Stereochemical relationships between phytol and phytanic acid, dihydrophytol and C18 ketone in Recent sediments

Geochimica et Cosmochimica Acta. Vol.42. pp I I75 to I 180 0 Pcrpnon PressLtd. 1978.Printed in Great Britam

00167037/78,@K01-I17SSO.2.OOP

Stereochemical relationships between phytol and phytanic acid, dihydrophytol and CIII ketone in Recent sediments* P. W. BROOKS. J. R. MAXWELL and R. L. PATIENCE Organic Geochemistry Unit. School of Chemistry. University of Bristol, Bristol BS8 ITS. U.K. (Received 9 January 1978; accepted

in recked form

31 March

1978)

Abstract-The relative stereochemistry of certain acyclic isoprenoid compounds has been determined from the following sources: two intervals from a core of bottom sediment of a eutrophic lake (Esthwaite Water. U.K.); a marshy shore sediment from a small lake on the watershed of Esthwaite; the bottom sediment of a tropical lagoon (U.S. Virgin Islands); a blue-green alga1 mat and its underlying reduced sediment (Shark Bay. Australia); a decayed blue-green alga (Microcysris aeruginosu) from Esthwaite Water and the products of alteration of phytol as sole carbon source by a bacterial culture from Esthwaite sediment. The stereoisomeric distributions indicate that (i) in the earliest stages of diagenesis of phytol in Esthwaite sediment, reduction to the dihydrophytyl skeleton (free, esterified or bound) is biological, probably microbial, (ii) by analogy with earlier studies of Esthwaite. the phytanic acid in the other sedimentary situations is compatible with an origin in phytol via biological processes; the variations in the distributions reflect the differences in the populations bringing about the conversion, (iii) for the samples where 6,lO,l4-trimethylpentadecan-2-one was examined, the stereochemistry is compatible with an origin in phytol but no distinction can be made between the pathways of oxidation (biological or chemical), and (iv) conversion of phytol by the bacterial isolate under conditions of low oxygen concentration produced 2(RS),6,10.14-trimethylpentadecan-2-01 as the major saturated alcohol; the likely route for its formation is via reduction of the corresponding ketone.

INTRODUCI’ION ACYCLICisoprenoid compounds with twenty or fewer carbon atoms are often relatively abundant and widespread components of ancient sediments and crude oils (for a review see inter &I MAXWELLet al., 197 1). There is a wealth of circumstantial evidence which indicates that these compounds originate in the main from phytol [E-3,7(R),l t(R),15-tetramethylhexadec-2enol] and the phytyl moiety of certain chlorophylls. The present study is a continuation of earlier work (BROOKSand MAXWELL,1974) and describes, in relation to an origin and pathway from phytol, the stereochemistry of certain acyclic isoprenoid compounds from: (i) the bottom sediment of a eutrophic lake (Esthwaite Water), (ii) a marshy sediment surrounding a small lake (Wray Mires Tarn) on the watershed of Esthwaite Water, (iii) the bottom sediment from a tropical lagoon (Great Pond, U.S. Virgin Islands), (iv) a core sample from a blue-green algal mat which included the living microbial community and the underlying reduced sediment (v) a decayed blue-green alga from Esthwaite Water, and (vi) the products of *Part VII of Stereochemical studies of acyclic isoprenoid compounds. For Part VI see BRCOKS P. W., MAXWELL J. R., CORNFORTHJ. W., BUTL~NA. G. and MILNE C. 8. (1977) The stereochemistry of famesane from crude oil. In Advances in Organic Geochemistry 1975 (editors R Cameos and J. Gonil. DD. 81-97. ENADIMSA. t &sent address: l\hkspec Analytical (Specialty Services) Ltd.. Woodchester, Stroud. Gloucester GL5 5HT. U.K.

