Crenarchaeol

GDGT isolation

For isolation of GDGT-0² and the core tetraether lipid of pelagic crenarchaeota, 700 g of surface sediments of the Arabian Sea (Netherlands Indian Ocean Program Site 311, off Yemen; 16°02'N, 52°46'E; water depth 1,087 m) was extracted by Soxhlet using dichloromethane (DCM)/methanol (9:1) as solvent. From this extract, apolar and polar fractions were obtained by column chromatography over aluminum oxide using hexane-DCM (9:1; v/v) and DCM-methanol (1:1, v/v) as eluents, respectively. Solvent was removed from the polar fraction by rotary evaporation under vacuum. The remaining solvent was removed under a stream of nitrogen, and the residue dissolved by sonication (10 min) in hexane-propanol (99:1, v/v). The resulting suspension was centrifuged (1 min, 3,500 rpm) and the supernatant filtered through a 0.45 μm, 4 mm diameter PTFE filter prior to injection.

GDGT-4 was isolated from a GDGT fraction of the thermophilic archaeon Sulfolobus solfataricus, prepared as previously described by Nicolaus et al. (12) and a kind gift of Dr. A. Gambacorta, Instituto di Chimica di Molecole di Interesse Biologico, Napoli, Italy. The GDGT fraction was dried, re-dissolved, and filtered as described above before injection.

GDGTs were isolated using an HP (Palo Alto, CA) 1100 series LC equipped with an auto-injector, and a fraction collector (Foxy Jr., Isco, Inc., Lincoln, NE). A first isolation was achieved on a semi-preparative Econosphere NH₂ column (10 × 250 mm, 10 μm; Alltech, Deerfield, IL), maintained at 30°C. Typical injection volume was 100 μl containing up to 10 mg material. GDGTs were eluted isocratically with 99% hexane and 1% propanol for 5 min, followed by a linear gradient to 1.8% propanol in 45 min. The flow rate was set at 2.5 ml/min. After each run the column was cleaned by back-flushing hexane-propanol (9:1; v/v) at 2.5 ml/min for 10 min. Column effluent was collected in 1 min fractions, which were screened for the presence of the target GDGT by HPLC/atmospheric pressure chemical ionization (APCI)-MS as described by Hopmans et al. (13). Fractions containing the target GDGT were pooled and further purified on an Econosphere NH₂ column (4.6 × 250 mm, 5 μm; Alltech, Deerfield, IL). Typical injection volumes were 50 μl containing up to 0.5 mg of material. GDGTs were eluted using an identical gradient and conditions as described above, but at a flow rate of 1 ml/min. After each run the column was cleaned by back-flushing hexane-propanol (95:5, v/v) at 1 ml/min for 10 min. Column effluent was collected in 0.5 min fractions and screened as described above.

GDGT extraction and analyses

In case of the sponge Axinella mexicana containing Cenarchaeum symbiosum (kind gift of Dr. E. DeLong, Monterey Bay Aquarium Research Institute, CA) direct solvent extraction did not yield significant amounts of GDGTs. The sponge was therefore extracted by refluxing with 2 N HCl in methanol for 8 h, followed by liquid/liquid extraction with DCM. Released GDGTs were subsequently analyzed by HPLC-APCI/MS as described previously (13).

Nuclear magnetic resonance

All GDGTs were solved in CDCl₃ at a concentration of 3–6 μmol/ml. NMR spectroscopy was performed on a Varian Unity Inova 500, a Bruker DRX600, and a Bruker AV-750 spectrometer equipped with an SWBB probe, an inverse TBI-Z probe with a pulsed field gradient (PFG) accessory, and a BBI-zGRAD probe, respectively. All experiments were recorded at 300 K in CDCl₃. Proton and carbon chemical shifts were referenced to internal CDCl₃ (7.24/77.0 ppm). In the 2D ¹H-¹³C correlated spectroscopy (COSY), the number of complex points and sweep widths were 2K points/6 ppm for ¹H and 512 points/150 ppm for ¹³C. In the 2D ¹H-¹H COSY the number of complex points and sweep widths were 2 K points/5.5 ppm. Quadrature detection in the indirect dimension was achieved with the time-proportional-phase-incrementation method. The data were processed with Varian or NMRSuite software packages. After apodization with a 90 shifted sinebell, zero filling to 512 real points were applied for the indirect dimensions. For the direct dimensions zero filling to 4 K real points, Lorentz transformations were used.

