PI2 was stirred at-15°C for 15 min and then a mixture of (Z)-3-penten-1-ol (2. 46 mg, 28. 6 mmol) and pyridine (6. 6 ml, 85. 7 mmol) in CH2C12 (100 ml) were added dropwise and the reaction proceeded as described in the general procedure for the preparation of iodides to afford the iodide as a colourless oil, which was used immediately in the next reaction.

A mixture of iodide (28. 6 mmol), PPh3 (13. 5 g, 51. 4 mmol) and K2CO3 (7. 88 g, 57. 1 mmol) in CH3CN (80 ml) was reacted as described in the general procedure for the preparation of phosphonium salts to give the phosphonium salt (6. 24 g, 48%) as a white solid. 1H NMR 8 7. 77 (m, 15H), 5. 49 (m, 2H), 3. 71 (m, 2H), 2. 44 (m, 2H), 1. 42 (d, 3H, J5 Hz).

(iv) 2- [ (3Z, 6Z)-3, 6-Octadienyloxy] tetrahydro-2H-pyran KOBut (13. 6 ml of a 1 M solution in THF, 13. 6 mmol) was added to a 0°C solution (Z)-3- pentenyl (triphenyl) phosphonium iodide (6. 24 g, 13. 6 mmol) and the aldehyde (1. 08 g, 6. 8 mmol) in CH2C12 (100 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a yellow oil. The oil was subjected to column chromatography (1% Et2O/pet. spirits to 5% Et2O/pet. spirits) to give the ether (224 mg, 16%) as a pale-yellow oil. 1H NMR 8 5. 43 (m, 4H), 4. 61 (t, 1H, J 3 Hz), 3. 78 (m, 2H), 3. 46 (m, 2H), 2. 82 (br t, 2H, J 6 Hz), 2. 40 (m, 2H), 1. 89-1. 34 (m, 9H) ; 13C NMR 8 129. 9, 128. 6, 125. 8, 124. 1, 98. 7, 66. 9, 62. 2, 30. 7, 27. 9, 25. 4, 19. 6 (two peaks superimposed or missing).

(v) (3Z, 6Z)-3, 6-Octadien-1-ol To a solution of 2- [ (3Z, 6Z)-3, 6-octadienyloxy] tetrahydro-2H-pyran (224 mg, 1. 07 mmol) in EtOH (10 ml) was added p-TsOH (40 mg, 0. 2 mmol) and the reaction proceeded as described in the general procedure for the deprotection of THP ethers to afford the alcohol (125 mg, 93%) as a colourless oil. 1H NMR 8 5. 46 (m, 4H), 3. 66 (t, 2H, J 6 Hz), 2. 83 (br t,

2H, J 6 Hz), 2. 39 (t, 2H, J 6 Hz), 1. 64 (d, 3H, J 5 Hz) ; 13C NMR 8 131. 0, 128. 4, 125. 4, 124. 3, 62. 1, 30. 7, 25. 3, 12. 7.

(vi) (3Z, 6Z)-3, 6-Octadienyl (triphenyl) phosphonium iodide To a solution of iodine (269 mg, 1. 06 mmol) in CH2C12 (10 ml), under N2, was added at -15°C a solution of PPh3 (296 mg, 1. 13 mmol) in CH2CI2 (10 ml). The yellow solution of Ph3PI2 was stirred at-15°C for 15 min and then a mixture of (3Z, 6Z)-3, 6-octadien-1-ol (89 mg, 0. 705 mmol) and pyridine (0. 16 ml, mmol) in CH2C12 (10 ml) were added dropwise and the reaction proceeded as described in the general procedure for the preparation of iodides to afford the iodide as a colourless oil, which was used immediately in the next reaction.

A mixture of iodide (0. 705 mmol), PPh3 (332 mg, 1. 27 mmol) and K2CO3 (194 mg, 1. 41 mmol) in CH3CN (10 ml) was reacted as described in the general procedure for the preparation of phosphonium salts to give the phosphonium salt (143 mg, 41%) as a yellow oil. 1H NMR 8 7. 72 (m, 15H), 5. 38 (m, 4H), 3. 68 (m, 2H), 2. 45 (m, 4H), 1. 45 (d, 2H, J 7' Hz).

(vii) Ethyl (lOZ, 13Z, 16Z)-10, 13, 16-octadecatrienoate KOBut (0. 3 ml of a 1 M solution in THF, 0. 3 mmol) was added to a 0°C solution (3Z, 6Z)- 3, 6-octadienyl (triphenyl) phosphonium iodide (143 mg, 0. 287 mmol) and ethyl 10- oxodecanoate (124 mg, 1. 15 mmol) in CH2CI2 (30 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a yellow oil. The oil was subjected to column chromatography (1% Et20/pet. spirits to 5% Et20/pet. spirits) to give the ester (13 mg, 15%) as a colourless oil. 1H NMR 8 5. 39 (m, 6H), 4. 12 (q, 2H, J7 Hz), 2. 81 (br t, 4H, J 6 Hz), 2. 28 (t, 2H, J 7 Hz), 2. 05 (br q, 2H, J 6 Hz), 1. 58 (m, 6H), 1. 26 (m, 12H).

