Preparation of common and unusual waxes

Chemistry and Physics o f Lipids, 24 (1979) 431-448 © Elsevier/North-Holland Scientific Publishers Ltd.

PREPARATION OF COMMON AND UNUSUAL WAXES

F. SPENER lnstitut far Biochemie, Universitiit Miinster, Orldans - Ring 23a, D-440 Miinster (F. R. G.)

This article reviews synthetic procedures available for the preparation of naturally-occurring wax esters, diester waxes and sterol esters, t.or each class of compounds syntheses are summarised in the following sections: salt method, acyl chloride method, anhydride method, direct esterification and interesterification. In addition, the preparation of ether analogues of diester waxes and sterol esters is described, as these compounds constitute useful compounds for biochemical research.

Introduction Originally, the term 'wax' referred to a natural product, composed o f a variety of fatty substances. This mixture has been defined by its 'waxy' appearance. However a more chemical sense the term has been restricted to esters o f long-chain alcohols with fatty acids. These wax esters are found naturally as constituents of protective coatings o f leaves and fruits. In some cases, for example in marine organisms or in jojoba, they are stored in place of or alongside triacylglycerols to function as energy reserves. In addition, wax esters occur in secretions of insects and other animals [ 1 - 4 ] . This article covers synthetic procedures for the preparation of species o f wax esters. The so-called 'diester waxes' which have gained attention as constituents of skin lipids and various sebaceous glands are also discussed. The common denominator o f the diester waxes is the presence o f three long aliphatic chains per moleo cule, held together by two ester linkages in the vicinal position. Because of the similarity of the chemical reactions involved in their synthesis, sterol esters will be dealt with in some detail, although their occurrence is not restricted to natural waxes. As an addendum to the latter two groups, the scope o f this review is broadened to include ether analogues o f some waxes. These compounds are isosteric to the corresponding esters, yet they cannot be attacked by hydrolytic enzymes and thus they constitute an interesting tool for enzymic studies. An aspect arising from the literature is the variation in physical constants reported for identical compounds synthesised by different methods and/or authors.

431

432

F. Spener, Preparation of waxes

One reason lies in the fact that prior to the development of chromatographic methods, pure starting compounds were rarely available, and by the same token, endproducts could not be isolated in sufficient purity. Moreover, the mode of synthesis of acyl chloride becomes important for the quality of final wax and sterol esters when they are prepared by the acyl chloride method. The synthesis of compounds containing polyunsaturated acyl chains and sterols is critical in terms of reaction time, temperature, acidic or basic catalysis, in order to r~inimise structural alterations and the formation of side-products. Last but not least, wax esters and sterol esters may or may not exhibit polymorphic forms, again giving rise to different physical data. Whenever possible the historic development of each mode of synthesis will be delineated briefly. This should facilitate a comparison of synthetic approaches and help to evaluate the quality of products synthesised.

Synthesis of wax esters

The earliest approaches to the preparation of individual wax esters employed the reaction of a long-chain alcohol with a salt or with an acyl chloride. Later the so-called 'salt method' and 'acyl chloride method' were supplemented by the direct reaction of long-chain alcohols with fatty acids in the presence of a catalyst and by interesterification reactions. Salt method

Based on the observation of Simonini [5], who treated the silver salt of caproic acid with iodine and obtained amyl caproate, pentadecyl palmitate and heptadecyl stearate have been prepared starting from silver palmitate and silver stearate, respectively [6-8]. The procedure is rather simple: Silver salts are mixed with iodine and heated slowly to 130--140°C. At IO0°C carbon dioxide begins to evolve and ceases after 4.5 h. The mixture is cooled and extracted with diethyl ether. Crystals precipitate in the cold and are recrystallised from 80% ethanol [8]. Silver salts of fatty acids are prepared best by the procedure of Whitby [9]. An alcoholic solution of a fatty acid is treated with 1 tool of ammonia and 1 tool of AgNO3 in water; after 2 - 3 h the precipitate formed is suctioned off at 50°C, yielding almost quantitatively the silver salt as a stable white powder. These products are not contaminated with fatty acids, in contrast to earlier preparations [10]. Similarly, the alkali salts are obtained by adding to a fatty acid, dissolved in hot ethanol, an equivalent amount of an alcoholic solution of sodium or potassium ethylate [11 ]. In 1926 Whitby [9] was the first to prepare a series of even-numbered esters of palmitic and stearic acids using the salt method. He reacted alkyl iodides and silver salts in the molten state at IO0°C for 30 rain. The reaction products are taken up in

F. Spener. Preparation of waxes

433

warm diethyl ether and then precipitated from the cold solvent. More recently, a procedure has been described as suitable for the preparation of wax esters, in which either the alkyl or acyl moiety, or both, are mono- and polyunsaturated [ 12 I. This method employs long-chain alkylmethanesulfonates, which are conveniently prepared by the reaction of long-chain alcohols with methanesulfonylchloride or anhydride in pyridine [13-15 ], and the alkali salts of fatty acids. Long-chain alkylmethanesulfonate and alkali salt (25% molar excess) are thoroughly powdered, mixed and kept at 120°C for 3 h. After cooling, the mixture is taken up in diethyl ether, refluxed for 20 rain and filtered. The filtrate is washed and dried and finally the solvent is evaporated. Crystallisation from diethyl ether affords pure wax ester in 90% yield. In cases where mixing of starting compounds is difficult, xylene is added to the reaction mixture. Reaction conditions are similar, but longer reaction times are required [12]. Acyl chloride method

The synthesis of wax esters by acylation of long-chain alcohols with acyl chlorides was first reported by Krafft in 1883 [ 16]. The conduct of this synthesis has long been hampered by the lack of methods generally applicable to the the facile synthesis of saturated and unsaturated (especially polyunsaturated) acyl chlorides. Long-chain saturated acyl chlorides have been prepared under rather drastic conditions, by heating the mixture of a fatty acid and PCIs in vacuo up to 150°C until all phosphorus had evolved as POC13 [16,17]. When thionyl chloride was introduced for activation of fatty acids, the preparation of compounds having a double bond such as oleoyl chloride [18,19], elaidinoyl chloride [20] and chaulmoogroyl chloride [19] became possible. According to this method, the fatty acid is dissolved in excess cold thionyl chloride and heated to around 70°C for 30 min, which is accompanied by development of HC1. After removal of excess thionyl chloride by evaporation, the product is either purified by distillation in high vacuum, or used as such for further reactions. Allowing milder conditions, Young et al. described the preparation of acyl chlorides, including monounsaturated species. by the action of PCls or PCI3 on fatty acids in inert solvents such as petroleum ether [21]. The reaction mixture is refluxed for 1 h and, upon cooling, quickly extracted with ice-water, leaving acyl chlorides in the organic phase. This solution is dried, and acyl chlorides with less than 1.5% unreacted fatty acid are obtained in almost quantitative yield after removal of the solvent. The method of choice involves the use of oxalyl chloride which broadens the spectrum to the preparation of polyunsaturated long-chain acyl chlorides [22]. The fatty acid and a three-fold excess oxalyl chloride are kept at 65-70°C for 1 h. The excess oxalyl chloride is then removed in vacuo at 100°C and the residue is distilled at 267-400 Pa giving yields up to 100%. Because of foaming, this tedious purification step usually can be deleted, as the product is of sufficient quality for further synthesis. A micro method for the

