Mono- and dicyclic unsaturated triterpenoid hydrocarbons in ...

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882 R. de Mesmay et al. / Organic Geochemistry 39 (2008) 879–893 Chemical shifts were referenced relative to the residual proton signal (7.24 ppm) or the central line of the 13 C multiplet (77.0 ppm) of CDCl 3. Assignment of individual resonances was achieved using a combination of 1D and 2D ( 1 H- 1 H and 1 H- 13 C) experiments. Multiplicity of each 13 C nucleus was determined using DEPT (enhanced polarisation transfer) spectra. 3. Results and discussion 3.1. Characterization of botryococcenes and reduced analogues 3.1.1. Composition of hydrocarbon fraction Examination of the hydrocarbon fraction isolated from the 1856–1871 cm depth sediment sample using GC-MS revealed a dominance (93% of total hydrocarbons; Table 1; Fig. 2A) of botryococcenes (CnH2n-10) ranging from C33 to C37 and of partially reduced counterparts (CnH2n-8, CnH2n-6 and CnH2n-2), together with a minor series of C17–C35 n-alkanes. The botryococcenes and related compounds accounted for more than 246 lg/g dry sediment and 4 mg/g TOC (total organic carbon). The major compound was an unknown C34 component (c) accounting for ca. 43% of the total hydrocarbons, each of the other components accounting for 87%). 3.1.2. Characterization of C34 masokocene c The high resolution EI spectrum of the dominant botryococcene c displays M +. at m/z 466.4530, consistent with aC34H58 compound (calcd. 466.4522, D +0.8 mmu). The Table 1 Composition, quantification and ozonolysis products of hydrocarbons from Lake Masoko sediment Hydrocarbon lg/g d.s. a Formula e MW Structure low resolution EI mass spectrum is characterised by a major ion at m/z 177, likely corresponding to a fragmentation at the central quaternary carbon (Fig. 2B). Catalytic hydrogenation led to the formation of four botryococcanes C 34H 66 (H3, Fig. 3A) occurring in a 3/23/28/4 ratio, and which exhibited identical mass and fragment ions (Fig. 3C). This indicated the presence of two rings in c and suggested that stereochemical isomerisation occurred during hydrogenation. The mass spectrum of H3 shows fragments at m/z 444/445 corresponding to the loss of the C-10 ethyl, at m/z 236/237 corresponding to loss of the long alkyl chain at C-10 and at m/z 292/293 corresponding to loss of the short alkyl chain at C-10. Detailed inspection of the 1 H, 13 C, DEPT, COSY (correlation spectroscopy), HMQC (heteronuclear multiple quantum correlation) and HMBC (heteronuclear multiple bond correlation) NMR spectra of c (Table 2) and comparison with NMR data from other botryococcenes indicated the presence of a terminal vinyl [dH 5.75 (H-26), 4.92 (H- 27b), 4.90 (H-27a); d C 147.3 (C-26), 110.9 (C-27)] bound to the quaternary carbon C-10 (dC 41.8). The HMBC experiment further established that the latter quaternary carbon is correlated with an olefinic proton (H-11, dH 5.26) of trans disubstituted unsaturation [J 11,12 = 16.0 Hz; H-12: d H 5.12]. Moreover, long range correlations showed that a methine carbon (CH-13, d H 2.03, d C 37.1) bearing a methyl (Me- 28, dH 0.94, dC 21.2) is connected to this olefin. This C-10 to C-13 substructure I (with methyls at C-10 and C-13 and a vinyl at C-10, Fig. 4), is characteristic for all botryococcene structures (Metzger et al., 1985b); it arises from the 1’-3 condensation of two farnesyl units (Huang and Poulter, 1989). The downfield region of the 1 H NMR spec- lg/g TOC b Relative% c Ozonolysis d C33H56 452 0.5 8.7 0.2 C34H58 466 0.3 4.4 0.1 C34H58 466 trace C34H66 474 a 0.8 13 0.3 O3 + O1 C34H62 470 b 1.1 17 0.4 O11 + O1 C34H58 466 c 114 1878 43.2 O11 + O9 C34H58 466 d 12 191 4.4 O11 + O9 C34H58 466 0.8 13 0.3 C34H58 466 0.5 8.7 0.2 C35H60 480 e 26 422 9.7 O11 + O10 C36H62 494 f1, f2 5.8 96 2.2 O11 + O6 C36H64 496 g1, g2 36 569 13.5 O11 + O4 C36H62 494 0.5 8.7 0.2 C37H64 508 h 10.6 174 4.0 O11 + O7 C37H64 508 7.9 130 3.0 O11 + O7 C37H66 510 i 18 291 6.7 O11 + O5 C37H64 +C37H66 508/510 3.2 52 1.2 C37H64 508 1.1 17 0.4 C36H62 494 j 6.6 109 2.5 O11 + ? C37H66 510 0.3 4.4 0.1 Other botryococcenes and n-alkanes 20 339 7.4 R hydrocarbons 266 4346 100.0 a Dry sediment. b Total organic carbon. c Composition determined using GC. d Compounds from ozonolysis. e Compounds listed in elution order.