the alteration of phytol by a bacterial culture isolated from Esthwaite sediment. EXPERIMENTAL. Sample collection

and description

intervals of Esthwaite Water sediment (O-5cm. were obtained from a 3Ocm core taken from the deepest part of the lake; the sample collection and descrip tion of the lake have been described in detail previously (BROOKSet al.. 1976) In August 1973 a heavy bloom of Microcysris aeruginosa (HEENY, personal communication) was present on Esthwaite Water: after extensive photoxidative decay had occurred over a period of intense‘sunlight (7 days), a sample (200 ml 6 g dry weight) was collected in a tow net. Isopropanol (30ml) was added to prevent growth of the alga and associated bacteria Wray Mires Tam (54”3O’N. 3”IO’W) is a small. eutroohic lake fmaximum depth co 1 m; water CIOT) in a marshy area in the watershed of Esthwaite Water. No road or domestic sewage effluent enters the tam. A surface sediment sample was collected from the shore. Isopropanol was added and the sample was stored at - 10°C until analysis. The Great Pond (Virgin Islands) sample was taken from black, reducing mud covered by an algal jelly. It was stored in an air-tight container and despatched to the laboratory (BROOKSet al., 1976). A description of the sample and the collection procedure used for the Shark Bay algal mat core (25cm) is given by Worn et a/. (1977). Two SlOcm)

r

Microbial

alteration

1--~-

-~

of phytol

Full details of the procedures are given by BRINKS (1974). To a solution containing mineral salts medium (1OOml) and phytol (3OOmg) was added an aliquot (I ml] of Esthwaite Water sediment ((Mcm). The sample was maintained (7 days, 25°C) on a rotary shaker to encourage growth of aerobic organisms; the procedure was repeated

1175

1176

P. W. BROOKS.J. R. MAXWELLand R. L. PATIENCE

with an aiiquot (I ml) of the mixture. Growth under more anaerobic conditions was also carried out. To the minerat salts medium (lOOmI) and phytol (300mg) was added an aliquot (I ml) of Esthwaite sediment (&6cm). The mixture was maintained under an atmosphere of N2 (14 days. 25‘ c1.

Detailed procedures for extraction of the samples from Esthwaite Water. Virgin Islands. Shark Bay and Wray Mires Tarn are given by BRWKS et al. (1976). Typically. the samples were extracted with hept~e/isopropanol (1:4). In the case of the Esthwaite and Virgin Islands samples. the residual sediment was acidified with concentrated HCI and the mixture heated under reflux; the hydrolysed sediment was extracted with heptane/isopropanol (1:4). The algal sample was filtered and the residue extracted (Soxhlet. 48 hr.) with toluene/methanol (3:l. ISOml). The microbial growth formed filmy sheets surrounding the phytol droplets: these were removed from the medium and extracted with toluene/methanol (3:1, 12 ml) in an ultrasonic probe. The extract was hydrolysed (methanolic KOH. 7Y,,. 5 ml) by heating under reflux (3 hr) and extracted with hexane (3 x 10ml) after addition of water (10 ml). Acidification of the aqueous layer (HCI. 6 N, to pH I) and extraction with hexane (3 x IOmI) yielded the acidic fraction.

A detailed description is given elsewhere (BROOKSer al.. 1976). Typically, the extract was fractionated into saturated and unsaturated fatty acids, saturated and unsaturated hydrocarbons. ketones and alcohols. The latter were not separated into saturated and un~turated components except for the products of microbial afteration of phytol under NL. individual classes were separated into straight and branched/cyclic fractions by urea adduction.

Routine analyses were carried out on a Varian 2700 or a Perkin-Elmer F17 instrument, each equipped with a flame ionisation detector. under the following conditions: 1.5 m (3.0 m) x 0.3 cm columns packed with 3% SE-30 (37; Dexsil] on Gas Chrom Q, programmed from 130 lo 250°C (160-310°C) at I”/min.. He 1s20 ml/min. The ketone fractions from the Esthwaite (O-5 cm) sediment extract, EsthWaite algae, Wray Mires sediment and the aerobic alteration of phytol were examined under these conditions, as were the branched/cyclic acids from the aerobic alteration of phytol, and the saturated branched/cyclic acids from the alteration of phytol under N1. All other fractions were analysed on the Varian 2700 using open-tubular capillary columns (50 m x 0.25 mm) coated with butanediol succinate (BDS) at 170°C isothermal and 40 psig He. Diastereoisomers of the compounds were separated on BDS (100 m x 0.25 mm) at 155 or 170°C an 40 or 45 psig He (ACKMAN et al.. 1972: Cox et al.. 1972).

2(b)

Gas chrotnarography-mass

spvctrotnrtry (CC-MS)

The system has been described by BROOKSrt al. (1976). Columns typically used were stainiess steel (3 m x 0.16 cm) packed with either 3% OV17 or 3% Dexsif on Gas Chrom Q. programmed from 180 to 280°C at 8”/min. and 180-310°C at 8”fmin.. respectively. with He at ca 3-5 ml/min.