434

F: Spener, Preparation of waxes

preparation of labelled acyl chlorides has been described by Borgstr6m and Krabisch [23]. [1-14C]Oleic acid is mixed with a large excess of cold oleoyl chloride in dry chloroform. After 2 h at room temperature equilibrium is achieved affording [1-~4C]oleoyl chloride in the gmolar range. Saturated wax esters have been synthesised by reacting long-chain alcohols with excess acyl chlorides in the molten state [16,17,24]. Mixtures are heated to 180°C and held at this temperature until HCI development has ceased. The cold melt is treated with water in order to destroy excess acyi chloride, and the residue is dissolved in diethyl ether/ethanol. The raw product is usually recrystallised from 96% ethanol. Scaled up preparations are facilitated by passing a slow stream of CO2 through the hot melt [25]. Upon cooling, the reaction mixture is neutralised with alcoholic KOH. Wax esters are separated from potassium salts of excess fatty acids by extracting them with petroleum ether/ethanol (1 : 1) whereas unreacted alcohols can be removed with hot ethanol. This method has served for the synthesis of high molecular weight fatty acid esters with sec-alcohols up to 13-pentriacontyl stearate [25]. The synthesis of unsaturated compounds has been achieved under milder conditions. Burschkies [19] has prepared octadencenyl chaulmoograte and 13-(2'cyclopentyl) tridecyl oleate by reactinglong-chain alcohol and excess acyl chloride in benzene for 6 h at 100°C, while a stream of nitrogen is bubbled through the solution. After evaporation of the solvent, the residue is taken up in diethyl ether and worked up in the usual way. The end products are purified by distillation in high vacuum.

Man et al. [26] have synthesised icosanyl icosanoate and pentacosanyl pentacosanoate by a method based on the observation that the preparation of phenyl esters is facilitated when Mg turnings are present in amounts equimolar to phenol. Thus,a long-chain alcohol and Mg turnings are placed in benzene and refluxed under constant stirring and a 15% excess of acyl chloride in benzene is added dropwise. The reaction is kept at reflux temperature until all HC1 has evolved. Upon cooling Mg turnings are removed by filtration and the filtrate is taken to dryness. Residual acyl chloride is destroyed by addition of water and the raw product is applied to an alumina column for final purification. Instead of letting gaseous HCI pass into the atmosphere, a weak base as catalyst may draw the reaction equilibrium towards synthesis by binding HC1 as insoluble salt and yet milder reaction conditions can be maintained. Brigl and Fuchs [271 have prepared saturated wax esters having chain lengths up to C~4 in either moiety. According to these authors, to a solution of a long-chain alcohol in quinoline (40% excess) and chloroform, a 40% excess of acyl chloride dissolved in chloroform, is added. This mixture is allowed to stand for 24 h at room temperature. After evaporation of the solvent, quinoline is removed by treatment with diluted H2SO4, whereas excess acyl chloride is converted to acid. The insoluble residue is dissolved in ethanol and excess fatty acid precipitates as salt with Ca acetate. Final recrystallisation of wax esters from abs. ethanol affords pure compounds. As early as 1914 pyridine has been used by Pickard and Kenyon [28] as catalyst

F. Spener, Preparation of waxes

435

in the preparation of esters of long-chain sec-alcohols. A series of esters of saturated, mono- and polyunsaturated alcohols with elaidic acid have been prepared in almost quantitative yields by Kaufmann and Pollerberg [20]. Long-chain alcohol and a 10% excess elaidinoyl chloride are dissolved in benzene and pyridine (25% excess) is added. Immediately, a white precipitate of pyridinium hydrochloride appears and the reaction is allowed to proceed for 15 h at room temperature. The benzene solution is then washed free of pyridine with 2 N H2SO4 and dried. Excess acyl chloride is separated by chromatography on alumina and the isolated wax ester is recrystallised from ethanol, methanol or acetone to remove traces of unreacted long-chain alcohol. Pyridine is now generally accepted as reagent when wax esters are synthesised by the acyl chloride method. Yet, as Mills [29] has pointed out, pyridine as solvent should be avoided, because it may partly solubilise pyridinium hydrochloride, thus leading to the formation of pyridinium ions. Under conditions of acid catalysis however, esterification reactions with unstable alcohols may lead to side products. It is recommenced that one works in inert solvents such as benzene and uses only a slight excess of pyridine over acyl chloride [29]. Iyengar and Schlenk [30] have synthesised a series of saturated and unsaturated wax esters by reacting long-chain alcohol and a 20% excess acyl chloride in anhydrous diethyl ether in the presence of pyridine (50% excess based on the alcohol). The mixture is stirred and gently refluxed for 3 h. Water and dilute sulfuric acid is added and the ethereal solution is washed free of pyridine and mineral acid. After further work-up according to standard procedures, the crude reaction product is purified by column chromatography on activated silicic acid and pure wax ester is finally recrystallised from diethyl ether. Physical data reported for wax esters synthesised by this method [30] are most reliable and have since been quoted as reference values. In a recent publication of Schlenk's group [31 ], the preparation of phytenyl esters of palmitic, oleic, linoleic and phytenic acids, some of which occur naturally in mosses [31 ] and bryophytes [32], was reported. Anhydrides have been used occasionally for the synthesis of wax esters [28,33], however this mode of synthesis has not gained wide acceptance. Direct esterification

The direct esterification offers a one-step procedure for the preparation of wax esters, but higher reaction temperatures are required thus causing the formation of various side-products. Nevertheless, chromatographic methods having become available for the isolation of pure end-products, wax esters having up to 4 double bonds have been synthesised by this approach. Earliest attempts were reported by Belluci [34], who synthesised hexadecyl stearate by passing a stream of CO2 through a mixture of hexadecanol and stearic acid, which was heated in the course of the reaction from 220°C to 270°C. Wax esters up to C6.~have been prepared by keeping a mixture of long-chain alcohol and fatty acid at IO0°C in the molten