P. Synthesis of (lOZ, 13Z, 16Z)-10, 13, 16-Octadecatrienoic acid Ethyl (10Z, 13Z, 16Z)-10, 13, 16-octadecatrienoate (13 mg, 0. 042 mmol) in THF (2 ml) and 0. 5 M aqueous LiOH (0. 17 ml) was reacted as described in the general procedure for the hydrolysis of esters to afford the acid (7 mg, 59%) as a yellow oil. 1H NMR 8 5. 39 (m, 6H), 2. 81 (br t, 4H, J 5 Hz), 2. 35 (t, 2H, J7 Hz), 2. 03 (m, 2H), 1. 64 (m, 6H), 1. 27 (m, 9H).

Q. Synthesis of Methyl (5Z, 8Z, 12Z, 15Z)-5, 8, 12, 15-octadecatetraenoate (i) 2- [ (4Z, 7Z)-4, 7-Decadienyloxy] tetrahydro-2H-pyran KOBut (15. 4 ml of a 1 M solution in THF, 15. 4 mmol) was added to a 0°C solution (Z)-3- hexenyl (triphenyl) phosphonium iodide (7. 29 g, 15. 4 mmol) and aldehyde (1. 33 g, 7. 71 mmol) in CH2Cl2 (100 ml), maintained under N2, and the reaction proceeded as described in ! the general procedure for the Wittig reaction with KOBut to give a yellow oil. The oil was subjected to column chromatography (1% Et20/pet. spirits to 5% Et20/pet. spirits) to give the ester (1. 07 g, 58%) as a pale-yellow oil. 1H NMR 8 5. 35 (m, 4H), 4. 58 (t, 1H, J 3 Hz), 3. 81 (m, 2H), 3. 45 (m, 2H), 2. 78 (t, 2H, J 6 Hz), 2. 11 (m, 4H), 1. 68 (m, 8H), 0. 97 (t, 3H, J 8 Hz) ; 13C NMR 8 131. 7, 129. 2, 128. 5, 127. 2, 98. 7, 66. 8, 62. 1, 30. 7, 29. 6, 25. 5, 25. 4, 23. 8, 20. 5, 19. 5, 14. 2.

(ii) (4Z, 7Z)-4, 7-Decadien-1-ol To a solution of the THP ether (1. 07 g, 4. 61 mmol) in EtOH (40 ml) was added p-TsOH (175 mg, 0. 92 mmol) and the reaction proceeded as described in the general procedure for the deprotection of THP ethers to afford the alcohol (498 mg, 70%) as a yellow oil.'H NMR 8 5. 37 (m, 4H), 3. 66 (t, 2H, J 6 Hz), 2. 80 (t, 2H, J 6 Hz), 2. 12 (m, 4H), 1. 65 (quint., 2H, J 7 Hz), 0. 97 (t, 3H, J 7 Hz) ; 13C NMR 8 131. 8, 129. 1, 128. 7, 127. 1, 62. 2, 32. 4, 25. 4, 23. 5, 20. 5, 14. 2.

(iii) (4Z, 7Z)-4, 7-Decadienal (4Z, 7Z)-4, 7-Decadien-1-ol (498 mg, 3. 23 mmol) in CH2C12 (20 ml) was added to a suspension of PCC (1. 04 g, 4. 84 mmol), NaOAc (132 mg, 1. 62 mmol) and molecular sieves in CH2C12 (20 ml) and the reaction proceeded as described in the general procedure for the PCC oxidation of alcohols to afford the aldehyde (397 mg, 81%) as a colourless oil. 1H NMR 8 9. 78 (t, 1H, J 1 Hz), 5. 36 (m, 4H), 2. 80 (t, 2H, J 6 Hz), 2. 45 (m, 4H), 2. 07 (quint., 2H, J7 Hz), 0. 97 (t, 3H, J7 Hz) ; 13C NMR 8 201. 8, 132. 0, 129. 7, 127. 3, 126. 7, 43. 6, 25. 4, 20. 5, 19. 9, 14. 1.

