THE separation of diastereoisomers of the methyl ester of methyl branched fatty acids by gas chromatography1 has recently been extended, using very efficient polar capillary columns, to isoprenoid fatty acids2. The relative stereochemistries (and in some cases, by supplementary use of rotation measurements, the absolute stereochemistry) of the asymmetric centres of such acids as phytanic (3,7,11,15-tetramethylhexadecanoic; I, J= H), pristanic (2,6,10,14-tetramethylpentadecanoic; II, _R = H) and 4,8,12-trimethyltridecanoic (III, J? = H) isolated from present day marine2-6 and terrestrial animal2'7-18 sources have been successfully determined. Speculation about the possible conversion of marine lipids to petroleums, bolstered by the discovery of pristanic and phytanic acids in a California petroleum19 and in recent20 and ancient (refs. 20-22 and unpublished results of Blumer, Douraghi-Zadeh, Douglas and Eglinton) sediments, has provoked interest in the source material and geological fate of these acids. We now report on the diastereoisomeric composition of four acids (I-IV, R = H) isolated from a sample of shale from the Green River Formation (Eocene, 50-60 million years). The results are consistent with the acids having arisen from the phytol (VI) portion of chlorophyll biosynthesized in Eocene times. A 100 g sample of the shale from the 334 m depth of a core taken from Sulphur Creek, supplied by Dr W. E. Robinson of the US Bureau of Mines, was pulverized, demineralized with concentrated hydrochloric and hydrofluoric acids (1 : 1), and hydrolysed with methanolic potassium hydroxide. Extraction of the shale with benzene/methanol (1:1) gave a dark brown residue from which the acidic lipids were isolated by chromatography on a silica/potassium hydroxide column23. Esterification with methanol/benzene/concentrated sulphuric acid produced a crude ester fraction, from which we isolated the methyl esters of the monocarboxylic fatty acids by thin-layer chromatography on silica gel. The methyl esters (78 mg) were further purified by thin-layer chromatography on silica gel impregnated with 20 per cent silver nitrate, and the straight chain fatty acid methyl esters were removed from the branched chain esters by urea adduction (three times)24. The branched methyl esters (15 mg in 0-5 mg portions) were gas chromatographed preparatively on an 'Aerograph A90-P' fitted with a column (7 m x'3 mm) of 3 per cent silicorie gum rubber (.27 52) on 100-120 mesh 'Gaschrom P'. The rate of flow of helium was 60 ml./min and the oven temperature was programmed from 100-300 C at 5/min. The methyl esters of the C14, C15, C16, C19 and C20 isoprenoid acids were collected and their structures were confirmed on an LKB 9000 gas chromatograph-mass spectrometer. The individual esters, which gave only single symmetrical peaks on packed column gas-liquid chromatography, were then analysed on highly efficient open tubular (capillary) columns coated with butanediol succinate polyester (BDS). Standard samples of the same esters, obtained synthetically or from biological sources, were studied similarly. Certain diastereoisomers of methyl pristanate, and of methyl phytanate, could be partially resolved on single columns (50 mx 0-25 mm) with an efficiency (calculated from methyl palmitate) of approximately 45,000 theoretical plates2. The lower isoprenoid fatty acids examined, specifically those possessing only two asymmetric carbon atoms, required two such columns operated in series (approximately 80,000 theoretical plates) for partial resolution of certain diastereoisomers25. The results are summarized in Table 1. Table 1. STERBOISOMEKIC COMPOSITION OF ISOPRENOID ACIDS (I)-(V) BY GAS-LIQUID CHROMATOGRAPHYNo, of diastereo-iBomers possibleNo. of peaks observedOrigin of sample Methyl Assignment t ester L-MenthylIsomer ratios and comments 3,7,11,15-Tetramethylhexadecanoic (phytanic) (I, E = H)(8) Fish and marine mammal oils6 2 Sheep13, ox12, butterfat10-15 \ 2Patient with Refsums syndrome16 f Halophilic bacteria29 1Geological2 