Energy & Fuels 1992,6, 553-559
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Steroid Moieties Attached to Macromolecular Petroleum Fraction via Di- or Polysulfide Bridges P. Adam,?B. Mycke,+J. C. Schmid,? J. Connan,! and P. Albrecht'lt Institut de Chimie, Universitk Louis Pasteur, 1 rue Blaise Pascal, 67000 Strasbourg, France, and Societk Nationale Elf-Aquitaine (Production),64018 Pau, France Received January 10, 1992. Revised Manuscript Received April 29, 1992
The mode of attachment of steroid moieties to a nonpolar macromolecular fraction (NPMF) of Rozel Point seep oil has been studied by selective chemical and thermal degradation procedures. The former comprised hydrogenolysisof C-S bonds with deuterated Raney nickel, cleavage of S-S bonds with lithium aluminum hydride, and thermal cleavage of S-S bonds. GC-MS study of the degradation products showed that sterane molecules formed in high concentration were essentially attached to the macromolecular networks by one linkage located in ring A or B. Careful analysis of the steroid monothiols formed in small amounts upon reductive or thermal cleavage, followed by synthesis of the two major c 2 7 homologs (cholestane-2a-thio1 and cholestane-38-thiol) indicated more accurately that some steroid moieties are linked to the macromolecularmatrix by di- or polysulfide bridges located at equatorial positions 2a and 30. This most probably implies that the greater part of the steroid molecules is attached to the network at the same positions via monosulfide bridges. It follows from these results that A2-sterenesare probably incorporated into a macromolecular network at an early stage of diagenesis or maturation in the source-rock by sulfur cross-linking with other mono- or polyalkenes. Catenated sulfur species produced by bacteria or formed by transformation of bacterial products could be active in these reticulation reactions by a mechanism involving homolytic cleavage of S-S bonds.
Introduction Sulfur-rich crude oils, especially immature ones, are mostly composed of high molecular weight fractions. Unravelling the chemical structure of these complex macromolecules is essential for understanding their origin and their mode of formation; it can bring a wealth of new geochemical informationwhen based on detailed structural characteri~ation.l-~ In a previous article4we have shown that sulfur-rich crude oils from highly anoxic evaporitic or upwelling areas contain a substantial proportion of a nonpolar macromolecular fraction (NPMF) which is hexane soluble and is often eluted from a silica gel column as a reddish band immediately after the aromatic fraction. This least polar part of the petroleum resins has a molecular weight ranging from less than one thousand to several thousand mass units and is indeed essentially composed of low molecular weight hydrocarbon units of planktonic or bacterial origin reticulated by sulfur. It constitutes a convenient model for the study of the role of sulfur as a cross-linking agent. In the present study we have focused our attention on the characterization of steroid moieties attached to NPMF from immature Rozel Point seep oil (Utah) and liberated by reductive cleavage or thermal treatment. We report, in particular, on the positions a t which the steroid skeletons are linked to the macromolecular network and on the
* To whom correspondence should be addressed. + Universitg
Louis Pasteur.
* SociBtg Nationale Elf-Aquitaine.
(1) Ignasiak, T.; Kemp-Jones, A. V.; Strausz, 0.P. J. Org. Chem. 1977, 42,312-320. (2) Schmid,J. C. Ph.D. Thesis, Universit4 Louis Pasteur, Strasbourg, 1986. (3) Sinninghe Damstg, J. S.;de Leeuw, J. W. Adu. Org. Geochem. 1990, 1077-1101. (4) Adam, P.;Schmid,J. C.; Mycke, B.;Strazielle, C.; Connan, J.; Huc, A,; Rive, A.; Albrecht, P. Submitted to Geochim. Cosmochim. Acta.
0887-0624/92/2506-0553$03.00/0
nature of the sulfide or polysulfide bridges by which these structures are attached to the latter. Results and Discussion The NPMF from Rozel Point seep oil is eluted from a silica gel column as a reddish band and represents 32% of the whole crude oil. Desulfurization of this fraction with Raney nickel leads to a high yield of saturated hydrocarbons (about 59% of the original hydrocarbon framework) in which steranes and to a lesser extent methylsteranes of probable plaktonic origin are major constituent~.~ Figure l a shows a gas chromatogram of the branched and cyclic alkanes separated from the total alkanes obtained by desulfurization, by inclusion of the linear alkanes in 5-8( molecular sieves. Steranes and methylsteranes are clearly the predominant class of compounds. The only other major peaks correspond to phytane, squalane, and carotane, whereas hopanes are rather weak.4 Fragmentogram mlz = 217 (Figure lb) shows the distribution of the steranes essentially ranging from c27 to CZS dominated by the C27 and C29 homologs. The high 20Rl 20s ratio of 5aH,14aH,17aH-steranes and the large predominance of 5aH,14aH,17aH over 5aH,14@H,170H isomers already observed in the steranes of the free alkanes confirm the high immaturity of the Rozel Point oil, which must have been generated at an early stage of maturation. Hydrogenolysis of C-S bonds with Raney nickel yields important information on the structures of the hydrocarbon subunits forming the base of the framework of the macromolecular matrix.3 However, this reagent is not specific and cleaves monosulfide as well as polysulfide bridges. It can therefore not bring any information on the nature of the sulfur cross-linking, nor can this method 0 1992 American Chemical Society
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554 Energy 8z Fuels, Vol. 6, No. 5, 1992 Sleranes
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Figure 1. Gas chromatogram of branched and cyclic alkanes formed by Raney nickel desulfurization of nonpolar macromolecular fraction of Rozel Point crude oil (a);mass fragmentogram m / z = 217 showing the distribution of the steranes (b). Conditions: SE 54,30m X 0.3 mm X 0.25pm; 40-100 "C, 10 OC/min; 100-300,4OC/min (a). DB5,30m X 0.25mm X 0.1pm; 55-100 "C, 10 OC/min; 100-300,4 OC/min; Finnigan MAT INCOS 50; IE, 70 eV (b): (1)aaaS c27; (2)@aaRCZ,;(3)a&3R c27; (4)a&% c27; (5) aaaR C27; (6) aaaS CB; (7) BaaR CZS;(8)aN3R Cze; (9) a&?S CB; (10)aaaR CZS,(11)aaaS C Z ~(12) ; BaaR CB; (13)a&?R Cm; (14)a&?S C,; (15)aaaR CB.