(iv) Methyl 5-hydroxy pentanoate Concentrated sulfuric acid (10 drops) was added to a magnetically stirred solution of valerolactone (10. 0 g, 0. 10 mol) in freshly distilled MeOH (150 ml) and the resulting mixture was refluxed for 5 h. The mixture was then cooled in an ice/salt bath and solid NaHCO3 (1. 0 g) was added. The mixture was stirred for 10 min, the excess solid was removed by filtration and the solvent removed under reduced pressure to afford the hydroxy ester (13. 1 g, quant.) as a colourless oil. 1H NMR 8 3. 67 (s, 3H), 3. 65 (t, 2H, J 6 Hz), 2. 36 (t, 2H, J 7 Hz), 1. 65 (m, 6H).

(v) Methyl 5-oxopentanoate Methyl 5-hydroxy pentanoate (13. 1 g, 0. 10 mol) in CH2C12 (60 ml) was added to a suspension of PCC (32. 0 g, 0. 15 mol) and molecular sieves in CHUCK (120 ml) and the reaction proceeded as described in the general procedure for the PCC oxidation of alcohols to afford the aldehyde (9. 20 g, 71%) as a colourless oil. 1H NMR 8 9. 77 (t, 1H, J 1 Hz), 3. 67 (s, 3H), 2. 54 (td, 2H, J7 & 1 Hz), 2. 38 (t, 2H, J7 Hz), 1. 95 (quint., 2H, J7 Hz).

(vi) Methyl (Z)-8- (tetrahydro-2H-pyran-2-yloxy)-5-octenoate KOBut (36. 7 ml of a 1 M solution in THF, 36. 7 mmol) was added to a 0°C solution of triphenyl [3- (tetrahydro-2H-pyran-2-yloxy) propyl] phosphonium iodide (20. 0 g, 37. 6 mmol) and methyl 5-oxopentanoate (2. 44 g, 18. 8 mmol) in CH2C12 (300 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a yellow oil. The oil was subjected to column chromatography (1% Et2O/pet. spirits to 10% Et20/pet. spirits) to give the ether (958 mg, 20%) as a colourless oil. IH NMR 8 5. 44 (m, 2H), 4. 59 (t, 1H, J 1 Hz), 3. 81 (m, 2H), 3. 67 (s, 3H), 3. 43 (m, 2H), 2. 32 (m, 6H), 2. 13-1. 43 (m, 8H).

(vii) (Methyl (Z)-8-hydroxy-5-octenoate To a solution of the THP ether (958 mg, 3. 74 mmol) in EtOH (40 ml) was added p-TsOH (140 mg, 0. 74 mmol) and the reaction proceeded as described in the general procedure for the deprotection of THP ethers to afford the alcohol (357 mg, 55%) as a yellow oil. tHb NMR 8 5. 44 (m, 2H), 3. 67 (s, 3H), 3. 65 (t, 2H, J 6 Hz), 2. 31 (m, 4H), 2. 12 (m, 2H), 1. 70 (quint., 2H, J 7 Hz) ; 13C NMR S 173. 7, 131. 5, 126. 5, 62. 1, 51. 5, 33. 6, 30. 7, 26. 6, 24. 7.

(viii) (Methyl (Z)-8-iodo-5-octenoate To a solution of iodine (789 mg, 3. 12 mmol) in CH2C12 (10 ml), under N2, was added at -15°C a solution of PPh3 (872 mg, 3. 32 mmol) in CH2C12 (10 ml). The yellow solution of Ph3PI2 was stirred at-15°C for 15 min and then a mixture of methyl (Z)-8-hydroxy-5- octenoate (357 mg, 2. 07 mmol) and pyridine (0. 47 ml, 6. 22 mmol) in CH2C12 (10 ml) were added dropwise and the reaction proceeded as described in the general procedure for the preparation of iodides to afford the iodide as a colourless oil, which was used immediately in the next reaction. 1H NMR 8 5. 46 (m, 2H), 3. 68 (s, 3H), 3. 14 (t, 2H, J 7 Hz), 2. 62 (m, 4H), 2. 32 (t, 2H, J7 Hz), 2. 08 (m, 2H), 1. 71 (m, 2H).

(ix) [ (Z)-8-Methoxy-8-oxo-3-octenyl] (triphenyl) phosphonium iodide A mixture of methyl (Z)-8-iodo-5-octenoate (2. 07 mmol), PPh3 (980 mg, 3. 73 mmol) and K2CO3 (570 mg, 4. 14 mmol) in CH3CN (20 ml) was reacted as described in the general procedure for the preparation of phosphonium salts to give the phosphonium salt (695 mg, 65%) as a yellow oil. 1H NMR 8 7. 77 (m, 15H), 5. 61 (m, 1H), 5. 39 (m, 1H), 3. 79 (m, 2H), 3. 60 (s, 3H), 2. 45 (m, 2H), 2. 22 (t, 2H, J7 Hz), 1. 89 (m, 2H), 1. 60 (m, 2H).