Synthetic (total synthesis)30 3Synthetic (from phytol)2r6,10-14-Trimethylpentadecanoic (pristanic) (II, jR = H) (8) Fish and marine mammal oils6 2Sheep14, butterfat11 2 Halophilic bacteria29 1Geological2 Fish oil (Kates/Sen Gupta)|j 5,9,13-Trimethyltetradecanoie(4) Synthetic % (phytol oxidation)1 4,8,12-Trimethyltridecanoic (III, E = H)(4) Herring, seal oil, etc.6-25 1 Sheep fat32 1Geological2 Synthetic (from farnesol)3 2Synthetic % (phytol oxidation)1 3,7,11-Trimethyldodecanoic (farnesanic) (IV, JJ = H)(4) Geological2 Synthetic (from farnesol)2 Synthetic % (phytol oxidation)1 2,6,10-Trimethylundecanoic (V, # = H) (4) Geological1 LDD,DDD LDD,DDDDDD LDD,DDD AllLDD, DDD LDD,DDD LDD,DDDDDD LDD,DDD AllDD DD LD and DD AllDD LD and DD AllLDD/DDD varies from ~ 1-0 to - 1.0 LDD/DDD variable with DDD always in slight excess. Samples exclusively from New Zealand LDD/DDD ~0-7Gas-liquid chromatography shows a triplet due to incomplete resolution of the eight diastereoisomers which would occur as four pairs of antimers Our sample gave a 1 :1 ratio of diastereoisomers, whereas samples from other sources25 all showed a slight DDD predominance. The L-menthyl esters increased the separation factor of the peaksLDD/DDD varied from 0-5 to 3-5 LDD/DDD ratio variable with general DDD predominance. Samples exclusively from New Zealand"Ratio of isomers ~ 1 : 1 An asymmetrical triplet was obtainedSuggests phytol origin Suggests phytol origin LD/DD ratio ~ 0-05 1 :1 ratio of peaks LD/DD ratio ~0-05The methyl esters give an overlapping doublet which is poorly resolved. The L-menthyl esters resolve into an incompletely separated quartet as a result of overlap of two of the diastereoisomers (Fig. 3D) Synthetic (from farnesol)Insufficient material available for informative examination 2 3 All An ill-defined twin peak for the methyl ester was resolvedinto a triplet on conversion to the L-menthyl ester (Fig. 3D) Synthetic % (phytol oxidation)I 1 ? DDDiscussed in text. t LDD phytanate refers to 3L,7D,llD-phytanate, which corresponds in the R andnomenclature to 3,7.R,1 Ifi-phytanate. J Phytol was oxidized with permanganate by the procedure of Murray40. Independently prepared by Drs A. K. Sen Gupta and M. Kates.il Unpublished results of Ackman. Natural phytol (VI), which is known26-27 to have the D configuration at carbons 7 and 11, provides a convenient source of reference acids of established absolute configuration. Thus phytanic acid (I, jR = H) was prepared from phytol (supplied by Stafford-Allen, Ltd) by catalytic hydrogenation (platinum oxide in methanol) and oxidation (chromic acid in acetic acid). The methyl phytanate (I, R - Me), obtained by treatment with diazo-methane and purified by preparative gas-liquid chromatography, is a mixture of two diastereoisomers (3L, TD, HD and 3D, TD, HD) which appear as twin peaks of equal intensity (Fig. 2A) when run on a gas-liquid chromato-graph with the BDS capillary column. The more slowly eluted isomer is known to be DDD by co-injection of a reference sample prepared from DDD dihydrophytyl glyceryl diethers which are produced by certain halophilic bacteria28'29.A mixture of all eight possible diastereoisomers of methyl phytanate (I, JR = Me) might be expected to give four peaks corresponding to the four pairs of enantiomers. Synthetic methyl phytanate30, however, which should have random stereochemistry, gives incomplete resolution resulting in a distorted triplet25. The extent of the interactions between the asymmetric centres is poorly understood. Fig. 1. Open-tubular (capillary) gas-liquid chromatography traces of the methyl esters of the branched chain fatty acids isolated from the Green River Shale. For details of chromatography see Table 2. A, Methyl ester of phytanic acid (I, R = Me). A small shoulder appears at the base of the leading edge. The largest component coincides with authentic 3D,7D,llD,15-tetramethylhexadecanoate. B, Methyl ester of pristanic acid (II, jR = Me). The two major components do not show shoulders and the second coincides with authentic 