Torsion angles in GDGT-4

To determine the average torsion angles in the cyclopentane rings of GDGT-4, a constant volume, constant temperature molecular dynamics (MD) simulation (14, 15) was performed at a system temperature of 298 K on a single GDGT-4 lipid. After equilibration at this temperature, a 25,000-iteration MD-simulation was performed. Atom positions were saved every 50 iterations, thus generating 500 conformational snapshots. Subsequently, average torsion angles in the five-membered rings and associated standard deviations were obtained from the analysis of these snapshots. The ReaxFF-force field (16) was used in these simulations.

Membrane volume determination

To determine the influence of molecular structure on membrane volume we performed constant pressure, constant temperature MD simulations (14, 15) on systems containing 3 × 3 GDGT-4 or crenarchaeol-lipid monomers. In accordance with work by Gabriel and Chong (6) we used β-d-glucopyranose and myo-inositolphosphate as the polar groups in both the GDGT-4 and crenarchaeol-lipids. Periodic images of the 3 × 3-lipid system were used in the y- and z-direction (parallel to the membrane), no periodicity was used in the x-direction (perpendicular to the membrane). The system was allowed to expand and contract in the y- and z-directions according to the intermolecular forces. By this means the initial membrane structure, containing the 3 × 3 lipid monomers evenly and symmetrically distributed in a periodic box of dimensions 21 × 30 Å in y- and z-directions, was allowed to relax until it reached a steady-state configuration, after which the periodic box dimensions (the yz-surface area) could be used as a direct measure for the membrane volume. The system temperature (298 K) and pressure (10 bar) were controlled using the algorithm described by Berendsen et al. (17), with temperature and pressure damping constants of 1,000 femtoseconds and a MD time-step of 1 femtosecond.

A modified version of the AMBER-protein force field (18), as described by van Duin and Larter (19), was used to evaluate the interatomic forces in these simulations. To test for potential biases associated with this choice of force field all simulations were repeated with the ReaxFF-potential (16).

HPLC-APCI/MS

Cenarchaeum symbiosum is an archaeon that lives in symbiosis with the sponge Axinella mexicana, originally in the Gulf of Mexico (10). Detailed molecular biological work has documented that this “culture” is uni-archaeal. It is the only archaeon available in culture belonging to the phylogenetic group of pelagic crenarchaeota (Marine Group 1) (2). Analysis of the H⁺-extract with an HPLC-MS technique, recently developed to analyze intact core GDGT membrane lipids (13), showed a base peak ion chromatogram dominated by GDGT-0 (structure I; see Fig. 1 for structures), the GDGT comprised of two biphytane chains containing no cyclopentane rings, and an unknown component, representing ∼60% of total GDGTs (Fig. 2A) . This latter component showed a mass spectrum typical for a GDGT (i.e., loss of water and glycerol) (13) but with a protonated molecular ion 10 daltons lower than that of GDGT-0, establishing the molecular formula as C₈₆H₁₆₂O₆ and indicating that this GDGT contained five rings. These results are in good agreement with ether cleavage studies of GDGTs of C. symbiosum (9), which revealed biphytanes with two and three cyclopentane rings (structures II and III) as major components.

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Fig. 1.

Structures of components listed in the text. For glycerol dibiphytanyl glycerol tetraether (GDGT)-0, GDGT-4, and crenarchaeol, the numbering of carbon atoms is indicated. The arabic numbering of carbon atoms was done after DeRosa et al. (22). The numbering with numerals was applied to make as much use as possible of the C₂ symmetry of the biphytanyl chains in the GDGT molecules to describe the NMR signals (Table 1–3) as efficiently as possible.

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Fig. 2.