436

F. Spener, Preparation of waxes

state for 30 min, while HCI gas is bubbled through the melt [7]. In 1936 Hilditch and Paul [35] were the first to synthesise unsaturated wax esters, using camphor-/~-sulfonic acid as catalyst, cis.9-Octadecenol, for example, mixed with a slight excess of oleic acid and a small amount of catalyst is heated to 180°C, while the reaction vessel is connected to a vacuum aspirator. After 1 h the formation of water has ceased and cis-9-octadecenyl oleate is obtained in 91% yield. This compound has been synthesised also by Swern et al. [36], employing a modified procedure. The reactants, with excess long-chain alcohol, and naphthalene-/~sulfonic acid as catalyst, are heated in benzene solution and the water formed during the reaction is constantly removed by azeotropic distillation. After 3 - 5 h the mixture is taken to dryness, the residue taken up in low boiling petroleum ether, filtered and finally recrystallised twice from the same solvent at -60°C. Using this procedure, Swern's group has prepared esters of a series of saturated alcohols [37], cis and trans 9-octadecenols [38] with isomeric 9,10-dihy4roxystearic acids and in addition, esters of 9,10-dihydroxyoctadecanol with different hydroxylated stearic acids [39]. These compounds are not only of interest as plasticisers and high melting waxes, but also as monomeric units of natural polymers such as cutins and suberins [40]. A similar approach was taken by Kaufmann and Pollerberg [20] who used benzene as solvent, but p-toluenesulfonic acid as catalyst. Pure products are obtained after chromatography on alumina, which removes unreacted fatty acids. This procedure proved suitable for the synthesis of cis, cis-9,12-octadecadienyl linoleate. The use of the BFa-diethyl ether compleX( as ~atalyst has been reported for the synthesis of phytanol or phytanyl phytanoate and phytanyl esters of saturated straight-chain fatty acids [41]. In a screw-capped glass tube equivalent amounts of phytanol and acid are dissolved in benzene, the catalyst is added, and the reaction carried out at 100°C for 7 h. Interestingly, as reported by Schlenk and coworkers [31 ], esterification by acidic catalysis with p-toluenesulfonic acid was not applicable to the synthesis of these unusual compounds. Saturated wax esters have been synthesised recently by the reaction of equimolar amounts of long-chain alcohol and fatty acid in the presence of thionyl chloride [42]. The reaction proceeds in benzene solution at room temperature overnight, affording yields of around 85%. In teresterification The interesterification reaction for the preparation of short-chain esters is well known, yet application of this method to long-chain compounds has surfaced rather late. in 1967, Phillips and Viswanathan [43] reported the synthesis of several wax esters, including unsaturated species. In a N2-atmosphere, a long-chain alcohol, the methyl ester of a fatty acid, and sodium methylate are placed in a reaction vessel linked to a vacuum aspirator. The mixture is heated to 70°C and vigorously stirred for 1 h, while methanol formed is removed by aspiration. After isolation of the raw product following established procedures, a pure compound is obtained in 85-90%

437

F. Spener, Preparation o f waxes

yield by means of chromatography on layers of silica gel. Wax esters can be prepared according to the method of Mahadevan and Lundberg [44] for the preparation of sterol esters, by interesterification of long-chain alkyl acetates and methyl esters of fatty acids, with the aid of sodium ethylate [45 ]. The reaction proceeds in vacuo at 90°C for 1 h, while methyl acetate is continuously suctioned off through a water aspirator. Upon cooling, the mixture is extracted with diethyl ether, the solvent evaporated and the raw product recrystallised from acetone affording pure compounds in 70-90% yields. Interesterification by acidic catalysis has been used for the preparation of octadecyl-[9,10-3H]oleate. A double molar quantity of inactive octadecyl oleate and [9,10-3H]oleic acid are heated in benzene solution together with p-toluenesulfonic acid for 2 h at 85°C. After work-up, pure radioactively-labelled wax ester is eluted from a Florisil column in 89% chemical yield [46]. Characterisation o f wax esters

Only two types of crystalline wax esters exist, dependent on the orientation of the aliphatic chains. In one type, the aliphatic chains are tilted towards the end d (00~)

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Fig. 1. Melting points (left) and long-spacings(right) of saturated wax esters [47]. The hgures denote the number of carbon atoms in the alcohol and acid part respestively.

-6

438

F. Spener, Preparation of waxes

group planes, whereas the other type exhibits chains in the vertical direction [47]. When crystals are sampled by crystallisation either from the melt or from acetone solution, in most cases the same types are obtained [48]. By plotting the melting points of a series of saturated wax esters against the difference in the number of carbon atoms in the alcohol and the acyl chains, Iyengar and Schlenk [30] observed a deviation to higher melting points in each series of homologues at a value of+2, as shown in Fig. 1. By studying X-ray diffraction patterns of wax esters, Aleby et al. [47] have found a good correlation of the data on long spacings of wax esters with those on melting points. At the value of +2, again a deviation to higher long spacings within a series ofhomologues was observed (Fig. 1). It was concluded that wax esters with longer spacings are those of the vertical type, i.e. with chains perpendicular to the planes of the methyl ends, whereas the other belong to the tilted type. If one assumes the chains in the tilted type to be straight, the angle of tilt must be close to 63 ° [47]. No differences between vertical and tilted types were found by infrared-spectroscopy. Although no chain-length specificity has been deduced, absorption at or near 1735 cm -I is typical for stretching vibrations of the ester C=O [47,49]. Using mass spectrometry, the identification of individual wax esters, even as constituents in mixtures, has been studied by Aasen et al. [50] in great detail. Of a wax ester having the general formula RCOOR', the fragments RCO2H÷, RCO2H~ and [ R ' - 1] ÷ form characteristic patterns, sufficient for accurate recognition of individual compounds. After reduction with tetradeuterohydrazine, even unsaturated species can thus be recognised.

