Variable stereochemistry in highly branched isoprenoids from diatoms

C(25) highly branched isoprenoid (HBI) alkenes are ubiquitous lipids found in geochemical samples around the globe. The origins of these widespread geochemicals are believed to be restricted to a limited number of diatoms, including Haslea ostrearia (and related species), Rhizosolenia setigera, and Pleurosigma intermedium. The unsaturation of the HBI alkenes ranges from 2-6 in different species and cultures. The number of stereogenic centres is usually limited to two in the HBI alkenes due to double bond positions. The relative and/or absolute configurations for these have been determined for a range of HBI alkenes produced from different diatoms cultured under a number of growth conditions. These determinations have involved a combined spectroscopic and chromatographic analysis using NMR spectroscopy and chiral gas chromatography, respectively. HBIs isolated from Haslea spp. belong to a specific structural type which exhibit configurational diastereoisomerism, while those isolated from P. intermedium and R. setigera represent a different structural type and usually exist as mixtures of geometric isomers only. HBIs are reported from a new species of diatom whose stereochemical properties lie between those found for Haslea spp. and P. intermedium.     

Novel polyunsaturated n-alkenes in the marine diatom Rhizosolenia setigera.

Four previously unknown n-C25 and n-C27 heptaenes of the marine diatom Rhizosolenia setigera were isolated and identified using NMR spectroscopy. They possess six methylene interrupted (Z)-double bonds starting at C-3 and an additional terminal or n-2 (Z)-double bond. Structural and stable carbon isotopic evidence suggests that these polyenes are biosynthesized by chain elongation of the C22:6n-3 fatty acid, followed by decarboxylation and introduction of double bonds at specific positions.     

OCCURRENCE OF AN ISOPRENOID C25 DIUNASATURATED ALKENE AND HIGH NEUTRAL LIPID CONTENT IN ANTRACTIC SEA-ICE DIATOM COMMUNITIES1

The lipid and hydrocarbon composition of natural populations of diatom communities collected during the austral spring bloom of 1985 in the sea-ice at McMurdo Sound, Antartica was analyzed by TLC-FID, GC and GC-MS. Sea-ice diatom communities were dominated by Amphiprora sp., Nitzschia stellata Manguin and Berkeleya sp. at Cape Armitage; N. stellata, Amphiprora, Pleurosigma, N. kerguelensis (O'Meara) Hasle and some small centric diatoms adjacent to the Erebus Ice Tongue; and Porosira pseudodenticulata (Hustedt) Jouse at Wohlschlag Bay. Lipid distributions of the sea ice diatom communities from the Cape Armitage and Ereus sites were characterized by high concentrations of tracylaglecycerol (triacylglycerolplar lipid = 1.0 to 1.5). The hydrocarbon n-C_21:6, common in temperate diatoms, and an isoprenoid C₂₅ diunsaturated alkene were the dominant hydrocarons detected at these two sites. Hydrogenation of the C₂₅ diene produced the known alkane 2, 6, 10, 14-tetramethyl-7- (3-methylpentyl)-pentadecane. The C₂₅ diene is one of several structurally related hydrocarbons reported in many estuarine, coastal and ocean ic sediments. We propose that certain species of diatoms are a likely source of these alkenes in sediments. The first reported biological occurrence of the C₂₅ diene in the green seaweed Enteromorpha prolifera may have been due to the presence of epiphytic microalgae in the field sample analysed.     

Phytotoxicity and ultrastructural effects of gymnopusin from the orchid Maxillaria densa on duckweed (Lemna pausicostata) frond and root tissues

The effect of life cycle on the distributions of C(25) and C(30) highly branched isoprenoid (HBI) alkene lipids has been investigated for the marine diatom Rhizosolenia setigera. The concentrations of the C(30) compounds are largely independent of the cell volume, though the ratios of the individual isomers possessing five and six double bonds show a dependence on the position of the cell during its life cycle, especially during auxosporulation. In contrast to the C(30) pseudo-homologues, the C(25) isomers are not always detected in cultures of R. setigera. The biosynthesis of the C(25) HBIs would appear to result from the onset of auxosporulation, with further changes to their distributions taking place after this phase, including the formation of more unsaturated isomers. The results of this investigation may be used in part to explain the large variations in these lipids reported previously.     

Anthrasesamones from roots of Sesamum indicum

Three anthraquinones, named anthrasesamones A, B and C, were isolated from the roots of Sesamum indicum, and their respective structures were determined to be 1-hydroxy-2-(4-methylpent-3-enyl)anthraquinone, 1,4-dihydroxy-2-(4-methylpent-3-enyl)anthraquinone and 2-chloro-1,4-dihydroxy-3-(4-methylpent-3-enyl)anthraquinone on the basis of spectroscopic evidence. Two known anthraquinones were also isolated for the first time from S. indicum roots and characterized as 2-(4-methylpent-3-enyl)anthraquinone and (E)-2-(4-methylpenta-1,3-dienyl)anthraquinone. Anthrasesamone C is a rare chlorinated anthraquinone in higher plants.     