Partial base peak chromatogram obtained by HPLC-atmospheric pressure chemical ionization (APCI)/mass spectrometry (MS) showing the distribution of GDGTs of (A) the H⁺-extract of the non-(hyper)thermophilic archaeon Cenarchaeum symbiosum, (B) the extract of water column suspended particulate matter obtained from a station (17°42'N, 57°51'E; 1,000 m water depth) in the Arabian Sea (20), and (C) the polar fraction of the solvent extract of surface sediment (Netherlands Indian Ocean Program Site 311, 16°02'N, 52°46'E; water depth 1,087 m) from the Arabian Sea used to isolate crenarchaeol. Key: 1, GDGT-0; 2, crenarchaeol; 3, GDGT-1, 4, GDGT-2; 5, isomer of crenarchaeol.

This major unknown GDGT is also the major GDGT in water column particulate organic matter and marine surface sediments (Fig. 2B, C) (3, 20), indicating that it is probably also the dominant GDGT of the pelagic crenarchaeaota, which represent 20% of the picoplankton in the ocean (4). This is in good agreement with earlier suggestions based on ether cleavage products of GDGTs (i.e., II and III) in the marine water column (8).

Isolation of the unknown GDGT

To fully elucidate the structure of this major unknown GDGT, it was isolated and its structure was determined by high-field 2D-NMR studies. As the source for isolation, surface sediments of the Arabian Sea were chosen. These sediments have a GDGT composition very similar to that of C. symbiosum (Fig. 2), contain high amounts of this GDGT, and are thus a good source for isolation. By solvent extraction, column chromatography over silica, and preparative HPLC a fraction significantly enriched in the unknown GDGT core membrane lipid was obtained. From this fraction, the unknown GDGT was isolated by repetitive analytical HPLC, resulting in ∼4 mg of isolate. HPLC-APCI/MS of this fraction indicated that the unknown GDGT was the only GDGT present in this fraction and did not reveal any other impurities. Consequently, this fraction was used for NMR studies.

For comparison of NMR data, GDGT-0 (I) and GDGT-4 (IV) were also isolated in high purity by HPLC from Arabian Sea sediments and cells of the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius, respectively. Both GDGTs (but especially GDGT-4) share structural similarities with the unknown GDGT of pelagic crenarchaeota and ¹³C-NMR data have been reported for the biphytane carbon skeletons of these components (21–23), thus assisting in the identification of the unknown GDGT. However, no high-resolution ¹H, ¹³C-NMR, and 2D-NMR correlation techniques have been applied to the intact GDGTs.

Basic skeleton

The ¹H-NMR spectrum is extremely complex even if measured at 750 MHz. In the 3.4–3.7 ppm region multiplets representing 18 protons are observed (Table 1). These represent the protons of the two glycerol units and the first and ultimate methylene units of the biphytane moieties bound via the ether linkages. The same signals are observed in the ¹H-NMR spectra of GDGT-0 and GDGT-4 (Table 2). At ∼2.2 ppm a broad singlet representing the two hydroxy groups is found. Between 0.82 and 0.88 ppm a complicated pattern of signals (mainly doublets) occurs in total representing eleven methyl groups. At 750 MHz, the resolution is high enough to separate a singlet at δ = 0.836 ppm, representing one methyl group, from a doublet at δ = 0.844 ppm, representing three methyl groups. In the 0.7–0.8 ppm region two “high-field” protons are observed; the remaining protons are all found in the 1.0–1.8 ppm region.