provide knowledge on the positions at which the steroid moieties are attached to the macromolecular matrix. Because of the limitations inherent to this method, we have used three other approaches aiming at a better understanding of these points: hydrogenolysis of C-S bonds with deuterated Raney nickel; reductive cleavage of S-S bonds with lithium aluminum hydride; thermal cleavage of S-S bonds. Hydrogenolysis of C-S Bonds with Deuterated Raney Nickel. Deuterated Raney nickel was prepared by digesting the Raney nickel alloy with sodium deuteroxide followed by washing with DzO and EtOD as described el~ewhere.~ The catalyst obtained by this method was slightly less active than the commercial hydrogencontaining catalyst, maybe due to the loss of some fine material during the decantation procedure; it led to a yield of total alkanes about 10% lower. However, despite some minor changes in the relative distributions of the various compounds, the deuterated catalyst basically yielded a very similar alkane fraction to that obtained with the nondeuterated catalyst. GC-MS study of the steranes gave interesting information on the number of linkages by which these molecules are connected to the macromolecular network, as well as on the possible positions of attachment. A comparison of the mass spectra of deuterated and undeuterated steranes clearly showed that the major components had incorporated only one deuterium.
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Figure 2. Mass spectrum of cholestane from hydrogenolysis of nonpolar macromolecular fraction of Rozel Point oil with deuterated Raney nickel (a); with Raney nickel (b); KRATOS MS 80; IE, 70 eV, GC-MS.
Figure 2 shows, for example, the mass spectral data obtained in the case of cholestane. It can be seen from the comparison between the deuterated and undeuterated molecule that our reagent was not fully deuterated; it is probable that this uncomplete deuteration can be explained by the large excess of catalyst along with a kinetic effect in favor of the minor but more reactive hydrogenated part of it. The catalyst was, however, good enough to serve the purpose. It is indeed obvious from Figure 2that the molecular ion [M+l = 372,fragment mlz = 367 (M+ - 151,as well as fragments I (mlz = 217)and I1(mlz = 149) corresponding to the left part of the molecule were essentially enhanced by one mass unit in the deuterated compound, whereas rearrangement fragment I11 (mlz = 262) corresponding to the right part of the molecule remained unchanged. These results were clear evidence that the incorporation of one deuterium did not occur randomly on the steroid molecule but was located either on ring A or ring B. The points of attachment of the steroid skeleton to the macromolecular network could therefore be restricted to one or several of the following positions 1,2,3,4,6,6, and 19 (Figure 3). However, further studies were necessary to locate these positions more precisely. Reductive Cleavage of S-S Bonds with Lithium Aluminum Hydride. Lithium aluminum hydride is a reagent well-known to selectively cleave di- and polysulfide bridges (but normally not monosulfides)6?6to give the corresponding thiols (Figure 4a). Treatment of the high (6)Gaylord, N. G.Reduction with Complex Metal Hydrides, Interscience Publishers: New York, 1956. ( 6 ) Micovic, V. M.; Mihailovic, M. L. Lithium Aluminium Hydride in Organic Chemistry; Naukna Knjiga: Belgrade, 1965.
Steroid Moieties Attached to Macromolecular Petroleum
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(Raney nickel, Dz)of nonpolar macromolecular fraction of Rozel Point oil and possiblepositionsof attachment. 4-Methylsteroids only tentatively attributed. a
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Figure 5. Gas chromatogram of steroid thiols obtained by reductive cleavage with LiAlHd of nonpolar macromolecular fraction of Rozel Point oil (a);steroid thiols obtained by thermal treatment (200 O C , 48 h) (b). Conditions: DB5,30 m X 0.25 mm X 0.1 Fm; 40-100 O C , 10 OC/min; 100-300 O C , 4 OC/min.