(x) Methyl (5Z, 8Z, 12Z, 15Z)-5, 8, 12, 15-octadecatetraenoate KOBut (1. 35 ml of a 1 M solution in THF, 1. 35 mmol) was added to a 0°C solution of phosphonium salt (695 mg, 1. 35 mmol) and (4Z, 7Z)-4, 7-decadienal (180 mg, 1. 18 mmol) in CH2C12 (30 ml), maintained under N2, and the reaction proceeded as described in the general procedure for the Wittig reaction with KOBut to give a brown oil. The oil was subjected to column chromatography (1% Et2O/pet. spirits to 5% Et20/pet. spirits) to give the ester (35 mg, 10%) as a colourless oil. 1H NMR 8 5. 36 (m, 8H), 3. 67 (s, 3H), 2. 78 (m, 4H), 2. 32 (t, 2H, J 7 Hz), 2. 09 (m, 8H), 1. 70 (quint., 2H, J 7 Hz), 0. 97 (t, 3H, J 7 Hz).

R Synthesis of (5Z, 8Z, 12Z, 15Z)-5, 8, 12, 15-Octadecatetraenoic acid Methyl (5Z, 8Z, 12Z, 15Z)-5, 8, 12, 15-octadecatetraenoate (38 mg, 0. 131 mmol) in THF (5 ml) and 0. 5 M aqueous LiOH (0. 58 ml) was reacted as described in the general procedure for the hydrolysis of esters to afford the acid (29 mg, 80%) as a yellow oil. 1H NMR 8 5. 35 (m, 8H), 2. 76 (m, 4H), 2. 37 (t, 2H, J8 Hz), 2. 06 (m, 8H), 1. 71 (quint., 2H, J7 Hz), 1. 25

Example 7 Comparison of the extent of protection against isoproterenol-induced asynchronous contractile activity in isolated adult rat cardiomyocytes by acutely added C18 polyunsaturated fatty acids (PUFAs) with 22 : 6n-3.

18 : 3n-3 (synthetic origin) versus 18 : 4n-3 and 18 : 5n-3 (both marine origin).

From Figure 4, it can be seen that at a final concentration of 20 micromolar added fatty acid, approximately 2% of cells were contracting asynchronously following addition of 18 : 4n-3 (of marine origin), compared with approximately 17% of cells with the acutely added 22 : 6n-3. For acute addition of 18 : 5n-3 (of marine origin), this figure was approximately 20%. In order to determine the rank order potency of 18 : 4n-3 and 18 : 5n-3 (both of marine origin) with acutely added 18 : 3n-3 (alpha linolenic acid-synthetic origin), data described in the publication (McMurchie, E. J., Leifert, W. R. and Head, R. J. (1998) was used.

Antiarrhythmic properties of polyunsaturated fatty acids in adult rat cardiomyocytes : A role for membrane fluidity ? in Essential Fatty Acids and Eicosanoids (Riemersma, R. A., Armstrong, R. A., Kelly, R. W. and Wilson, R., eds.) American Oil Chemists Society Press,, Champaign, Chapter 6, pp 284-289). The method used in this publication differs from the method outlined for determining antiarrhythmic effects of PUFAs outlined in this patent application. However, from the data shown in Figure 1 of the above publication, it has been found that in comparison with 22 : 6n-3 (at 25 micromolar final concentration) which gave 37% cells developing asynchronous contractile activity, 18 : 3n-3 gave a value of 73% (Figure 1-McMurchie et al-reference as above). Therefore, when comparing these different C18 PUFAs using 22 : 6n-3 as the reference, it can be concluded that 18 : 3n-3 is not as effective as 18 : 4n-3 or 18 : 5n-3 (of marine origin) in protecting cardiomyocytes from developing isoproterenol-induced asynchronous contractile activity.

18 : 4n-3 (synthetic origin) 18 : 4n-3 (synthetic origin) was found to produce a value of about 25% asynchronous cells compared with a value of about 20% asynchronous cells for 22 : 6n-3 when both PUFAs were tested acutely at 20 micromolar final concentration in the cell based assay. It can therefore

be concluded that 18 : 4n-3 from synthetic origin is as effective as 18 : 4n-3 obtained from marine origin, using 22 : 6n-3 as the reference.

18 : 4n-3 (iso-branched-synthetic origin) 18 : 4n-3 (iso-branched ; synthetic origin) was found to produce a value of about 30% asynchronous cells compared with a value of about 30% asynchronous cells for 22 : 6n-3 when both PUFAs were tested acutely at 20 micromolar final concentration in the cell based assay. We can therefore conclude that 18 : 4n-3 iso-branched from synthetic origin is as effective as 18 : 4n-3 obtained from marine origin (and by synthetic means), using 22 : 6n-3 as the reference.

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Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps of features.