The ¹³C-NMR spectrum of the unknown GDGT shows 11 primary, 53 secondary, 21 tertiary, and 1 quaternary carbon atoms (Table 1). Attached proton test (APT), distorsionless enhancement by polarization transfer (DEPT)90, and DEPT135 experiments were used to assess the multiplicity of carbon atoms. The ¹³C-NMR spectrum did not show 86 resolved signals because many carbon atoms are either strictly, or effectively, equivalent. Assignments of the carbon atoms is partially based on literature data (22, 23) and the ¹³C-NMR data of GDGT-0 and GDGT-4, in combination with an heteronuclear multiple quantum correlation (HMQC) experiment. This established one of the diether-bound biphytane moieties as structure II, well known from the membrane lipids of hyperthermophilic crenarchaeota. The other proposed dibiphytanyl moiety (structure III) (8, 11) is, however, inconsistent with the NMR data as it does not contain a quaternary carbon atom. The ¹³C-NMR data, however, do match with the position of the two cyclopentane rings as in II (Table 1). The presence of the quaternary carbon atom suggests that the third ring in the second biphytanyl moiety is not a cyclopentane (as in III) but a cyclohexane ring if we infer that the additional ring is biochemically formed through ring closure of a biphytanyl skeleton. This would also be consistent with the observed methyl group at δ = 0.836 ppm as a singlet in the ¹H-NMR spectrum, which is absent in the ¹³C-NMR spectrum of GDGT-4 (Fig. 3) . There are two possible structures (Va and Vb) for the second moiety through ring closure of the biphytanyl chain. Both are consistent with mass spectrometry data (11). Inverse long-range heteronuclear multiple bond correlation (HMBC) experiments enabled discrimination between these two possibilities since the singlet at δ = 0.836 ppm did not show correlation with the neighboring carbon atom (i.e., A10′) of the cyclopentane ring, as would be expected for skeleton Vb, but instead with carbon atom A16′ (Fig. 3). The remaining NMR data (Table 1) are also in agreement with this assignment. Furthermore, this structure is also in better agreement with published MS data of the biphytane moiety with three rings released after ether bond cleavage (11) because cleavage of the C-C bond between A15′ and A16′ explains why the fragment at m/z 263 is relatively abundant. This established that the abundant unknown GDGT membrane lipid in pelagic crenarchaeota is VI. We propose to call this component crenarchaeol, in analogy to the nomenclature of other archaeal ether lipids (24). Of the 86 carbon atoms of crenarchaeol, 23 are chiral and below we will explore literature and our NMR data to determine their stereochemistry.

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Fig. 3.

Heteronuclear multiple bond correlation (HMBC) experiments (at 750 MHz) for GDGT-4 (left panel) and crenarchaeol (right panel). A selected range of the spectrum is displayed to show the correlations between the methyl groups and specific carbon atoms. Partial proton spectra (750 MHz) and attached proton test (APT) (125 MHz) spectra are plotted above and beside, respectively, the contour plot. Peak labeling refers to carbon numbering indicated in Fig. 1. Correlations between methyl groups and carbon atoms are indicated by stippled lines. In the HMBC spectrum of crenarchaeol, only the correlations of the methyl groups different from those in GDGT-4 (A20 and A20′) are indicated.

Stereochemistry of the glycerol moieties and the acyclic chiral centers

A significant feature of archaeal ether lipids is that glycerol is sn-2,3-di-O-alkylated but not sn-1,2-diacylated as in bacteria and eukaryotes. The unusual (R) configuration at the sn-2 position has been confirmed in case of the GDGTs of Sulfolobus acidocaldarius by appropriate incorporation experiments (25). The NMR data (both chemical shift and splitting pattern) of crenarchaeol, GDGT-0, and GDGT-4 (Table 3) of the protons and carbon atoms of the glycerol units and the ultimate and penultimate carbon atoms of the biphytanyl moieties and their attached protons are identical. This indicates that the stereochemistry of the glycerol units of crenarchaeol is the same as in GDGT-4 of Sulfolobus acidocaldarius and, thus, (R), as all other archaeal diethers and GDGTs.

Heathcock et al. (26) have established the full stereostructure of GDGT-0 [2,3,2′,3′-tetra-O-di-(3R,7R,11S,15S, 18S,22S,26R,30R-3,7,11,15,18,22,26,30-octamethyldotriacontanyl)-di-sn-glycerol; I]. Since all cyclopentane ring-containing GDGTs are biosynthesized by internal cyclization reactions of GDGT-0 (I), it is assumed that the remaining acyclic stereocentres in cyclopentane-containing GDGTs are also as in I (23). This assumption is also likely for crenarchaeol. This establishes the stereocentres of A3, A11, A3′, A15′, B3, B11, B3′, B11′ as (R) and A15, B15, and B15′ as (S). Note that due to changes in the priorities of the groups on chiral carbon atoms according to the Cahn-Ingold-Prelog convention, the naming of the configuration may be different although the absolute stereochemistry remains the same.