in steranes for the right part of the molecule, were consistent with the results obtained in the reductive cleavage with deuterated Raney nickel. They confirmed the presence of one thiol function located either on ring Figure 4. Cleavage of di- or polysulfide bonds with LiAlHd (a). Cleavage of di- or polysulfide bonds by thermal treatment (b). A or B of the steroid skeleton. Moreover, the existence Cleavage of monosulfide bonds by thermal treatment (c). of fragments mlz = 370 + n X 14 corresponding to the loss of H2S from the molecular ions, along with the occurrence molecular weight fraction from Rozel Point oil with an of fragmenta at mlz = 316 + n X 14 and mlz = 215 bring excess of this reagent in tetrahydrofuran under reflux more accurate information on the positions of attachment indeed yielded a small (l%), but very informative low of the steroid molecule. Indeed these fragmentsare typical molecular weight fraction dominated by steroid thiols with features displayed by A2-sterenes.' Fragmentation of the a carbon number distribution ranging from c27 to C ~ O latter leading to mlz = 316 + n X 14 results from a retro(Figure 5). Diels type rearrangement8 operating at ring A, which Mass spectral data obtained by GC-MS indicated that implies without any doubt that the thiol function must be the fraction was essentially composed of thiosteranes (Czr located either at position 2 or position 3 or at both. Ca) and some methylthiosteranes (essentially (3%and Cw). To confirm this hypothesis, we have synthesized the The major sterane thiols display similar mass spectra following three c27 thiols: cholestane-2a-thiol(l),choles(Figure 6) with molecular ions at mlz = 404 + n X 14 ( n tane-38-thiol (2), and cholestane-3a-thiol (3). The syn= 0-2) and the fragment ions at mlz = 249 and 181,which thetic schemes used for this purpose are shown on Figure correspond to the sulfurated counterpart of those shown 7 and described in detail in the experimental part. by steranes at mlz = 372 + n X 14,217 and 149. These thiols can be conveniently studied by using the mlz = 249 (7) Dastillung, M.; Albrecht, P. Nature 1977,269, 678-679. fragmentogram (Figure 6). These data, along with the (8) Budzikiewin, H.; Djerassi, C.; Williams,D. H. Mass spectrometry presence of fragment mlz = 262, the same as that observed of organic compounds; Holden-Day: San Francisco, 1967.
666 Energy & Fuels, Vol. 6,No. 5, 1992
Adam et al.
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Figure 6. Mass spectra of the two major C27 steroid thiols obtained by reductive cleavage of a nonpolar macromolecular fraction of Rozel Point oil. Conditions: Finnigan MAT INCOS 50; IE, 70 eV; GC-MS. (a) Cholestane-2a-thiol. (b) Cholestane38-thiol. (c) Mass fragmentogram of ion mlz = 249 showing the distribution of the steroid thiols obtained by reductive cleavage of the nonpolar macromolecular fraction (eame conditions as Figure lb).
The mass spectra of the three synthetic standards are shown in Figure 8. Desljite their overall similarity, they display some significant differences. Indeed in compound 3, formation of cholest-2-ene (mlz = 370) by elimination of HzS is obviously favored due to the axial position of the thiol function as compared to cholestane-3@-thiol(2),where the function is located in an equatorial position. In the latter, elimination of HzS also takes place (as can be judged from the occurrence of fragment mlz = 215) but intermediates at mlz = 370 and mlz = 316 are formed in small amounta only. These fragments appear to be more favored in cholestane-2a-thiol (1) despite the unfavorable equatorial position of the thiol function. Comparison of mass spectral (Figures 6 and 8) and chromatographic data of the synthetic standards with those of the thiols obtained by reductive cleavage of the macromolecular fraction of Rozel Point oil with lithium aluminum hydride showed that the two major CZ, thiols are cholestane-2a-thiol (1) and cholestane-3@-thiol (2) which both bear the thiol function in the more stable equatorial configuration. Details concerning the identification of these thiols have been described elsewhere.9 (9) Adam, P.; Schmid, J. C.; Connan, J.; Albrecht, P. Tetrahedron Lett. 1991, 32, 2955-2958.
Figure 7. Synthetic scheme used for the preparation of cholestane-3fl-thiol(a);cholestane-2a-thiol(b);cholestane-3a-thiol(c). (a) MsC1; (b) AcOCs 18-crown-6;(c) KOH MeOH; (d) l-methyl2-fluoropyridiniumtosylate;(e) sodium Nfl-dimethyldithiocabamate; (f) LiAlH4; (g) Ip CF3COZAg.
Cholestane-Ba-thio1(3),which displays a mass spectrum similar to that of cholestane-2a-thiol(l)but a different GC retention time, could not be detected in significant amounts in our sample. From mass spectral data and the results from Raney nickel hydrogenolysis, it can be inferred that the CZSand C29 steroid thiols correspond to homologs of the C27 thiols alkylated at (2-24. Furthermore it is clear from the gas chromatogram (Figure 5) and GC-MS studies (Figure 6) that the mixture contains minor amounts of other yet unidentified steroid thiols which accompany the 2a and 30 thiols. They mostly correspond to other isomers of the steroid skeleton bearing the thiol function a t the same positions, as suggested by their mass spectra. ThermalCleavageof S-S Bonds. Thermal treatment (200 "C, 48 h, sealed tube under argon) of NPMF from Rozel Point oil,which is susceptibleto break preferentially the more fragile S-S bonds (Figure 4b),l0leadsto a mixture of steranes, sterenes, and thiols which are again dominated by steroid thiols. In fact, the distribution of the latter shows great similarities with those obtained by reductive cleavage with lithium aluminum hydride (Figure 5). This is obvious from the respective gas chromatograms and has been confirmed by GC-MS. The slightly higher yields of steroid thiols (1.3%) obtained by thermal cleavage as compared to reductive (10) Houben-Weyl Methoden der organischen Chemie, Band I X Schwefel, Selen, Tellur- Verbindungen; G.Thieme Verlag: Stuttgart, 1955; p 80.