Structures were assigned as follows: (i) comparison of retention data and/or coinjection with standards on 100 m BDS (al1 compounds), (ii) comparison of equivatent chain length (ECL) values on 100 m BDS with a standard (phytanic acid from Esthwaite 0-Scm extract and sediment residue), and (iii) comparison of mass spectra with a standard run under the same conditons (phytanic acid in EsthWaiteO-5 cm extract and from aerobic alteration of phytol; dihydrophytol from Esthwaite O-5 cm extract and sediment residue. and from hydrolysis of the ester fraction in the extract; 6.10,14-trimethylpentadecan-2-one from Esthwaite &5 cm extract, Wray Mires Tarn, Esthwaite alga, and aerobic alteration of phytol; 6,10,14trimethylpentadecan-2-01 from alteration of phytol under N,).

RESULTS

The fractions containing phytanic acid, dihydrophytol or 6, IO,14-trimethylpentadecan-2-01, and 6,10,14-trimethylpentadecan-2-one, were examined by gas chromatographic analyses of the methyl esters. acetates and free ketones, respectively. Only the reiative stereochemistry was therefore determined since enantiomers are not separated on the opticaly inactive stationary phase (BDS) used. It is assumed that the chiral centres corresponding to those at the 7and ll-positions in phytol each have the original R conjuration rather than the opposite con~guration. Table 1 summarises the stereochemical assignments.

Figure 1 shows the glc record of the dihydrophytyl acetate obtained by hydrolysis of the esters from the &Scm layer of Esthwaite sediment and subsequent with the acetylation; the singlet coincides 3(R).7(R),l l(R) isomer (la) in a standard obtained by catalytic hydrogenation of phytol. The elution order in the standard was obtained by comparison with a sample of the RRR acetate (KATESef al., 1967). In the other fractions the RRR (la) isomer predominated markedly over the SRR (1b) isomer (Table I). The ratio of the two isomers in the free alcohol from the

Stereochemical relationships

1177

in Recent sediments

Table 1. Relative abundances of stereoisomers of acyclic isoprenoid compounds*

Phytanic Acid

Sample

: RRR

SRR

Esthwsite O-5 cm Extract Esters fhydrolysed) Residual sediment (hydrolysed)

3

2 na

Esthwaite 5-10 cm Extract Residual sediment fhydrolysed) Virgin Islands Extract Residual sediment (hydrolysed) Shark Bav Extra&

Qihydrophytol SRR

: RRR

1

105

0

All

‘18

ketone

cl8

alcohol

SRR (RRR RR

3

2

2

5

ne

3

2

2

5

na

3

2

2

5

na ~---

: RRR : SRR) Jr nk.

nd' t nd t nd t nd ---_

14

na

na

na

14

na

na

na

na

na

na

RR

0

All

Wray Mires Tarn Extract

na

nd

Esthwaite

na

nd'

RR

2. nd' t nd

? nd na

RR na

t nd 1:l

alga

Microbial alteration of phytol Aerobic Under N2

0

All na

t

-

* Onlyrelative configuration determined; enantiomers not separable under conditions used. 5 Ratio not accurate; very iittk materiai present. t Alcohol fraction not separated into saturated and unsaturated fractions. nd Not detected. ne Present but stereochemistry not examined. na Fraction not analysed. O-5 cm interval could not be accurately measured because of the low concentration of dihydrophytol

No dihydrophytol

could be detected in the marshy

shore sediment from Wray Mires Tarn, the decayed

algal sample, or in the products of microbial alteration of phytol. Phytanic acid The dis~bution of phytanic acid in the sediment from the Virgin Islands is shown in Fig. 2. The ratio of the SRR and RRR isomers (2a,b) is the same for the free acid and the acid obtained by hydrolysis of the extracted sediment (Table 1). The same situation applies to both intervals of Esthwaite sediment, although the ratio differs. The free acid in the algal mat from Shark Bay and in the products of microbial alteration (aerobic) of phytol comprises solely the RRR isomer. 6,10,14Trimethylpentadecat&one

-1

I M E (mins)

s-b

Fig. 1. Gas chromato~~s of dihydrophytol (a~tate). A. From hydrolysis of esters in extract of Esthwaite sediment (O-5 cm). B. A 3(S),7(Rf,l l(R) and 3(R),?(R),ll(R) isomer standard. Conditions: 100 m x 0.25 mm capillary coated with BDS; 170°C. 4Opsig He.