Stereochemistry of the cyclopentane rings

The absolute stereochemistry of the cyclopentane-ring has not yet been established. De Rosa et al. (22) reported, on basis of the chemical shifts of the carbon atoms of the cyclopentane rings in comparison with ¹³C-NMR data of dimethylcyclopentanes (27), that the 1,3-substitution pattern of the cyclopentane ring in archaeal GDGTs is probably trans.

To determine the full stereochemistry of the cyclopentane rings, it was decided to first concentrate on the symmetrical GDGT-4 (IV), where no interference of signals from the cyclohexane ring occurs. HMQC, HMBC, COSY, and total correlation spectroscopy (TOCSY) experiments resulted in the assignment of all protons of the cyclopentane rings (Table 2). The two protons of both the A8 and A9 methylene groups showed a large difference (0.6–0.7 ppm) in chemical shift, whereas this difference for the two protons of the A18 methylene unit was only small (∼0.1 ppm) (Table 2). This suggested for the protons at A8 and A9 a situation comparable to that of cyclohexane rings, where the chemical shifts of protons strongly depend on their axial or equatorial position: the axial protons often resonate at much higher field than their equatorial counterparts. On the other hand, the protons at A18 seem to be more in eclipsed than in staggered positions. This assignment is, however, complicated by the fact that much conformational freedom exists in the cyclopentane compared with the cyclohexane ring. Therefore, we simulated the conformation of GDGT-4 using molecular dynamics and determined the average torsion angle of the protons and alkyl substituents of the cyclopentane ring. The results show that i) there is indeed a significant degree of conformational freedom in the cyclopentane ring and ii) the protons at A8 and A9 have a pronounced axial/equatorial character, whereas the torsion angles of the two protons at A18 with the ring are similar (Fig. 4A) , consistent with our assignments of the NMR signals. The shifts of protons at the more substituted, tertiary carbon atoms A7 and A10 are at 1.79 and 1.68 ppm, respectively, not allowing any conclusion on whether they are axially or equatorially substituted.

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Fig. 4.

A: Average (over four rings) torsion angles of substituents of the cyclopentane rings in GDGT-4 calculated by molecular dynamics. The standard deviation is indicated. Because the cyclopentane ring is not planar but is in an “envelope” form, two torsion angles have to be taken into account. The calculations indicate that the protons of carbon atoms A8 and A9 are in equatorial-like and axial-like positions. B: The calculated 3D-structure of the “average” cyclopentane partial stucture in GDGT-4. Indicated are the nuclear Overhauser effect spectroscopy interactions which determine the trans substitution of the alkyl side-chains.

A definite stereochemical assignment was revealed by a nuclear Overhauser effect spectroscopy (NOESY) experiment, which showed nuclear Overhauser effect (NOE) interactions of the proton at A10 with the axial proton at A9 and one of the protons at A18, and NOE interaction of the proton at A7 with the axial proton at A8 and the other proton at A18 (Fig. 4B). This proved that the stereochemistry of the cyclopentane ring is indeed trans, as suggested by DeRosa et al. (22). The full stereochemistry is subsequently determined by the original stereochemistry of GDGT-0 (26), in combination with the fact that biosynthesis of cyclopentane moieties in GDGTs occurs through internal cyclization (22): only one of the two possible ring closures results in an 1,3-alkyl trans substituted cyclopentane ring. This stereochemical assignment is confirmed by the observed coupling constants for the equatorial protons at A8, A9, and the proton at A10 (Table 2). This establishes the stereochemistry at the chiral centers A7 and A10 to be (S).

Based on these assignments, the stereochemistry of the cyclopentane rings of crenarchaeol was assessed to be identical to those in GDGT-4. All the protons and carbon atoms resonate at identical field strength (Tables 1 and 2), except those of the cyclopentane ring attached to the cyclohexane ring. In this cyclopentane ring, most chemical shifts are also identical except for the shift of the proton at carbon atom A10′, which is at slightly higher field (1.47 ppm vs. 1.68 ppm in GDGT-4). This is attributed to the attached cyclohexane ring, which forces the proton in a slightly more “axial” position.