Synthesis of diester waxes Diester waxes have been detected in skin lipids of mammals [51 ], in the vernix caseosa of new-born babies [52], in sebaceous glands of rodents [53] and in preen glands of birds [54]. One group of molecules, the so called 'diester waxes type I', consists of a 2-hydroxy fatty acid esterified with both a fatty acid and a long-chain alcohol. Compounds of the 'diester wax type II' are composed of a long-chain 1,2alkanediol esterified with two fatty acids. In addition, lipid extracts of preen glands from many birds contain 'uropygial esters', a class of compounds having a longchain 2,3-alkanediol backbone, whose two hydroxy groups are esterified to fatty acids. These unusual diols, the 'uropygiols', occur naturally as threo and erythro isomers [55,56]. Recent investigations have shown that both 2-hydroxy fatty acids and long-chain 1,2-alkanediols of diester waxes from rat skin possess D-configuration [57]. Logani et al. [58] published data allowing the expeditious identification of all types of diester waxes by high resolution NMR.spectroscopy. The literature covering occurrence, composition and biosynthesis of various diester waxes (Fig. 2) has been thoroughly reviewed in a book published recently [4]. Few reports have appeared on the synthesis of diester waxes. Lack of knowledge and lack of availability of stereochemically-defined starting compounds resulted mostly in the preparation of racemic end-products, unless rather tedious steps were undertaken to isolate the naturally-occurring building blocks in amounts sufficient for synthesis.

F. Spener, Preparation of waxes

7'

,',

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CH --OCOR 2

CH --OCOR 2

I

I

I

COOR 3

CH2--OCOR 3

CH - - O C O R 3

439

I

CH 3

Fig. 2. Diester wax type I, Diester wax type 1I, Uropygiol ester.

Diester waxes type I

Nikkari and Haahti in 1968 were the first to report a synthesis of a racemic diester wax of this type [51 ]. 2-Hydroxy palmitic acid is reacted with an equivalent amount of stearoyl cbioride in toluene containing pyridine as catalyst [59]. The intermediate 2-octadecanoyloxy hexadccanoate is isolated by TLC, converted to the corresponding acyl chloride with SOC12 in toluene and subsequently treated with cis-9-docosenol. Pure diester is finally recovered in 38% overall yield after elution from a Florisil column. When 2-hydroxy ester intermediates have been synthesised via the salt method, the overall yield could be raised to around 65% [12,45]. A long-chain alkyl methanesulfonate and the sodium salt of a 2-hydroxy fatty acid, which is readily prepared [11], are thoroughly mixed and kept for 4 h at 120°C. Upon cooling of the melt, the mixture is taken up in diethyl ether, processed the usual way and the resulting crude 2-hydroxy ester is purified by preparative TLC to give a pure compound in 85% yield. This intermediate is dissolved in benzene and small amounts of pyridine, and acyl chloride in benzene is added dropwise at room temperature. Acylation is completed after refluxing the solution for 4 h. Upon isolation of the crude diester wax, a pure product is obtained in 71% yield, following preparative TLC on silica gel and crystallisation from acetone [45]. Diester waxes type H

Only racemic molecules having identical acyl groups have been prepared. The procedure is straightforward, long-chain 1,2-alkanediols are reacted with acyl chlorides in benzene solution in the presence of pyridine. After chromatographic purification and crystallisation, products are obtained in 60-70% yields [45,51]. Long-chain 1,2-alkanediols can be obtained by hydroxylation of long-chain 1-alkenes [60] or by reduction of methyl esters of 2-hydroxy fatty acids with LiAIH4. Uropygiol esters

Mixtures of uropygiols, i.e., threo and erythro 2,3-alkanediols, have been obtained after chemical degradation of naturally-occurring uropygiol esters [55,58]. These mixtures are acylated with palmitic anhydride in benzene using perchloric acid as catalyst [58]. Under the conditions used [61] no epimerisation of diols is observed, however, threo diols succumb to some dehydration, resulting in lower

440

F. Spener, Preparation of waxes

yields (63%). The total synthesis of uropygiols has not been reported as yet. However, Riley and Kolattukudy, pursuing the biosynthesis of uropygiols, reported the chemical synthesis of a precursor Cis-acyloin, namely, 3-hydroxy-[3-14C]octadecane-2-one [62]. Obviously, chemical reduction of this intermediate results in the formation of a long-chain 2,3-alkanediol, thus justifying a closer look at this experimental approach. [1-14C]Hexadecanol in toluene is oxidised with CrO3 on graphite [63] to the aldehyde. 2-Methyl-l,3-dithiane is obtained by treating 1,3-dithiane with excess of both methyl iodide and n-butyl lithium [64]. To the anion of 2-methyl-l,3dithiane, generated with n-butyl lithium in tetrahydrofuran at -30°C, [ 1-14C]hexadecanol is added and the reaction allowed to proceed for 14 h at 0°C. Water is added and the mixture stirred at room temperature. After extraction with chloroform, the resulting intermediate is purified by TLC. Desulfuration of 2-methyl-2(l'-hydroxy[l'-14C]hexadecyl)-l,3-dithiane with HgCI2 and HgO in refluxing 85% methanol, followed by purification on layers of silica gel, yields pure 3-hydroxy[3-14C]octadecane-2-one in 15% yield, based on [ 1-~4C]hexadecanal. Certainly, this reaction merits attention, because of its versatility with regard to variation of starting products, either by using different long-chain alcohols or 2-alkyl dithianes. Synthesis of ether analogues of diester waxes Ester ether waxes type I

The direct alkylation of 2-hydroxy fatty acids with KOH and long-chain alkyl methanesulfonates in xylene according to standard procedures [13] results in the formation of only minute amounts of the desired 2-alkoxy fatty acid intermediates. Good results have been obtained, however, when methyl esters of 2-bromo fatty acids have been reacted with long-chain sodium alcoholates [65]. Simultaneous transesterification of the methyl ester group in course of the reaction led directly to ester ether waxes having identical alkyl chains. For the preparation of 2-alkoxy wax esters having three different chains per molecule, these compounds have been subjected to methanolysis, followed by interesterification with long-chain alkyl acetates of the intermediate methyl ester of 2-alkoxy fatty acids. This general route of synthesis has served for the preparation of a series of homologous ester waxes type I, starting from C14, C16 and Cls compounds in various combinations [65,66]. To a hot solution of long-chain sodium alcoholate in xylene half molar amounts of the methyl ester of a 2-bromo fatty acid in xylene is added dropwise and the reaction allowed to continue for 5 h at reflux temperature. Upon cooling, addition of water and diethyl ether, the organic layer is washed and dried. The solution is taken to dryness and the residue recrystallised twice from abs. ethanol and once from acetone/hexane (5 : I) affording pure ester ether wax in 4 0 ~ 5 % yield. For further variations ofalkyl chains, this compound is treated with 6% HCI in methanol in the presence of benzene, to aid solubilisation of the sample, at 90°C for 24 h in