Saturated hydrocarbons in bovine liver

A homologous series of n-alkanes (C(14)-C(33)) and two isoprenoid hydrocarbons, 2,6,10,14-tetramethylhexadecane (phytane) and 2,6,10,14-tetramethylpentadecane (pristane) have been identified in bovine liver. Another branched but non-isoprenoid alkane and three isomers of molecular formula C(20)H(40) were partially identified. Phytane and the C(18)-C(22) and C(29)-C(33)n-alkanes were found to be the major components in liver, suggesting that at least the main hydrocarbon components were derived from various plants in the diet. The hydrocarbons were separated and identified by a series of steps involving solvent extraction, saponification, elution chromatography on alumina and silica gel columns, molecular sieving and by infrared and ultraviolet spectroscopy, followed by combined capillary gas chromatography-mass spectrometry.     

Factors influencing the distributions of polyunsaturated terpenoids in the diatom, Rhizosolenia setigera.

Polyunsaturated highly branched isoprenoid (HBI) hydrocarbon distributions of laboratory cultures of five strains of the planktonic diatom Rhizosolenia setigera (Brightwell) are shown herein to be highly variable. Some strains produced both haslenes with from three to five double bonds and rhizenes. The haslenes comprised not only Delta5 alkenes but also those with C7(20) unsaturation, including hasla-7(20),9E,Z, 23-trienes and hasla-7(20),9E,Z-13, 23-tetraenes. The rhizenes contained C7(25) unsaturation and the vinyl moiety common to all algal haslenes so far characterised. The effects of temperature and salinity on HBI composition, along with isotopic content, were determined in strain CS 389/A. Increase in growth temperature from 18 to 25 degrees C increased the degree of unsaturation in the haslenes and E to Z isomerisation in the triene. There was also an increase in unsaturation in the rhizenes at the highest growth temperature, with hexaenes dominant over the pentaenes but in the rhizenes, Z to E isomerisation increased. Increased salinity from 15 to 35 psu increased cell growth and rhizene production but decreased haslene production. Unsaturation in haslenes was not changed by increased salinity but unsaturation in the rhizenes decreased. These may reflect growth rate differences. The carbon isotopic compositions of the haslenes and rhizenes were similar to that of the major sterol at 18 degrees C, but the major HBI isomers were 3-4 per mil depleted relative to phytol released by saponification from chlorophyll a. This suggests biosynthesis of HBIs from a different isotopic pool of isopentenyl biphosphate to that from which phytol is biosynthesised. At 25 degrees C, further isotopic differences were observed. The variables controlling HBI distributions in R. setigera are still not fully understood and rationalisation of the environmental controls on the sedimentary distributions of the HBIs from R. setigera may only be possible once such factors are established.     

Kombic acid,a hydroquinone polyisoprenoic carboxylic acid from Pycnanthus kombo seed fat

The fat of the seeds from the West African tree Pycnanthus kombo contains ca 20% of a hitherto undescribed compound. This compound was identified as 16(2′,5′-dihydroxy-3′-methylphenyl)-2,6,10,14-tetramethyl-2,6,10,14-hexadecatetraenoic acid, for which the name kombic acid is proposed.     

Long-chain alkenones and related compounds in the benthic haptophyte Chrysotila lamellosa Anand HAP 17

The neutral lipid compositions of the coastal haptophyte Chrysotila lamellosa HAP 17 grown in batch culture at 10 and 20 degrees C have been determined. A comparison was also made between the lipid compositions of cells harvested in early and late stationary phase. This species contains a suite of very long-chain C(37)-C(40) alkenones and alkenoates as found in a few microalgae from the Haptophyta. The distributions of these compounds show some differences to earlier reports of different strains of this alga, which are only in part attributable to culture conditions. A suite of long-chain alkenols, the reduced form of the alkenones, was characterized for the first time. The abundance of these compounds was only 1.5% of that of the corresponding alkenones, and the relative proportion of C(37)-C(38) constituents depended on growth temperature. These data show that haptophyte algae are a possible source of the alkenols found in some marine sediments, but the small amounts found suggest that other sources such as bacterial reduction of alkenones are more likely in highly reducing sediments. A mixture of C(29)-C(33) n-alkenes, dominated by the C(31:1) monoene, was found in marked contrast to previous analyses of other strains which reported only the presence of a C(31:2) diene. The sterol distribution included the common haptophyte sterol 24alpha-methylcholesta-5,22E-dien-3beta-ol (epi-brassicasterol) as well as significant amounts of Delta(5)- and Delta(5,22)-C(29) sterols.     