2D,6D,10D,14-tetra-methylpentadecanoate. C, Methyl esters of reference myristic (14 :0) acid and of 4,8,12-trimethyltridecanoic acid (III, .R = Me). A very small shoulder appears at the base of the leading edge of the major component; the latter coincides with authentic 4D,8D,12-trimethyl-tridecanoate. D, Methyl ester of 3,7,11-trimethyldodecanoic acid (IV, JJ = Me). A small shoulder is apparent on the leading edge. The major component coincides with the corresponding acid prepared by oxidation of phytol.Oxidation of phytol with potassium permanganate gave an acid fraction containing the DD isomers of the acids III, IV and V (# = H). The methyl esters of these acids were identified by combined gas chromatography-mass spectrometry (GO-MS) and on subsequent capillary gas chromatographic examination (Fig. 4) exhibited the expected sharp single peaks. The equivalent chain length (ECL) values of these esters were used in our comparative study of the corresponding geological samples. The separations of the diastereoisomeric pairs of methyl 4,8,12 trimethyltridecanoate (III, .R = Me) and methyl 3,7,11-trimethyldodecanoate (IV, jR=Me) prepared with mixed stereochemistry are shown (Fig. 2(7 and D) for comparison. It should be noted that on optically inactive gas-liquid chromatography liquid phases and supports, no resolution of optical antimers (that is, LD from DL or LL from DD) can be obtained. The preparation involves the nonspecific hydrogenation of farnesol and gives samples showing two barely resolved peaks (respectively DL, LD and DD, LL). Methyl phytanate (I, JR = Me) and methyl pristanate (II, J? = Me) from the Green River Shale were each resolved by capillary gas-liquid chromatography into two peaks (Fig. IA and B), and in the case of the phytanate showed a predominance of the DDD (or LLL) isomer. Geological methyl 4,8,12-trimethyltridecanoate (III, R = Me) and methyl 3,7,11-trimethyldodecanoate (IV, Jf? = Me), on the other hand, gave major peaks (Fig. 1(7 and D) with only about 5 per cent of the faster running LD (or DL) diastereoisomers, indicating that they consist essentially of the DD (or LL) isomers. Geological methyl 2,6,10-tri-methylundecanoate (V, jR = Me) was obtained in such a low yield that the chromatograms are not informative. Capillary gas-liquid chromatography examination of these methyl esters also does not resolve enantiomeric pairs into optical antimers and so assignments such as DDD for one peak in the chromatogram of the geological methyl phytanate are made only for simplicity: it may be correct, but the other (LLL) enantiomer remains a possibility. Further confirmation of the absolute configuration at asymmetric centres in the a or p positions can normally be obtained from optical rotations which are significant in sign and magnitude for these positions25'29'31. Results for other asymmetric centres provide useful supporting evidence when the amount of sample is adequate31'32. The [MJD of the isoprenoid acids, however, is so low that at the concentrations available from geological sources no definite conclusions can be drawn.In the isoprenoid series of acids, methyl branching at all positions except that giving a terminal iso structure to the fatty acid chain gives rise to centres of asymmetry. Variations in the properties of the diastereoisomers will necessarily be small, for any two asymmetric centres along the chain are separated by four freely rotating C-C single bonds. The eight diastereoisomers of synthetic methyl 2,4,6-trimethyloctanoate, which have been separated by gas-liquid chromatography on an open-tubular column into four racemic pairs1 with greater facility than isoprenoid acids, probably owe the greater differences in physical properties to the greater proximity of the asymmetric centres to the carboxyl group and to each other. The contribution of [M]o from the anteiso (o-2) asymmetric centre is also significant25'31.Esterification with an optically active alcohol is the classical approach to resolution of racemates in the differing enantiomeric pairs