F. Spener, Preparation of waxes

441

tightly closed screw-capped vials. After work-up the crude product is recrystallised twice from abs. ethanol yielding 45-55% of pure methyl ester of 2-alkoxy fatty acid. This ester is then interesterified with excess long-chain alkyl acetate in the presence of sodium ethoxide according to the procedure described above. The reaction product is taken up in diethyl ether, filtered and the solvent evaporated. Excess long-chain alkyl acetate is removed by crystallisation from abs. ethanol. Purification is achieved by preparative TLC and type I ester ether wax with chains of different lengths is finally precipitated from cold acetone; yields range from 4 0 50% [65]. Diether waxes type H and ester ether waxes O'pe I1

Direct alkylation of 1,2-alkanediols with long-chain alkylmethanesulfonates has led to the formation of diether waxes having identical alkyl chains [65,66]. The potassium salt of diols is prepared with the aid of KOH in xylene, and water formed is continuously removed by ~etropic distillation. Long-chain alkylmethanesulfonates in xylene are added and the solution refluxed for 12 h. In course of the reaction, small amounts of monohydroxy compounds and dialkyl ethers are formed as side products as monitored by TLC. Thus, upon isolation of the crude material, diether waxes are obtained in 40-50% yields after preparative TLC and precipitation from cold acetone [65]. The conduct of synthesis for the preparation of type II diether waxes and ester ether waxes having different alkyl- and acyl chains is outlined in Scheme 1 [65,66].

I CH2--OH

=-

,

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R2-OMs

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R1 ~

ICH~OR 2 CH2--OR3

2 -Tr-OH _--- CH--OR I CH2--OH ell--OR 2 ~H2--OCORa Scheme 1. Route of synthesis for ether analogs of diester waxes type I1

Tritylchloride proved to be a specific blocking agent for primary hydroxyl groups of long-chain 1,2-alkanediols. This has been ascertained by acetylafing the hydroxyhexadecyloxy-hexadecane intermediate and comparing the resulting acetate with l-acetoxy-2-hexadecyloxy-hexadecane and 1-hexadecyloxy-2-acetoxy-

442

F. Spener, Preparation of waxes

hexadecane obtained by different routes. The former has been prepared by alkylating methyl 2-bromopalmitate with hexadecanol. The intermediate hexadecyl-2hexadecyloxy palmitate has been reduced with LiAIH4 to l-hydroxy-2-hexadecyloxyhexadecane and subsequently acetylated to l-acetoxy-2-hexadecyloxy-hexadecane. The other compound has been prepared in 5 steps: Methyl-2-hydroxypalmitate has been converted to methyl-2-trityloxy-palmitate and subjected to hydrogenolysis with LiAIH4 affording 1-hydroxy-2-trityloxy-hexadecane. Alkylation to l-hexadecyloxy-2-trityloxy-hexadecane and detritylation gave 1-hexadecyloxy-2-hydroxy-hexadecane, which in turn has been acetylated to l-hexadecyloxy-2acetoxy-hexadecane. The mass spectrum of 1-acetoxy-2-hexadecyloxy-hexadecane agreed with that obtained from the compound derived according to the synthetic scheme outlined above [65]. The synthesis of diethers and ester ethers starts with the action of tritylchloride on 1,2-alkanediol (in equimolar amounts) in pyridine solution. The mixture is kept at reflux temperature for 5 h. At the end of this time, the mixture is cooled, water and diethyl ether are added and the ethereal layer worked up following standard procedures. After evaporation of the solvent, l-trityloxy-2-hydroxyalkane is freed by chromatography on silica gel from unreacted diol and minute amounts of the ditrityloxy compound. The pure intermediate is subsequently precipated from cold hexane in yields between 45-50%. Alkylation of the tritylated compound with long-chain alkylmethanesulfonates is achieved as outlined earlier. The crude 1-alkoxy-2-trityloxy-alkane is dissolved in a mixture of 95% methanol and diethyl ether and HC1 is passed through the solution until saturation. For complete removal of the trityl group, the solution is refluxed for 5 h and finally taken to dryness. Addition of water and extraction with diethyl ether is followed by isolation of the crude product, which recrystallises from hexane and from acetone affording pure l-hydroxy-2-alkoxy-alkane in 75-81Y~ yield. This compound is either alkylated with long-chain alkylmethanesulfonate to give diether waxes with different alkyl chains, or acylated with acyl chlorides in benzene/pyridine to afford 1-acyloxy-2-alkoxy-alkanes. Pure end products are obtained in either case after preparative TLC and crystallisation from acetone in 5 0 70% yields [65].

Synthesis of sterol esters

The chemistry of sterol esters, in particular cholesterol esters, has been spawned by their important role in biochemistry and pathogenesis of living organisms [67]. In man, cholesterol esters occur as structural components of serum lipoproteins [68] and they are found as constituents of atherosclerotic lesions as well [691. On the one hand, cholesterol esters are known to function as acceptors for acyl chains from phosphatidyl cholines [70], on the other hand they may be regarded as storage molecules for cholesterol, forming subcellular droplets surrounded by a

F. Spener, Preparationo[ waxes

443

monolayer membrane. Upon action of an esterase, cholesterol is quickly released to serve its function as precursor of steroid hormones [71]. In Wolman's disease, a lipidosis caused by low or even complete lack of esterase activity, large accumulations of cholesterol esters are observed [72]. In contrast to man and animals, /3-sitosterol and other 'phytosterols' are the main sterols of sterol esters occurring in plants [73]. For synthetic purposes sterols are available commercially in sufficient purity. Cholesterol can be purified on a large scale by the bromination-debromination procedure of Fieser [74]. Acyl chloride method