In the four ketone fractions examined (Table l), the C,, ketone showed only one peak (Fig. 3). The configuration was assigned in each case as 6(R),lO(R) by comparison with a standard synthesised from allisomer phytol or by coinjection. 6,10,~4T~met~yl~ntadec~-2-o1 The branched/cyclic alcohol fraction from the alteration of phytol under aerobic conditions was not

P. W.

BROOKS. .I.

R. MAXWELL and R. L.

V.I. RESIDUE

V.I.EXTRACT

ALL

C

(mans)

_j)

separated into saturated and unsaturated fractions, and contained mainly residual phytol. In one experiment, in which an inoculum directly from the mud

was allowed to stand under nitrogen in the presence of phytol. the alcohols were separated into saturated and unsaturated components. The unsaturated fraction contained solely residual phytol, whereas the saturated fraction comprised more than 80% of the C,s alcohol by comparison with an authentic standard. The acetate showed a doublet with a slight preference in favour of the first eluting peak (Fig. 4). Comparison of retention times and coinjection with (2RS)-6(R),lO(R),l4-trimethylpentadecan-2-o1 (4a,b), synthesised from phytol (Cox. 1971) indicated that the bacterial product is 4a,b. The elution order has not been established so it is not known whether the 4mlns

d

w VI $ a ul w

a

a

A n

EX ESTHWAITE 0-5cms

:

a

0 0 w a ALL

I

TIME

(minsl

z” :! w

AM

EX

MICROBIAL ALTERATION

EX

PHYTOL

49

I

Fig. 2. Gas chromatograms of phytanic acid (methyl ester). A. In extract from hydrolysis of residual sediment after extraction of Virgin Islands surface sediment. B. In solvent extract. C. Synthetic all isomer mixture. Conditions as for Fig. 1.

T

w

ISOMER

li0 TIME

PATIENCE

ISOME

R

I

Fig. 3. Gas chromatograms of 6,10,14-trimethylpentadecan-2-one. A. In extract of Esthwaite sediment (G5cm). B. Synthetic all isomer mixture. Conditons as for Fig. 1 except 45 psig He.

-TIME

(mins)

___)

Fig. 4. Gas chromatograms of 6,10,14-trimethylpentadecan-2-01 (acetate). A. From microbial alteration of phytol under conditions of low oxygen concentration. B. Synthetic (2RS)-6(R).lO(R),lCtrimethylpentadecan-2-01 from phytol. Conditions as for Fig. 3 except 155°C.

slight stereochemical preference is in favour of the 2(R)- or 2(S)-isomer (4a or 4b). The C,s alcohol was not detected in any other sample, although the alcohols were not separated into saturated and unsaturated components, and it could be present in low concentrations. DISCUSSION

Phytanic acid, dihydrophytol and the Crs ketone have all been reported to occur in Recent sediments (BLUMERand COOPER,1967; SEVERand PARKER,1969; KAPLAN and BAEDECKER,1970: CRANWELL, 1973; IKAN ef al., 1973; SIMONEITand BURLINGAME, 1973; BRINKS and MAXWELL, 1974; SIMONEIT, 1974; BRINKS et al., 1977, DE LEEUWer al.. 1977).

Laboratory thermal alteration studies at relatively low temperatures (DE LEEUWer al.. 1974, 1977; IKAN et al., 1975) and incubation of radio-labelled phytol with recent lacustrine sediments (BROOKSand MAXWELL, 1974; DE LEEUW et al., 1974. 1977) indicate that phytol is a likely precursor of these sedimentary compounds, although the dihydrophytol and phytanic acid (free and esterified) in Dead Sea sediments more likely derive from the di-0-phytanylglycerol contributed by halophilic bacteria (ANDERSON er al., 1977). The relative stereochemistry of free dihydrophytol and phytanic acid and of those obtained from hydrolysis of the residual sediment has already been reported for the O-5 cm interval of Esthwaite sediment (BROOKSand MAXWELL,1974) but the results are included in Table I for comparison purposes. Table 1 shows that the relative configurations of these two compounds are the same for both sediment intervals, with a preference for the SRR isomer of phytanic acid and for the RRR isomer of dihydrophytol. The