of isoprenoid acids. In any event, the separation factor between individual diastereoisomers should be increased. A particularly appropriate ester or other derivative might permit the separation of all the possible diastereoisomers for that carbon skeleton. This approach to the analysis of mixtures of branched acids in nature is being investigated. Optical rotation measurements alone are useless when applied to a situation where a number of diastereoisomers differing little in rotation are possible. The gas-liquid chromatographic technique is in principle much more definitive and is applicable to smaller (ug) quantities. Fig. 2. Open-tubular gas-liquid chromatography traces of the methyl esters of synthetic branched chain fatty acids. For details of chromatography see Table 2. A, Methyl ester of phytanic acid (I, jR = Me) prepared by oxidation of dihydrophytpl of plant origin. The second component coincides with authentic 3D,7D,llD,15-tetramethylhexa-decanoate. C, Methyl esters of reference myristic (14 : 0) acid and of 4,8,12-trimethyltridecanoic acid (III, U=Me) prepared by chain elongation from farnesol of plant origin. The second component coincides with authentic 4D,8D,12-trimethyltridecanoate and present-day samples of biological origin. D, Methyl ester of 3,7,11-trimethyldodecanoic acid (IV, .R=Me) prepared from farnesol of plant origin. The second component coincides with the corresponding acid prepared by oxidation of phytol and therefore of DD structure.We have accordingly prepared the L-menthyl esters of a number of isoprenoid acids by heating the acid chlorides under reflux in benzene containing L-menthol. The L-menthyl group should increase the conformational rigidity of the system, and its asymmetrical bulk would be expected to interfere with the diastereoisomeric interactions further along the chain from the carboxyl group. L-Menthyl phytanate (I, _R = L-menthyl) prepared from phytol was more clearly resolved into two (LDD and DDD) gas-liquid chromatographic peaks than was the corresponding methyl ester (Fig. 3A and 2A, respectively), even though a double column was used for the methyl esters. In addition, the L-menthyl esters of DD acids III, IV and V, obtained from oxidation of phytol, again showed single sharp peaks (compare Fig. 4). In contrast, the L-menthyl ester of farnesanic acid (IV, J? = H), prepared from farnesol by reduction, oxidation and esterifica-tion, gave a composite quartet (Fig. 3-D), which must correspond to the four diastereoisomers (the L-menthyl esters of the DD, LL, DL and LD acids). The last peak to elute coincides exactly with that of the ester of IV prepared from phytol and is therefore DD. In the same chromatogram a triplet of lower retention time was assigned to the incompletely resolved diastereoisomers of the L-menthyl esters of 2,6,10-trimethylundecanoic acid (V) which is formed in small yield in the oxidation step. These results encourage us to prepare the L-menthyl esters of the geological isoprenoid acids, but results of this study are not yet available. We have also investigated the possibility of separating the diastereoisomers of di-hydrophytol on gas-liquid chromatography by conversion to their L(+) lactates. Both the lactates and the acetoxy-propionates (formed by acetylation of the lactates) gave resolution on the BDS or 'Apiezon L' capillary column inferior to that obtained with the menthyl esters.Fig. 3. Open-tubular gas-liquid chromatography traces of the L-menthyl esters of synthetic branched chain fatty acids. For details of chromatography see Table 2. A, L-Menthyl ester of phytanic acid sample illustrated in Fig. 2A. D, L-Menthyl ester of 3,7,11-trimethyldodecanoic acid illustrated in Fig. 2D with added reference methyl stearate (18 : 0) and arachidate (20 : 0). The minor triplet peak is believed to be the L-menthyl ester of a secondary oxidation product, 2,6,10-trimethylundecanoic acid (V). Fig. 4. Open-tubular gas-liquid chromatography trace (omitting lengthy blank sections) of an examination of the methyl esters of the