Steroid Moieties Attached to Macromolecular Petroleum
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Energy & Fuels, Vol. 6, No. 5, 1992 557
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Figure 8. Mass spectra of synthetic steroid thiols: (a) cholestane2a-thiol; (b) cholestane-3j3-thiol; (c) cholestane-3cu-thiol. Conditions: Finnigan MAT INCOS 50; IE, 70 eV; GC-MS. cleavage with lithium aluminum hydride could be due to a small contribution of compounds formed by cleavage of C-S bonds (Figure 4c) or to a lack of access of the reducing agent. Our results from reductive and thermal cleavage of S-S bonds demonstrate that steroid moieties are attached to the macromolecularnetwork via di- or polysulfide bridges at position 2a and 3j3 of the steroid skeleton. Kohnen et al.11 have tentatively confirmed these results, based on preliminary communications by us,12J3 in the case of steroid moieties formed upon selective cleavage of di- or polysulfides occurring in macromolecular fractions of an immature sulfur-rich shale. Moreover,our data obtained from hydrogenolysiswith deuterated b e y nickel indicate that the bulk of the steroid moieties are monoattached. They must be connected to the macromolecular network via monosulfide bridges located at these very positions (Figure 9). A rough estimate of the proportion of di- or polysulfide bridges can be deduced from the various experiments. Indeed steranes represent about one-third of the alkanes neoformed upon desulfurization of NPMF. Since they are monoattached, it would mean that a t least 3 % of the steroid skeletons are connectedvia di- or polysul(11) Kohnen, M. E. L.; Sinninghe Damst4 J. S.; Kock-van-Dalen, A. C.; de Leeuw, J. W. Geochim. Cosmochim. Acta 1991,55, 1375-1394. (12) Mycke, B.; Schmid, J. C.; Albrecht, P. Oral presentation at the 197th American Chemical Society Meeting, Symposium on Geochemistry of Sulfur in Fossil Fuels, Dallas, 1989; Geoc Abstract 24. (13) Mycke,B.;Schmid,J. C.;Albrecht,P.;Connan,J. Oralpresentation at the 14th International Meeting on Organic Geochemistry, Paris, 1989, Abstract 338.
Figure 9. Mode of attachment of sterane skeleton to macromolecular matrix in sulfur cross-linkedmacromolecularfraction of Rozel Point oil. n > 1, R = H, CH3, C~HE. fide bridges. This low percentage practically implies that a hydrocarbon subunit which is di- or multiattached is certainly not connected to the macromolecularmatrix via more than one di- or polysulfide bridge. This explains why the thiols obtained by hydrogenolysis with lithium aluminum hydride or by thermolysis are essentially composed of steroid structures and confirmstherefore that the long alkyl or polyisoprenoid chains occurring in the NPMF macromoleculesare most probably multiattached as already indicated by deuteration experiments. The positions of attachment of the steroid moieties to the macromolecular matrix almost certainly imply that A2-sterenes must play a key role as intermediates susceptible to react with active catenated sulfur species formed by bacterial processes.4 This assumption is quite reasonable given the fact that A2-sterenesare well-known transformation products of sterols and stanols, formed in recent sediments at the early stage of diagenesisq7 It implies and confirms, moreover, as has been mentioned in a previous article,4 that the cross-linking reaction with sulfur species leading to the attachment of the steroid moieties to the macromolecular matrix occurs in the sediment as long as A2-sterenesare still present, e.g., during the early stage of diagenesis or early maturation. Reductive cleavage of NPMF of Rozel Point seep oil has shown that it is mainly composed of linear, branched, and cyclic alkane subunits essentially interconnected with mono- but also with some di- or polysulfide bridges. Our results therefore suggest that the cross-linking process is likely to take place between A2-sterenes and linear, branched, or cyclic mono- or polyalkenes. The catenated sulfur species (elemental sulfur or polysulfides) which are most likely to be operative in these reactions could be directly produced by bacterial activity (e.g., photosynthetic sulfur bacteria)l4or by chemical processes resulting from various equilibria between reduced sulfur species (e.g., H2S and elemental sulfur) or from oxidation of H2S (producedby sulfate reducingbacteria).'& Under the effect of maturation sulfur would be gradually eliminated from S, bridges, increasingly leading to the formation of the more stable C-S-C linkagesm4 Polysulfides have been mentioned as being the active species in the formation of di- and trisulfides with the (14) Brock, T. D.; Madigan, M. T. Biology of microorganisms;Prentice Hall: New York, 1988. (15) Postgate, J. R. The sulphate reducing bacteria; Cambridge University Press: Cambridge, 1979.