Stereochemistry of the cyclohexane ring

A prominent feature in the ¹H-NMR data of crenarchaeol are the two high-field protons, which are absent in the spectrum of GDGT-4, and obviously related to the presence of the cyclohexyl moiety. The assignment of these high-field protons is based on COSY, TOCSY, HMQC, and HMBC correlations. The proton at δ = 0.70 represents a quasi triplet with a coupling constant of J = 12.5 Hz. This signal must be assigned to the axial proton at A19′; in addition to the relatively large geminal coupling there must be an equally large, axial-axial coupling with the proton at carbon atom A11′. The other high field proton absorbs at δ = 0.72 and forms a quasi-double quartet with coupling constants J = 13.0 and 4.5 Hz. This is the axial proton at carbon atom A12′, which couples with the axial protons at A11′ and A13′, the geminal proton (all with large coupling constants of ∼13 Hz), and with the equatorial proton at carbon atom A13′ with a much smaller coupling constant. The proton at carbon atom A11′ absorbs at δ = 1.17 and is not well resolved from other signals. However, in 2D-NMR spectra it shows up as a double doublet with two relatively large coupling constants, in agreement with this assignment. Coupling with the proton at carbon atom A10′ is only weak, indicating that the dihedral angle is probably close to 90°C. These results indicate that the cyclopentane ring is equatorially substituted at carbon atom A11′ and that the stereochemistry at position A11′ is thus (S).

The stereochemistry at position A15′ follows from two observations. First, the chemical shift of the methyl group at A15′ (A20′) in the ¹³C-NMR spectrum is at relatively low field (22.39 ppm), indicative for equatorially substituted methyl groups of cyclohexane rings (28). Second, remarkably, both axial protons at carbon atoms A14′ and A19′ show a strong long-range (four bonds) correlation with methyl group A20′ in the COSY spectrum. This established the (R) stereochemistry at A15′.

The stereochemistry of the cyclohexane ring is consistent with its presumed biosynthetic formation through ring closure via A15′ and A19′; the resulting stereochemistry is “inherited” from the stereochemistry of the GDGT-0 (I) precursor. This also determines the equatorial/axial positions of the alkyl substituents of the cyclohexane ring. If the cyclohexane ring is in the more stable chair configuration, this fits with the stereochemical configuration of the cyclohexane ring in crenarchael as determined by NMR.

Regioisomerism

It has been assumed for a long time that archaeal GDGTs were characterized by an antiparallel arrangement of glycerol units as in I (29). However, Gräther and Arigoni (30) showed by selective chemical degradation for three archaeal species that GDGT-0 is in fact a 1:1 mixture of the regioisomeric components I and VII.

During isolation of GDGT-4 from S. solfataricus, a fraction enriched in a less abundant, slightly later eluting (13) (∼35% of GDGT-4) isomer (GDGT-4′) was also isolated. This isomer had virtually identical ¹H- and ¹³C-NMR spectra, indicating that the four cyclopentane rings must be in the same position and have the same stereochemistry. Detailed comparison of the ¹³C data indicated, however, a subtle difference; the carbon atoms A3, A4, A5, and A6 showed two signals in case of GDGT-4 but only one in case of GDGT-4′ (Table 3). This observation led us to the conclusion that GDGT-4 is the antiparallel isomer whereas GDGT-4′ is the parallel isomer. This latter isomer has a plane of symmetry and only one stereoisomer exists. For GDGT-4 there is no plane of symmetry and its mirror image is therefore different. This explains why for some carbon atoms two close but not identical signals are observed.

For crenarchaeol an even more complicated situation exists since for some carbon atoms even four different signals are observed (Table 3). This indicates that the isolated isomer probably has the antiparallel configuration of glycerol units like in GDGT-4. Indeed, a minor isomer of crenarchaeol (presumably the parallel regioisomer) elutes later on the HPLC column, just as with GDGT-4 and GDGT-4′. In case of crenarchaeol there are, however, two additional regioisomers (VI and VIII) both with the antiparallel configuration of glycerol units, since the two biphytanyl chains in crenarchaeol are not the same, resulting in four stereoisomers. This explains the even more complex ¹³C-NMR spectrum.