elative intensity (%) 100 50 0 relative intensity n-C 27 40 42 44 69 33 34 a b trum also shows a broad singlet for two protons (d H 5.03) of two trisubstituted double bonds [dC 138.3 (quaternary carbons C-6 and C-17) and 132.7 (tertiary carbons C-24 and C-29)] belonging to two identical substructures II (Fig. 4). The HMBC correlations (Table 2 and Fig. 4) established that II contain cyclohexenyl rings bearing three methyls, of which two are geminal [for example, in the left hand moiety of the molecule: Me-1 (dH 0.78, dC 23.4) and Me-23 (d H 0.95, d C 29.5)] at C-2 (d C 34.5) and a third at C-3 [Me-31 (dH 0.86, dC 16.2)]. Such a trimethylated cyclohexenyl moiety was previously found in cyclobotryococcene k, isolated from a strain of B. braunii from the Ivory Coast and grown under laboratory conditions (David et al., 1988). The relative stereochemistry of the methyls in c was deduced from the correlations observed in the NOESY (nuclear Overhauser enhancement spectroscopy) NMR spectra (Fig. 4), which suggested that Me-31 is pseudo-axial. The connection of each substructure II with the rest of the molecule was drawn from the HMBC experiment. So, in the ‘‘left” moiety of the molecule, cross peaks of H-7 and protons of Me-32 at C-7, to C-6 indicated the connection C-6/C-7. Furthermore, the two bond HMBC correlations H-7/C-8, H-8/C-9, and H-9/C-10 and the long range correlations H-7/C-9 and H-25/C-9 allowed us to connect the ‘‘left” structural moiety of c to the quaternary 95 R. de Mesmay et al. / Organic Geochemistry 39 (2008) 879–893 883 123 c 149 n-C 28 d 177 178 34 34 203 n-C 29 231 e 259 g1 f2 f1 36 177 285 123 -2H -2H 231 h i j c 259 -2H -2H 285 n-C 31 retention time (min.) 50 100 150 200 250 300 350 400 450 g2 285 37 37* 37 37 M+. -CH 3 314 341 355 396 423 123 M +. 451 466 Fig. 2. (A) Total ion chromatogram of hydrocarbon fraction from 1856–1871 cm depth sediment interval from Lake Masoko [C xx are n-alkanes with xx carbon atoms; numbers indicate the carbon number of botryococcenes and reduced counterparts whose structures have not been established in this work, cf. Table 1, * = coelution between one C37 botryococcene (C37H64) and one partially reduced counterpart (C37H66)], (B) EI mass spectrum of C34 masokocene c. m/z carbon C-10. In the right hand part of the molecule, 1 H NMR data and HMBC correlations showed that the cylohexenyl ring is bound at C-17 to a tertiary methine carbon C-16 which bears the methyl group Me-33. Finally, crosspeaks of H-15 to C-14 and C-13 allowed connection of the second cyclohexenyl moiety II. Major fragment ions at m/z 177 (base peak) and 123 in the mass spectrum of c support this structural pattern (Fig. 2B). Ozonolysis has proved to be a powerful tool for the structural determination of botryococcenes and derivatives (Huang et al., 1996). Application to an aliquot of the total hydrocarbons resulted in two predominating compounds: O9 (C 16H 28O 3)andO11 (C 17H 28O 4) resulting from oxidation of masokocene c (Table 1; Figs. 5 and 6A). Mass fragmentation patterns of O9 and O11 (Fig. 6E and G) were helpful for the characterization of other dicyclic triterpenoid hydrocarbons (see below). The generic name masokocene is proposed for all these dicyclobotryococcenes. HPLC purification of c afforded a coeluting minor botryococcene d (ca. 10% of the mixture) which exhibited a mass spectrum identical to that of c. It is very likely that d is a stereoisomer of c. 3.1.3. C 35 and C 36 masokocenes e and j GC-MS analysis of the intact and the hydrogenated hydrocarbon fractions clearly indicated that compounds e
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