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558 Energy & Fuels, Vol. 6,No. 5, 1992
phytane skeleton occurring in recent sediment.l6 The authors suggest an anionic attack on the double bonds of phytadiene. This type of mechanism, however, does not seem very likely in order to explain incorporation of Azsterenes into a macromolecular network. Indeed, polysulfides exist in natural environments in a rather limited range of pHl7 which is a priori not compatible with the severe acidic conditions required for their addition on an isolated double bond.ls Another possible mechanism of incorporation of polysulfides HS,-or Sn2-intoorganic matter is a Michael addition on activated double bonds.lg Such a mechanism could indeed explain connection of a certain type of molecules based, for instance, on the phytane skeleton (e.g., additions on phytenal or phytenic acid) but cannot be considered in the case of sterenes in which the double bond is not activated. It seems likely therefore that we have to deal with a reaction between a double bond and catenated sulfur species which could proceed via a radical type mechanism. Radicals would be formed by homolytic cleavage of S-S bonds occurring in polysulfides or elemental sulfur and further react with alkenes in a cross-linking, vulcanization type p r o ~ e s s .Such ~ a radical type mechanism has already been s u g g e ~ t e in d ~order ~ ~ ~to~explain incorporation of sulfur into geological organic matter (protokerogen and humic acids, respectively) at early stages of diagenesis. However, at this stage a heterolytic cleavage of S-S bonds followed by an electrophilic attack of a double bond cannot be excluded.21 Conclusion
Investigation of NPMF of Rozel Point seep oil by reductive and thermal cleavage of C-S and S-S bonds has led to the elucidation of the mode of linkage of steroid moieties to the macromolecular matrix. Indeed, hydrogenolysis of C-S bonds with deuterated h e y nickel liberated a great proportion of steranes. The latter were, as shown by GC-MS,essentially monodeuterated on ring A or B, indicating one point of attachment to the macromolecular network. Furthermore, selective reductive cleavagewith lithium aluminum hydride, as well as thermal cleavage of S-S bonds, yielded small amounts of steroid monothiols, the two major homologs of which have been identified by synthesis as cholestane-2a-thiol and cholestane-3~-thiol.These results show that some steroid moieties (about 3%) are monoattached to the macromolecular network via di- or polysulfide bridges located either at position 2 or 3 and that by far the greater part of the steroid molecules are probably attached at the same positions via a monosulfide linkage. They further imply that the formation of these macromolecular fractions probably takes place at early stages of diagenesis or maturation by cross-linking A2-sterenesand other monoor polyalkenes with catenated sulfur species (polysulfides, elemental sulfur) produced by bacterial processes. Radicals formed by homolytic cleavage of s-S bonds could (16) Kohnen, M. E. L.; Sinninghe Damst4, J. S.; Ten Haven, H. L.; de Leeuw, J. W. Nature 1989, 341, 640-641. (17) Aizenshtat, Z.; Stoler, A.; Cohen, Y.; Nielsen, H. Adu. Org. Geochem. 1983, 279-288. (18) Prilezhaeva, E. N.; Shostakovskii, M. F. Rum. Chem. Rev. 1963, 32, 399-425. (19) LaLonde, R. T.; Ferrara, L. M.; Hayes, M. P. Org. Geochem. 1987, 11, 563-571. (20) FranCois, R. Ceochim. Cosmochim.Acta 1987,51, 17-27. (21) Bateman, L.; Moore, C. G.; Porter, M. J. Chem. SOC.1958,28662879.