In 1910 Abderhalden and Kautzsch [75] reported the synthesis of saturated cholesterol esters. At room temperature, cholesterol is reacted with equivalent amounts of acyl chloride in chloroform and the solution is warmed up until HCI development ceases. The product is precipitated by adding methanol to the reaction mixture and recrystallised. Unsaturated cholesterol esters have been synthesised by Page and Rudy [76] by heating a mixture of cholesterol and an acyl chloride to 70-80°C under nitrogen. When all HC1 has evolved, the melt is briefly heated to 100°C, cooled and extracted with diethyl ether. After work-up, the ethereal solution is taken to dryness and cholesterol esters are obtained after final drying in vacuo. More moderate conditions were employed by Front and Daubert [77], who reacted cholesterol and linoleoyl chloride in chloroform in the presence of quinoline for 3 h at reflux temperature. Upon cooling and evaporation of the solvent, the residue is dissolved in diethyl ether, washed free of base and dried. After concentration of the solution, ethanol is added until slight turbidity, and the product is obtained after several recrystallisations at 0-5°C. As in the synthesis of wax esters, pyridine is the basic catalyst commonly used for the syntl-.esis of sterol esters using acyl chlorides. Thus, cholesteryl laurate was prepared in pyridine solution, using a small excess of acyl chloride over cholesterol [79]. The solution is heated to boiling and cooled after 1 min. The resulting brown solid is dissolved in diethyl ether, washed free of pyridine and lauric acid, and dried. Three recrystallisations from chloroform/methanol afford pure cholesteryl laurate in 80% yield, with the highest melting point (78-78.5°C) thus far reported for this compound. A modification of this approach was used by Swell and Treadwell in 1955 [79], who synthesised cholesterol esters with acyl chains from C2Cla. Cholesterol is added in small portions to a warmed excess of acyl chloride; after the initial heavy reaction has subsided, the mixture is treated with pyridine in amounts equivalent to cholesterol and heated to 80°C for 20 min. Upon cooling, the solid is taken up in abs. ethanol, or in acetone, for esters having chain lengths over C1o. After two recrystallisations from acetone at 0°C, pure cholesterol esters are obtained in yields ranging from 75 to 80%. Labarr6re et al. [80] published infrared-spectra and the separation by paper chromatography of saturated, monounsaturated and polyunsaturated cholesterol esters they had synthesised accordingto

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b~ Spener, Preparation o f waxes

Swell and Treadwell [79]. The same procedure has been used by Deykin and Goodman [81] for the synthesis of tritium-labelled cholesterol esters. After chromatography on alumina, [7a-3H]cholesteryl oleate and [7aPH]cholesteryi linoleate are obtained in 82% and 77% yields, respectively. When cis-polyunsaturated compounds are prepared, less than 2% trans-isomers are formed. [1,2-3H]Cholesteryl oleate has been prepared recently also by Momsen and Brockman [82]. The pyridine-catalysed reaction proceeds under very mild conditions in benzene solution affording yields up to 90% [83]. Cholesterol, dissolved in benzene and pyridine is added dropwise to a solution of acyl chloride in benzene at 0°C during 15 rain, then the reaction is allowed to proceed for 2 h at room temperature, while pyridinium hydrochloride precipitates. Water and diethyl ether are added and the raw product is isolated according to standard procedures. Saturated esters are crystallised from acetone, whereas cholesteryl arachidonate, for example, is purified by chromatography on alumina. An extensive study on the synthesis of plant sterol esters was undertaken by Kuksis and Beveridge in 1960 [84]. These authors took great pains to have weldefined starting products not only with regard to fatty acids, but with regard to sterols as well, a point overlooked in many previous publications. Esters of/3- and 3,-sitosterol, stigmasterol, their saturated analogues and of ergosterol with acids ranging from C2 to C22 and with unsaturated C~s-fatty acids were synthesised under conditions where undesirable effects such as dehydration, isomerisation and polymerisation have been systematically minimised. The sterols are dissolved in a mixture of benzene and petroleum ether, and excess acyl chlorides, dissolved in benzene, toluene or xylene - depending on their solubility - plus pyridine are added. Pyridine is added only in amounts sufficient to bind the HCI formed in the course of the reaction. After refluxing for 30 rain, the solution is cooled, diluted with petroleum ether and directly applied to a silicic acid column. In this way, pyridinium hydrochloride and any undecomposed acylpyridinium stays at the top of the column, whereas pure sterol esters are eluted in 60-95% yield. By using inert solvents and carefully avoiding any formation of free acid this approach is especially suited for preparations involving acid labile ergosterol. The synthesis of saturated and unsaturated cholesterol esters on a micro scale has been worked out by Pinter et al. [85], A 20-200-fold excess acyl chloride is prepared in a small vessel with oxalyl chloride and [4-14C]cholesterol, dissolved in a small volume of diisopropyl ether, is added. This mixture is placed under vacuum at 60°C and allowed to boil until no visible amount of solvent remains. The residue is taken up in hexane and poured onto a silicic acid column and cholesterol esters, chemically and radioactively pure, are eluted from the column with hexane/benzene in yields from 30 to 70%. Recently, Stoffel and Michaelis have synthesised fluorescent-labelled cholesterol esters [86]. The mono- and diunsaturated acyl chlorides used for synthesis are tagged at the w-position with the fluorescent probe, an anthracene group. Stoffel's group had been also the first to synthesise cholesterol esters with 13C-label in both the cholesterol and acyl moieties [87].

17. Spener, Preparation of waxes

445

Anhydride method Although acid anhydrides are known to be the mildest acylating agents, attempts at synthesising long-chain sterol esters have not proved successful under the mild conditions necessary for the preparation of many phytosterol esters [84]. Recently, however, Lentz et al. [88] have worked out an acylation technique on a semimicro and micro scale by simply mixing cholesterol and acid anhydrides at elevated temperature, thus avoiding refluxing at higher temperature, which is necessary in the presence of solvents. Cholesterol and a three-fold excess anhydride are dissolved in CC14for mixing. Upon evaporation of the solvent, the residue is kept for 5 - 1 0 h in inert atmosphere between 60 and 90°C, depending on the chain length of the anhydride. The reaction product is taken up in heptane and purified on a column of silicic acid. [4-14C]Cholesteryl esters of palmitic, oleic and linoleic acids are prepared in yields ranging from 70 to 90%. The starting material, the anhydrides of fatty acids, can be conveniently prepared by the method of Selinger and Lapidot [89]. Direct esterification While cholesterol esters of unsaturated fatty acids were prepared by Page and Rudy in 1930 by the acyl chloride method, more drastic, though less cumbersome, conditions have been employed for the preparation of saturated esters [76]. A mixture of cholesterol and fatty acid is kept at 200°C for 3 h and a stream of CO2 is continuously passed through the melt. Upon cooling and extraction, the desired compounds are obtained after three recrystallisations from ethanol. Stigmasteryl pahnitate and stearate have been prepared by a similar approach [90]. Direct esterification has been achieved also by Kaufmann et al. [91] using p-toluenesulfonic acid as catalyst. Cholesterol and a 25% excess of saturated fatty acid are dissolved in benzene and p-toluenesulfonic acid 0 % of cholesterol) is added. The solution is heated and water formed in the course of the reaction is removed by azeotropic distillation. After 3 - 4 h most of the benzene is distilled off and the residue is taken up in diethyl ether. Upon isolation of the crude product, pure cholesterol esters are obtained after recrystallisation from ethanol, acetone or diethyl ether/acetone. The use ofthionyl chloride as catalyst has been reported recently by Prabhudesai [92]. Cholesterol and a fatty acid in 10% excess are reacted in benzene in the presence of the catalyst, affording yields up to 90%. Reaction times required are 12 h at room temperature, or 1.5 h at 70°C. In teresterification Starting from equimolar amounts of cholesteryl acetate and methyl esters of fatty acids, cholesterol esters have been prepared in the presence of sodium