branched chain fatty acids derived from permanganate oxidation of phytol. The sample was run on a BDS capillary (100 m x 0-25 mm inner diameter) of 80,000 theoretical plates at 130 with a helium pressure of 50 psi. Shaded peaks are added reference normal C10, C12 and C14 acid methyl esters. Dotted lines reproduce peaks for III and IV of geological origin examined in identical GLC conditions. Identification of 3,7-dimethyloctanoate (DM0), 4,8-dimethylnonanoate (DMN) and 5,9-dimethyldecanoate (DMD) by calculation from GLC retention data39. Other labelled components, including 5,9,13-trimethyltetradecanoate (TMTD), identified also by combined GLC-MS.Our interpretation of the stereochemical data (Table 1) in terms of the isoprenoid acids (I-IV, .K = H) isolated from the shale may be related to the established occurrence2"18 of these and other isoprenoid compounds in present day organisms and recent sediments. Three of the acids (I-III, R = H) are fairly widely distributed in marine animal lipids, although the actual quantities are seldom in excess of 1 per cent of the total fatty acid content. The ultimate source of the carbon skeletons of these acids is likely to be the phytol side chain of the chlorophyll molecule produced by the photosynthetic organisms such as phytoplankton at the beginning of the marine food web. Phytol (VI) obtained from chlorophyll is known26'27 to have the D configuration at both asymmetric centres and it may be provisionally assumed that this applies to chlorophyll from all sources. Phytanic acid (I, R = tL) derived from phytol by reduction and oxidation would therefore have the D configuration at carbons 7 and 11. Unless, however, a stereospecific reduction step is invoked the product will be a pair of acids enantiomeric at carbon 3 (that is, 3D, 7o, HD, and 3L, 7r>, HD). Using the present technique of capillary column gas-liquid chromatography, surveys have revealed a range of values for the phytanate LDD/DDD ratio2'6. Pristanic acid (II, # = H) exhibits the same situation in regard to carbon atom 2. (Pristanic acid and 2,6,10-trimethylundecanoic acid (V) with an a-methyl group will be more susceptible to epimerization at this position.) The results to date for fat-rich tissues indicate that depot fats tend to reflect the ratios of diastereoisomers in dietary fats when preformed pristanic and phytanic acids are ingested, but it is not yet known with any certainty what selective stereochemical effects are involved in the conversion of ingested phytol to phytanic acid or in the eventual elimination or catabolism of these acids in the animal2'6'33. A sufficient variety of samples of marine origin have, however, been analysed to establish that in this milieu LDD phytanate normally exceeds DDD. This may be a result of preferential conversion of the DDD diastereoisomer to pristane in zooplankton where this hydrocarbon is widely observed34'35. In addition to biological processes leading to degradation of pristane and derivatives, there may be less specific chemical oxidation of isoprenoid acids and hydrocarbons in the surface layers of the oceans and products therefrom may be recycled back into the marine food web, complicating assessment of the origin of minor components. Table 2. GAS-LIQUID CHROMATOGRAPHY CONDITIONS EMPLOYED IN FIGURES (WITH RETENTION DATA)Fig. Sample and component1 A (phytanate) B (pristanate) C (14 : 0) C (4,8,12-TMTD) D (3,7,11-TMDD)- A (phytanate) C (14 : 0) C (4,8,12-TMTD) D (3,7,11-TMDD)3 A (menthyl phytanate) D (above) (18 : 0) D (above) (menthyl 2,6,10-TMUD) D (below) (menthyl 3,7,11-TMDD) D (below) (20 : 0)4 3,7-DMO 4,8-DMN 10:0 5,9-DMD 2,6,10-TMUD 12:0 3,7,11-TMDD 14:04,8,12-TMTD 5,9,13-TMTDMethyl esters unless otherwise stipulated. Column No. (BDS)7 77 + 10 7 + 10 7 + 10 7 + 10 7 + 10 7 + 10 7 + 1012 12 12 12 12 7 + 10 7 + 10 7 + 10 7 + 10 7 + 10 7 + 10 7 + 10 7 + 10 7 + 10 7 + 10Temperature 150 150 140 140 130150 140 140 140 170 170 170 170 170130 130 130 130 130 130 130 130 130 130 Adjusted ECL value toPressure (psig) retention time (min) to leading edge leading edge (based on methyl esters) 40 61-7 16-9840 36-5 15-76 80 47-3 14-0080 50-3 14-17 80 38-6 12-9280 82 17-04 80 47-3 14-0080 50-3 14-17 80 28-9 12-9450 62-4 23-85 20 27-1 18-0020 30-3 18-32 20 51-5 19-7220 57-4 20-00 50 7-3 8-6850 13-3 9-94 50 13-6 10-0050 20-7 10-87 50 30-2 11-6950 35-1 12-00 50 54-4 12-9350 89-7 14-00 50 97-5 14-1850 150 15-09 The lower acids (III-V, i? = H) have only two asymmetric centres which correspond to the D centres at carbons 7 and 11 of phytol. If racemization of these tertiary carbons is excluded, a phytol origin requires that each acid be present as a single diastereoisomer, DD. A comparison of acid III of geological origin may be made with present day samples isolated from biologically active systems. In suitable conditions of gas-liquid chromato-graphy methyl esters of synthetic acids III and IV have been partially resolved into two components of nominally equal size (Fig. 2(7 and D)25. The second peak to elute has a retention time identical with III isolated from herring oil, seal blubber and sheep fat and also coincides with III prepared by the oxidation of phytol. In all three natural samples there was no evidence of the earlier eluting peak and the (+) optical rotation of the sheep fat sample32 is believed to confirm a DD structure for the peaks which elute later. Further support for this view is found in observations indicating a general principle that "all D" (or "all L") isoprenoid fatty acid methyl esters will be the last diastereoisomers to elute from the gas-liquid chromatography columns used in this work25. An origin in phytol is therefore most likely for acid III of biological origin. Amounts of acid III are quite substantial in marine lipid systems6, possibly because it may be treated to some extent by esterases and other depot fat biochemical processes as though it were palmitic (hexadecanoic) acid, the saturated fatty acid most prevalent in depot fat systems4'5'36. In contrast, acids IV and V are present in only trace amounts in marine lipid systems (unpublished results of Ackman), although myristic (tetradecanoic) acid is fairly plentiful. The reason for this differentiation, and the nature and details of the diastereoisomers of 3,7,11-trimethyldodecanoic (IV, R = H) and 2,6,10-trimethylundecanoic (V, jR = H) acids in present day biological systems, remain to be studied. For geological samples the findings for acids III and IV (.R = H) are important in another respect, for they show that the asymmetric centres have not been racemized appreciably during the 50-60 million years of the entombment of the compounds themselves or their immediate precursors. Older rocks await study.Many natural products based on the isoprenoid skeleton could conceivably have given rise to the isoprenoid fatty acids in the Green River Shale. Thus a dihydrophytyl glyceryl ether is an important lipid constituent of certain halophilic bacteria28'29, but is known to be entirely DDD. Again, polyunsaturated compounds such as the carotenoids would give one isomer only by stereospecific reduction, or all possible stereoisomers by non-stereospecific reduction. Selective metabolic breakdown could be a further complicating factor, as already mentioned. The C15 isoprenoid acid (IV, J? = H) could be produced by stereospecific reduction of farnesoic (3,7,ll-trimethyldodeca-2,6,10-tri-enoic) acid, which is synthesized in the liver of mammals37, or by oxidation of phyta-2,4-diene which occurs in zoo-plankton38. These results concerning the stereochemistry of the asymmetric centres of these acids from the shale conform best to a phytol derivation, however, presumably originated by the profuse phytoplanktonic growth in the shallow waters of the lakes of that period. This work was supported in part by the US National Aeronautics and Space Administration and the Natural Environment Research Council. The GC-MS instrument was obtained through a grant from the Science Research Council to Drs G. Eglinton and C. J. W. Brooks at the University of Glasgow.