eventually be the reactive species in the buildup of the macromolecular network. Simulation experimentsaiming at a better understanding of these mechanisms will be reported elsewhere. Experimental Part Sample Description. Rozel Point seep oil (Utah)is a surface sample containing 15% sulfur.22 The source rock of the oil is supposed to be a hypersaline deposit of Miocene age.23 The separation scheme of the sulfur cross-linked nonpolar macromolecular fraction has been described in a previous article.4 Analytical Techniques. Gas chromatographicanalyseswere performed on Carlo Erba 4160 gas chromatograph with an oncolumn injector. Gas chromatography-mass spectrometry analyses were mostly performed on a Varian 3400 gas chromatograph equipped with an on-column injector and a DB5 column (30 m x 0.25 mm; film thickness 0.1 wm) connected to a Finningan MAT INCOS 50 mass spectrometer operating at 70 eV in E1 mode. Fractions were injected at 55 OC in hexane. The temperature was programmed from 55 to 100 "C at 10 OC/min and then from 100 to 300 "C at 4 OC/min. The temperature was then held at 300 OC for 20 min. Several mass spectrometric measurements were carried out under similar conditions on Kratos MS 80, LKB 9OOOS and Finnigan MAT TSQ7O instruments by GC-MS or direct introduction. NMR measurements were made on Bruker AM-400 and Bruker WP-200-SY. Deuteration of Raney Nickel. The procedure used has been described el~ewhere.~ Reductive Cleavage of S-S Bonds with LiAlH,. The high molecular weight fraction from Rozel Point oil (424 mg) was treated for 8 h with LiAlH4 (200 mg) in anhydrous THF under reflux. After hydrolysis (pH = I), the mixture was extracted with ether. The ether phase was then washed with water. The extract (100 mg) was separated by chromatography on silica thin layer with hexane as eluent (Rr0.15-0.85), yielding 1 mg of a mixture of steroid thiols (1% 1. Thermal Treatment. The macromolecularfraction (190mg) was heated at 200 OC for 48 h in a glass vessel purged with argon and sealed under vacuum. The pyrolysate was chromatographed on a short silica gel column by extensive elution with hexane yielding a fraction which was further separated by thin-layer chromatography on silica gel (elution with hexane; Rf0.15-0.85). A mixture (2.4 mg, 1.3%)containing mainly steroid thiols was obtained. Preparation of Reference Compounds. Cholestan-Sfl-ol Mesylate. To a solution of 6.45 g of cholestan-3fl-ol(16.6mmol) in 500 mL of anhydrous methylene chloride was added 5 mL of triethylamine and 2 mL (25.8mmol) of methanesulfonyl chloride under argon. The mixture was allowed to stand for 16 h. The solution was then poured on crushed ice and extracted with methylene chloride. The organic phase was dried with MgSO,. After removal of the solvent, 7.65 g (98%) of the mesylate was obtained. 1H NMR (400 MHz, CDC13) 6 (ppm) 0.65 (s,3 H, H-18), 0.83 (e, 3 H, H-19), 0.867 (d, 3 H, J = 6.6 Hz, H-26 or H-271, 0.872 (d, 3 H, J = 6.6 Hz, H-26 or H-27), 0.90 (d, 3 H, J = 6.5 Hz,H-21), 3.00 (s,3 H, CH3S03), 4.62 (m, 1 H, H-3). SM (EI, 70 eV), m/z (re1 intensity): 466 [M+] (52%),451 (8),370 (38), 316 (15), 312 (39), 215 (loo), 147 (28), 84 (33). Mp 119 "C. Cholestan-Sa-01Acetate. Cholestan-3j3-01methanesulfonate (6.58 g, 14.1 mmol) and 18-crown-6(4 g, 15.2 mmol) were added to a suspension of cesium acetate (15 g, 78.2 mmol) in 120 mL of anhydrous toluene. The mixture was kept under argon at 80 O C for 30 h. The suspension was then poured in water and extracted with CHC13. The organic extracts were washed with water and dried, and the solvent was removed under reduced pressure. Chromatography on a silica gel column with hexaneethyl acetate (95/5) yield cholestan-3a-01acetate (4.47 g, 73%). IH NMR (400 MHz, CDC13) 6 (ppm) 0.66 (s,3 H, H-18),0.80 ( 8 , (22) Eardley, A. J. Oil seeps at Rozel Point. Special studies No. 5, Utah Geological and Mineralogical Survey, 1963; pp 5-26. (23) Bortz, L. C. Exploration for heavy crude oil and natural bitumen, AAPG Studies in Geology 25, Meyer, R. F., Ed.; 1984; pp 555-563.