Stereochemical relationships

configuration of free dihydrophytol in the O-5 cm interval could not be accurately determined (Table 1) but compares with the other Esthwaite fractions in showing a strong preference for the RRR isomer. In all the sediment situations a preference was found for one isomer of phytanic acid; these observations extend the earlier work (BROOKS and MAXWELL, 1974) in showing that biological, probably microbial, processes are responsible for the production of the acid from phytoL Chemical processes would be expected to be non-specific and would yield the two diastereoisomers in equal proportions. The wide variation in the ratios in all the samples-from a slight preference of the SRR isomer (2a) in Esthwaite sediment to the sole presence of the RRR isomer (2b) in the products of aerobic alteration of phytol by bacteria isolated from Esthwaite sediment-probably reflects the difference in the populations carrying out the conversion. In the case of dihydrophytol, the results of the present study also extend earlier work (BROOKSand MAXWELL1974) in showing that, in the earliest stages of diagenesis of phytol in Esthwaite sediment, the conversion to the dihydrophytyl skeleton is stereoselective whether it is present as the free alcohol, or as dihydrophytyl esters, or is released only on hydrolysis of residual sediment. These observations present further evidence that the process of double bond reduction is microbiological. The presence of esterified dihydrophytol in EsthWaite sediment, and of phytanic acid in the Shark Bay core and in the products of the aerobic alteration of phytol (Table l), with only the R configuration at C-3 is noteworthy. The only previous report of such a situation relates to a hypersaline environment, i.e. Dead Sea sediment. This contains both free dihydrophytol and free and esterified phytanic acid, comprising only the RRR isomer in each case (la,2b) which derive from the lipids of extreme halophiles of the Halobacterium genus (ANDERSONet al., 1977). In the present study it is significant that the presence of only the RRR isomer of phytanic acid was associated with the simplest microbial ecosystems (Table 1). Preliminary examination of the bacteria isolated from Esthwaite sediment after five subculturing cycles on phytol as sole carbon source showed that the major species was a Lactobacillus or Corynebacterium sp. The Shark Bay core was collected from an intertidal pool (salinity average ca 60ym; DAVIES,1970). The restricted population in the algal mat includes Lyngbya aestuarii and Schizothrix splendida (blue-greens) as the major microbial species, with a layer of unidentified pink filamentous bacteria and pockets of sulphur bacteria also present (GOLUBICand AWRAMIK, 1974). It appears likely, therefore, that in this hypersaline (salinity > SO%,,)environment, the phytanic acid has originated from chlorophyll-derived phytol unlike the extremely hypersahne environment (> 300X below 70 m water depth) of the Dead Sea (ANDERSON er al., 1977) where lipids of Halobacterium

in Recent sediments

1179

spp. are the main source of the phytyl skeleton. The presence of only the RRR isomer of phytanic acid and dihydrophytol in a sediment does not necessarily preclude an origin from phytol. The absence of dihydrophytol in the marshy shore sediment from Wray Mires Tarn, the decayed EsthWaite alga and in the products of the aerobic alteration of phytol possibly reflects the suggestion that its ‘formation from phytol is only favoured in an oxygen-poor environment (DE LEEUW et al., 1977). In the samples examined, 6,191~trimethylpentadecan-Zone (C,s ketone) was present as only the RR isomer (Table 1). The configuration is compatible with a phytol origin but the loss of the centre at C-3 in phytol does not allow a distinction to be made between the pathways of formation of the ketone (biological or chemical oxidation). Its presence in the decayed alga from Esthwaite Water shows that this degradation product can be contributed to the sediment as such in addition to its formation (BROOKS and MAXWELL,1974) in the sediment. The corresponding alcohol, 6,10,14-trimethylpentadecan-2-01, was only found in the products of microbial alteration of phytol under conditions of low oxygen concentration. The most likely route to the alcohol is reduction of the Cis ketone formed by oxidation of phytol. The only reported occurrence of the alcohol in sediments is its presence as the major free alcohol in the Green River shale (Cox et al., 1972). The present study does not provide information about the pathway to the alcohol in this ancient sediment where chemical or biological processes may have been responsible. It does demonstrate, however, that it can be formed by catabolism of phytol. If it occurs in Recent sediments, the latter is a likely route for its formation. Acknowledgements-We thank the Natural Environment Research Council and the Science Research Council for Research Studentships (RLP and PWB respectively). Financial support from the National Aeronautics and Space Administration (Subcontract from NGL-05-003-003) and the Natural Environment Research Council (GR3/655) is gratefully acknowledged.

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P. W. BROOKS.J. R. M~U~WELLand R. L. PATIENCE

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