446

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ethoxide [44]. The reaction is conducted under nitrogen at 80-90°C, while methyl acetate formed is removed continuously by aspiration. The end of the reaction is indicated by a gradually subsiding effervescence. After isolation of the product following established procedures, cholesterol esters of saturated fatty acids are crystallised from acetone, unsaturated species, including cholesteryl arachidonate are purified by silicic acid chromatography. No conjugation and cis-trans isomerisation of double bonds could be detected by the authors in the course of this synthesis; yields range from 70 to 80% [44]. Phillips and Viswanathan [43] have reported the preparation of cholesteryl palmitoleate by reacting cholesterol and methyl palmitoleate in the presence of sodium methoxide in a manner as described for the synthesis of wax esters (see above). Synthesis of sterol ethers Ether analogs of cholesterol esters occur naturally in bovine cardiac muscle, as reported by Funasaki and Gilbertson [93]. The synthesis of long-chain alkyl ethers of cholesterol is achieved by alkylating the potassium salt of cholesterol in benzene with long-chain alkyl methanesulfonates [94]. Pure products are obtained after chromatography on silicic acid and crystallisation from acetone in yields around 5(~o. The configurational integrity of the cholesterol moiety has been ascertained by NMR-spectroscopy.

Epilogue It is obvious that a restriction of this review to compounds composed of alcohols, sterols and fatty acids, having a maximum of two functional groups, must be an arbitrary one. For example, the so-called 'coloured waxes', having a chromophore of isoprenoid origin [95] may be classified as wax esters, and their synthesis involves chemical reactions similar to those described in this article. The 'estolides' constitute another class of naturally-occurring compounds [96], which may be regarded, from a chemical point of view, as part wax ester, part t riacyglycerol. However, as the number of functional groups per molecule increases, the chemistry of these compounds affords the use of blocking agents, as known from the well worked out synthetic procedures for triacylglycerols [97].

References 1 A.H. Worth, The Chemistry and Technology of Waxes, 2nd ed., Reinhold Publ. (1956) p. 1. 2 H.P. Kaufmann, Analyse der Fette und Fettprodukte, Vol. 1, Springer (19~8) p. 305. 3 F.D. Gunstone, An Introduction to the Chemistry and Biochemistry of Fatty Acids and their Glycerides, Chapman and Hall (1967} p. 2.

F. Spener, Preparation o f waxes 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

447

P.E. Kolattukudy (Ed.),Chemistry and Biochemistry of Natural Waxes, Elsevier (1976). A. Simonini, Monatsh. Chem., 13 (1892) 320, A. Simonini, Monatsh. Chem., 14 (1893) 81. A.Gascard, Ann. Chim., 15 (1921) 332. A. Heiduschka and J. Ripper, Bet., 56 (1923) 1736. G.S. Whitby, J. Chem. Soe., (1926) t458. L. Panicz, Monatsh. Chem., 15 (1894) 9. J.W. McBain, R.D. Void and M. Frick, J. Phys. Chem., 44"(1940) 1013. F. Spener and H.K. Mangold, Chem. Phys. Lipids, 8 (1972) 180. W.I. Baumann and H.K. Mangold, J. Org. Chem., 29 (1964) 3055. G. Cegla and H.K. Mangold, Chem. Phys. Lipids, 10 (1973) 354. F. Spener, Chem. Phys. Lipids, 11 (1973) 229. F. Krafft, Bet., 16 (1883) 3018. R. Pummerer and H. Kranz, Bet., 62 (1929) 2620. K.N. Meyer and R. Liihdemann, Helv. Chim. Acta, 18 (1935) 307. K. Burschkies, Ber., 73 (1940) 405. H.P. Kaufmann and J. Pollerberg, Fette, Seifen, Anstrichm., 64 (1962) 908. C.G. Young, A. Epp, B.M. Craig and H,R. Sallans, J. Am. Oil Chem. Soc., 34 (1957) 107. T.R. Wood, F.L. Jackson, A.R. Baldwin and H.E. Longenecker, J. Am. Chem. Soc., 66 (1944) 287. B. Borgstr6m and L. Krabisch, J. Lipid Res., 4 (1963) 357. H. Rheinboldt, O. K6nig and R. Otten, Ann., 473 (1929) 249. A. Griin, E. Ulbrich and F. Krcil, Z. Angew. Chem., 39 (1926) 421. E.H. Man, F.W. Swanner and C.R. Hauser, J. Am. Chem. Soc., 73 (1951 ) 901. P. Brigl and E. Fuch, Hoppe-Seyler's Z. Physiol. Chem., 119 (1922) 280. R.H. Pickard and J. Kenyon, J. Chem. Soc., 105 (1914) 830. J.A. Mills, J. Chem. Soc., (1951) 2332. B.T.R. lyengar and H. Schlenk, Lipids, 4 (1969) 28. J.L. GeUerman, W.H. Anderson and H. Schlenk, Lipids, 10 (1975) 656. J.L. Gellerman, W.H. Anderson, D.G. Richardson and H. Schlenk, Biochim. Biophys. Acta, 277 (1975) 388. W. Bleyberg and H. Ulrich, Ber., 64B (1931) 2504. I. Belluci, Chem. Ztg., 35 (1911 ) 669. T.P. Hilditch and H, Paul, J. Chem. Soc., (1936) 644. D. Swern, G.N. Billen and H.B. Knight, J.Am. Chem. Soc., 69 (1947) 2439. D. Swern and E.F. Jordan Jr., J. Am. Chem. Soc., 67 (1945) 902. D. Swern, E.F. Jordn Jr. and J.B. Knight, J. Am. Chem. Soc., 68 (1946) 1673. H ~ . Knight, E.F. Jordan Jr. and D. Swern, J. Am. Chem. Soc., 69 (1947) 717. P.E. Kolattukudy, R. Croteafi and J.S. Buckner, in P.E. Kolattukudy (Ed.), Chemistry and Biochemistry of Natural Waxes, Elsevier, (1976) p. 290. D.R. Body, Biochim. Biophys. Acta, 380. (1975) 45. A.V. Prabhudesai and C.V. Viswanathan, Chem. Phys. Lipids, 22 (1978) 71. F. Phillips and C.V. Viswanathan, Lipids, 2 (1967)437. V. Mahadevan and W.O. Lundberg, J. Lipid Res., 3 (1962) 106. F. Spener and H.K. Marigold, Chem. Phys. Lipids in press. J.S. Patton, J.C. Nevenzel and A.A. Benson, Lipids, 10 (1975) 575. S. Aleby, I. Fischmeister and B.T.R. lyengar, Lipids, 6 (1971) 421. D.A. Lutz, C.R. Eddy and J.J. ttunter, Lipids, 2 (1967) 204. WJ. Baumann and H.W. Ulsh6fer, Chem. Phys. Lipids, 2 (1968) 114. A.J. Aasen, H.H. Hofstetter, B.T.R. lyengar and R.T. Holman, Lipids, 6 (1971) 502. T. Nikkari and E. Haahti, Biochim. Biophys. Acta, 164 (1968) 294. H.C. Fu and N. Nicolaides, Lipids,4 (1969) 170.