Steroid Moieties Attached to Macromolecular Petroleum 3 H, H-19), 0.870 (d, 3 H, J = 6.6 Hz, H-26 or H-271, 0.875 (d, 3 H, J = 6.6 Hz, H-26 or H-27), 0.91 (d, 3 H, J = 6.5 Hz, H-21, 2.06 (s,3 H, CH3CO2), 5.02 (m, 1H, H-3). SM (EI, 70 eV), mlz (re1 intensity) 430 [M+] (20%),415 (41,370 (1001,355 (551,316 (16), 275 (16), 257 (E),230 (25), 215 (99),201 (22), 147 (39). Mp 95 "C. Anal. Found: C 80.99,H 11.95. Calcd for CaHmO2: C 80.87, H 11.70. Cholestan-3a-01. Cholestan-3a-01 acetate was treated with a solution of KOH (30 g) in methanol (900 mL). The mixture was allowed to stand for 12 h at room temperature. The solvent was removed under reduced pressure, and water added to the residue. This mixture was then extracted with CHCl3. The extract was washed with 2% HCl and water, dried, and evaporated to give cholestan-3a-ol(3.65g, 97%) which can be used without further purification. 1H NMR (200 MHz, CDCl3) 6 (ppm) 0.64 (s,3 H, H-18), 0.77 (8, 3 H, H-19), 0.86 (d, 6 H, J = 6.9 Hz, H-26 and H-27), 0.89 (d, 3 H, J = 7.0 Hz, H-211, 4.04 (m, 1 H, H-3). SM (EI, 70 eV), mlz (re1 intensity): 388 [M+l (35%),373 (21), 370 (2), 355 (E),316 (2), 262 (19), 234 (74), 233 (1001, 217 (39, 215 (88), 165 (46), 147 (23). Mp 187 OC. Anal. Found C 83.44, H 12.57. Calcd for C27HeO: C 83.44, H 12.45. Cholestan-38-01N,N-Dimethyldithiocarbamate.To a suspension of 1-methyl-2-fluoropyridiniumtosylate (3.24 g, 11.4 mmol) in anhydrous chloroform (75 mL) was added a solution of cholestan-3a-ol(3.41 g, 8.78 mmol) and triethylamine (2 mL) in dry chloroform (75 mL). The mixture was stirred under argon at room temperature for 1 h. A homogeneous yellow solution was then obtained. Chloroform was evaporated under reduced pressure and the remaining solid dissolved in dry DMF (150 mL). Anhydrous sodium N,N-dimethyldithiocarbamate (2.73 g, 19.1 mmol), dried in an oven at 125 OC, was added and the mixture was heated at 80 "C for 2.5 h. After extraction with chloroform, the organic extract was washed with water, dried, and evaporated under reduced pressure. Chromatography over silica gel (hexanelethyl acetate, 85:15) afforded pure cholestan38-01NJV-dimethyldithiocarbamate(3.13g, 72 % 1. lH NMR (200 MHz, CDCl3)6 (ppm) 0.64 (s,3 H, H-18),0.81 (s,3 H, H-19),0.86 (d, 6 H, J = 6.8 Hz, H-26 and H-27), 0.89 (d, 3 H, J = 7.0 Hz, H-21), 3.33 (s,3 H, -NMe2), 3.53 (s,3 H, -NMeZ), 3.81 (m, 1H, H-3). SM (EI, 70 eV), m/z (re1intensity): 491 [M+l (17%),371 (8), 122 (loo), 121 (93),95 (10). Mp 197 "C. Anal. Found: C 73.21, H 10.95, N 2.84. Calcd for C N H ~ ~ S ZCN73.26, : H 10.86, N 2.85. Cholestane-38-thiol. LiAlH4 (500 mg, 13.2 mmol) was added to a solution of cholestan-38-01 N,N-dimethyldithiocarbamate (2.83 g, 5.76 mmol) in 200 mL of dry ether. The suspension is allowed to stand at room temperature, under argon, for 18 h. After hydrolysis and extraction with ether, the organic layer was washed with water,dried, and evaporatedunder reduced pressure. Chromatography of the residual solid over silica gel with hexane as eluent yielded cholestane-38-thiol (2.10 g, 90%). 'H NMR (200 MHz, CDCl3) 6 (ppm) 0.64 (s,3 H, H-18),0.79 (s,3H, H-19), 0.86 (d, 6 H, J = 6.5 Hz, H-26 and H-27), 0.89 (d, 3 H, J = 6.4 Hz, H-21), 2.75 (m, 1 H, H-3). GC-MS (EI, 70 eV), m/z (re1 intensity): 404 [M+] (25%),389 (111,316(I),249 (loo),250 (66), 215 (25), 216 (S),217 (25), 182 (20), 181 (25). Mp 103 OC (lit. 103-105 OC). Anal. Found: C 79.89, H 11.89. Calcd for C27HtsS: C 80.12, H 11.95. 3a-Iodo-cholestan-2~-olTrifluoroacetate. Iodine (1.44 g, 5.67 mmol) and cholest-2-ene(600 mg, 1.62 mmol) are added, under argon, alternativelyand by small portions, to a stirred suspension of silver trifluoroacetate (3.18 g, 14.4 mmol) in 180 mL of dry methylene chloride. The mixture is allowed to stand at room temperature for 15 min. The green insoluble silver salts were removed by centrifugation. The organic layer was washed with sodium thiosulfate (0.1 N) and water and dried, and the solvent was evaporated. The residue was rapidly chromatographed on a short silica gel column with hexane as eluent, and the crude product immediately used without further purification. lH NMR (400 MHz, CDC1,) 6 (ppm) 0.64 (s, 3 H, H-18), 0.862 (d, 3 H, J = 6.6 Hz, H-26 or H-27), 0.866 (d, 3 H, J = 6.6 Hz, H-26 or H-27), 0.90 (d, 3 H, J = 6.5-7.5 Hz, H-21), 0.91 (s,3 H, H-19),4.58 (m, 1H,H-2), 5.40 (m, 1H, H-3). MS (EI, 70 eV), mlz (relintensity):