448

F. Spener, Preparation of waxes

53 C.O. Rock, V. Fitzgerald, W.T. Rainey, Jr. and F. Snyder, Chem. Phys. Lipids, 17 (1976) 207. 54 E. Haahti, K. Lagerspetz, T. Nikkari and H.M. Fales. Comp. Biochem. Physiol., 12 (1964) 435. 55 I I.O.A. Haahti and H.M. Fales, J. Lipid Res., 8 (1967) 131. 56 I.A. Hansen, B.K. Tang and E. l~dkins, J. Lipid Res., 10 (1969) 267. 57 P.C. Bandi and H.H.O. Schmid, Chem. Phys. Lipids, 17 (1976) 267. 58 M.K. Logani, D.B. Nhari and R.E. Davies, Chem. Phys. Lipids, 16 (1974) 80. 59 1.. Haahti, Scand. J. Clin. Lab. Invest. 13, Suppl., 59 (1961 ) 13. 60 D. Swern, Org. Reactions, VII (1953) 399. 61 I .H. Matsson, R.A. Volpenhein and J.B. Martin, J. Lipid Res., 5 (1964) 374. 62 R.G. Riley and P.E. Kolattukudy, Arch. Biochem. Biophys., 171 (1975) 276. 63 J.M. Lalancctte, G. Rollin and P. Dunas, Can. J. Chem., 50 (1972) 3058. 64 I.J. Corey and D. Seebach, Angew. Chem., Int. Ed. Engl., 4 (1965) 1075. 65 F. Spener, U. Cramer and H.K. Mangold, Chem. Phys. Lipids in press. 66 1'. Spener and H.K. Mangold, Fette, Seifen, Anstrichm., 73 (1971) 679. 67 D.S. Goodman, Physiol. Rev., 45 (1965) 747. 68 V.P. Skipski in G.J. Nelson (Ed.) Blood Lipids and Lipoproteins, Wiley-lnterscience (1972) p. 471. 69 N. Tuna and H.K. Marigold, in R.J. Jones (Ed.) Evolution of the Atheroselerotic Plaque, University of Chicago Press (1963) p. 85. 70 L. Wallentin and O. Vikrot, J. Clin. Lab. Invest., 35 (1975) 669. 71 S. Wallat and W.-H. Kunau, Hoppe-Seyler's Z. Physiol. Chem., 357 (1976) 949. 72 G. Assmann. D.S. Fredrickson, H.R. Sloan, H.M. I:ales and R J . Highct, J. Lipid Res., 16 (1975) 28. 73 E. Heftmann, Lipids, 6 (1971) 128. 74 L.F. Fieser, Org. Synthesis, 35 (1955)43. 75 E. Abdcrhalden and K. Kautzsch, Hoppe-Seyler's Z. Physiol. Chem., 65 (1910) 69. 76 I.H. Page and H. Rudy, Biochem. Z., 220 (1930) 304. 77 J.S. Front and B.F. Daubert, J. Am. Chem. Soc., 67 (1945) 1509. 78 D. Kritchevsky and M.E. Anderson, J. Am. Chem. Soc., 74 (1952) 1857. 79 L. Swell and C.R. Treadwell, J. Biol. Chem., 212 (1955) 141. 80 J.A. Labarr6re, J.R. Chipault and W.O. Lundberg, Anal. Chem., 30 (1958) 1466. 81 D. Deykin and D.S. Goodman, J. Biol. Chem., 237 (1962) 3649. 82 W.E. Momsen and H.L. Brockman, Biochim. Biophys. Acta, 486 (1977) 103. 83 L.F. Fieser and W.P. Schneider, J. Am. Chem. Soc., 74 (1952) 2254. 84 A. Kuksis and J.M.R. Beveridge, J. Org. Chem., 25 (1960) 1209. 85 K.G. Pinter, J.G. Hamilton and J.E. Muldrey, J. Lipid Res., 5 (1964) 273. 86 W. Stoffel and G. Michaelis, Hoppe-Seyler's Z. Physiol. Chem., 357 (1976) 7. 87 W. Stoffel, O. Zierenberg, B. Tunggal and E. Schreiber, Proc. Natl. Acad. Sci. U.S.A., 71 (1974) 3696. 88 B.R. Lentz, Y. Barenholz and T.E. Thompson, Chem. Phys. Lipids, 15 (1975) 216. 89 Z. Selinger and Y. Lapidot, J. Lipid Res., 7 (1966) 174. 90 A. Heiduschka and H.W. Gioth, Arch. Pharm., 253 (1915) 415. 91 H.P. Kaufmann, Z. Makus and F. Deicke, Fette, Seifen, Anstrichm., 63 (1961) 235. 92 A.V. Prabhudesai, Lipids, 12 (1976) 242. 93 H. Funasaki and J.R. Gilbertson, J. Lipid Res., 9 (1968) 766. 94 F. Paltauf, Monatsh. Chem., 99 (1968) 1277. 95 W. Karrer, Konstitution und Vorkommen der Organischen Pflanzcnstoffe, BirkhauserVerlag (1976)p. 716. 96 H.W. Sprechcr, R. Maier, P. Barber and R.T. Holman, Biochemistry, 4 (1965) 1856. 97 F.H. Matsson and R.A. Volpenhein, J. Lipid Rcs., 3 (1962) 281.