Energy & Fuels, Vol. 6, No. 5, 1992 569 610 [M+] (85%), 595 (91,456 (loo), 455 (961,483 (ll),387 (48), 369 (83), 341 (53), 215 (64), 315 (12), 229 (30). Cholestan-28-01. 3a-Iodo-cholestan-2~-01 trifluoroacetate was reacted with LiAlH4 (300 mg, 7.89 mmol) under argon in anhydrous ether (100 mL) at reflux for 2 h. The suspension was poured in water and extracted. The organic extra- were washed with 4% HC1 and water and dried, and the solvent was removed under reduced pressure. Purificationon silicagel column afforded cholestan-26-01 (131 mg, 21%/cholest-Bene). 'H NMR (200 MHz, CDCl3) S (ppm) 0.65 (5, 3 H, H-l8),0.86 (d, 6 H, J = 6.6 Hz, H-26 and H-27), 0.89 (d, 3 H, J = 6.6 Hz, H-21), 1.02 (8, 3 H, H-19), 4.14 (m, 1 H, H-2). GC-MS (EI, 70 eV), mlz (re1 intensity): 388 [M+] (47%),373 (50),370 (51,355 (16), 316 (6), 315 (12), 262 (18), 234 (72), 233 (77), 217 (30), 215 (loo), 165 (44), 147 (38). Mp 146 OC. Anal. Found: C 83.31, H 12.19. Calcd for C27HeS: C 83.44, H 12.45. Cholestan-2a-01 N,N-Dimethyldithiocarbamate. The procedure described for the synthesis of cholestan-38-01N,N-dimethyldithiocarbamate was applied to cholestan-28-01 (130 mg, 0.335 mmol) yielding cholestan-2a-01N,N-dimethyldithiocarbamate (40 mg, 24%). lH NMR (200 MHz, CDCls) 6 (ppm) 0.64 (s, 3 H, H-18), 0.856 (d, 3 H, J = 6.5 Hz, H-26 or H-27), 0.859 (d, 3 H, J = 6.5 Hz, H-26 or H-27),0.89 (d, 3 H, J = 6.1 Hz, H-21), 0.94 (8, 3 H, H-19), 3.33 (8, 3 H, -NMez), 3.53 (8 3 H, -NMez), 3.81 (m, 1H, H-2). SM (EI, 70 eV), mlz (re1intensity): 491 [M+l (6%),458 (5),370 (21), 122 (50),121 (loo),88 (37). Mp 195-197 "C. Anal. Found: C 73.32, H 10.77, N 2.85. Calcdfor CNHDSZN: C 73.26, H 10.86, N 2.85. Cholestane-2a-thiol. A solution of cholestan-2a-01N,N-dimethyldithiocarbamate (20 mg, 0.041 mmol) was treated with LiAlH4 following the same method as for cholestan-38-thiol. Purification on silica gel afforded cholestane-2a-thiol. lH NMR (200 MHz, CDC13)6 (ppm) 0.64 (s,3 H, H-18), 0.80 (s,3 H, H-19), 0.87 (d, 6 H, J = 6.7 Hz, H-26 and H-27), 0.91 (d, 3 H, J 6.9 Hz, H-21), 2.92 (m 1 H, H-2). GC-MS (EI, 70 eV), m/z (re1 intensity): 404 [M+] (29%), 389 (111,316 (31,249(1001,250 (50), 215 (31), 216 (31), 217 (331,182 (6), 181(29). Mp 110 OC. Anal. Found C 80.33, H 11.68. Calcd for C27H4S: C 80.12, H 11.95. Cholestan-3a-01NJV-Dimethyldithiocarbamate.Cholestan3a-01N,N-dimethyldithiobamate was synthesized from cholestan-38-01 using the same procedure as for cholestan-38-01NJVdimethyldithiocarbamate. Yield 87 % 'H NMR (200 MHz, CDC13)6 (ppm) 0.65 (s,3 H, H-18), 0.81 (s,3 H, H-19), 0.858 (d, H, J = 6.6 Hz, H-26 or H-27), 0.862 (d, H, J = 6.6 Hz, H-26 or H-27), 0.89(d, 3 H, J = 6.6 Hz, H-21),3.38 (8,3 H, -NMe2), 3.54 (s, 3 H, -NMe2), 4.38 (m, 1 H, H-3). SM (EI, 70 eV), mlz (re1 intensity): 491 [M+] (6%),371 (14), 370 (281,122 (100),121(83), 95 (15). Mp 159 OC. Anal. Found: C 73.08, H 10.79, N 2.86. Calcd for C N H ~ ~ S ~CN73.26, : H 10.86, N 2.85. Cholestane-3a-thiol. Cholestan-3a-01N,N-dimethyldithiocarbamate was reacted with LiAlH4using the method described for the synthesis of cholestane-3/3-thiol. Cholestane-3a-thiolwas obtained in 35% yield. lH NMR (200 MHz, CDCls) 6 (ppm) 0.65 ( ~ , H, 3 H-18), 0.78 ( ~ , H, 3 H-19), 0.87 (d, 6 H, J 6.8 Hz, H-26 and H-27), 0.91 (d, 3 H, J = 6.9 Hz, H-21), 3.53 (m, 1 H, H-3). GC-MS (EI, 70 eV), mlz (re1 intensity): 404 [M+l (21%), 389 (14), 316 (3), 249 (loo), 250 (641,215 (36), 216 (19), 217 (48),182 (IO), 181 (37). Mp 79-80 OC. Anal. Found: C 80.26, H 12.05. Calcd for C2,HaS: C 80.12, H 11.95.
.
Acknowledgment. We thank the Socibte Nationale Elf Aquitaine (Production) and the CNRS for a doctoral fellowship (P.Adam) and financial support; the European Economic Community for financial support (Contract No. ST2-0225);G. Ryback, Shell Research Limited, Sittingbourne, UK, for a sample of Rozel Point seep oil; E. Krempp, Universite Louis Pasteur, Straebourg, for NMR analysis; G. Teller and P. Wehrung, Universitb Louis Pasteur, Strasbourg, for mass spectral measuremente. Regietry No, Phytane, 238-36-8; squalane, 111-01-3; carotane, 17161-33-0; cholestane-2a-thiol, 134932-55-1;cholestane3P-thiol, 134932-54-0.