SALINITY TOLERANCE OF DIATOMS FROM THALASSIC HYPERSALINE ENVIRONMENTS

Thirty-four benthic diatom strains were isolated from thalassic hypersaline marine environments and their salinity tolerance characterized in growth experiments conducted at salinities ranging from 0.5% to 17.5% (weight of total salts per volume, g·100 mL ^{− 1}). The results were compared with the patterns of diatom species distribution and abundance in hypersaline evaporation ponds and tidal channels of Guerrero Negro, Baja California Sur, Mexico. The isolated strains were representative of the diatom assemblages present in the saltern ponds but were less so of natural assemblages in tidal channels. In general, we found a clear decreasing trend of diatom diversity in the field and in the isolated strains with increasing salinity. With some exceptions, the upper limit of salinity tolerance in cultivated strains corresponded to their distribution in field samples. However, the relative abundance of species in the field was not correlated with growth rates achieved in culture for the same salinities. Most cultured strains exhibited extreme euryhalinity growing well from brackish to hypersaline conditions, but the particulars of salt tolerance were quite diverse among strains. The most halotolerant taxa, two Amphora species, Amphora cf. subacutiuscula Schoeman, Nitzschia fusiformis Grunow, and Entomoneis sp., grew well in salinities ranging from 0.5% to 15%. Three strains of Pleurosigma strigosum W. Smith that were unable to grow in salinities less than 5% total salts represent the only true halophilic diatoms ever reported. The fact that many strains displayed a remarkable halotolerance, with optimal or near-optimal growth rates at salinities as high as three times that of seawater, implies that diatoms from hypersaline environments are evolutionarily highly adapted to such environments.     

Champanones, yellow pigments from the seeds of champa (Campomanesia lineatifolia)

Chemical investigation of the methanol extract of the seeds of Campomanesia lineatifolia Ruiz and Pav. (Myrtaceae) led to the isolation of two new beta-triketone type compounds, named champanones A (1) and B (2), together with the known 2,3-dihydro-5-hydroxy-6,8,8-trimethyl-2-phenyl-4H-1-benzopyran-4,7(8H)-dione (champanone C) (3). The structures of 1 and 2 were determined to be 2,2,4,4-tetramethyl-6-(1-oxo-3-phenylprop-2-enyl) cyclohexane-1,3,5-trione (occurs as an enol form) and 2,2,4-trimethyl-6-(1-oxo-3-phenylprop-2-enyl)cyclohexane-1,3,5-trione (occurs as an enol form), respectively, by means of spectroscopic analysis. The three compounds showed mild antimicrobial activity.     

Microbial Oxidation of Isoprenoid Alkanes,Phytane, Norpristane and Farnesane

Rhodococcus sp. BPM 1613, a pristane oxidizing microorganism, grows on isoprenoid hydrocarbons such as phytane (2,6,10,14-tetramethylhexadecane), norpristane (2,6,10-trimethylpentadecane) and farnesane (2,6,10-trimethyldodecane) as the sole carbon source, resulting in accumulation of oxidation products in the culture broth. The oxidation products of phytane, norpristane and farnesane in the respective culture broth were isolated and purified by the use of silica gel column chromatography. Their chemical structures were determined by instrumental analyses such as IR, NMR and mass spectrometry. The oxidation products of phytane were identified as 2,6,10,14-tetramethyl-1-hexadecanol and 2,6,10,14-tetramethylhexadecanoic acid, the product of norpristane as 2,6,10-trimethyl-1-pentadecanol, and that of farnesane as 2,6,10-trimethyl-1-dodecanol. All these oxidation products were either monoalcohols or monocarboxylic acids derived through oxidation of the isopropyl terminus of each alkane.

In addition, the relationship between the terminal structure of isoprenoid hydrocarbons and microbial oxidation was explored on the basis of these results.     

Fatty Acids in Bacillus larvae, Bacillus lentimorbus, and Bacillus popilliae

The types of fatty acids produced by two strains each of Bacillus larvae, B. lentimorbus, and B. popilliae, and their distribution patterns, were studied by gas-liquid chromatography. All six organisms produced eight major fatty acids: six branched (iso-C(14), -C(15), -C(16), and -C(17), and anteiso-C(15) and -C(17)), two normal (n-C(14) and -C(16)), and two minor (n-C(15) and monounsaturated n-C(16)). In addition, some other trace acids were produced. Branched-chain fatty acids accounted for 54 to 85% of the total fatty acids. These compositions are similar to those previously found with 26 strains of 12 species of the genus Bacillus. Thus, an abundance of branched-chain fatty acids seems to be a characteristic of the biochemical nature of the genus Bacillus. It is noteworthy that marked differences between the nutritional requirements of the three insect pathogens used in the present study and those of the other 12 species of the genus Bacillus studied previously are not significantly reflected in their fatty acid composition.     

The diatom flora of the Salton Sea, California

We report on diatom species of the Salton Sea, a highly saline (43 g l^–1) inland lake in California. We identified and photographed all diatom taxa encountered in the phytoplankton and benthos of the Salton Sea and its immediate tributaries. Ninety-four taxa were distinguished based on their morphological features using light- and electron microscopy. In the Salton Sea, there are four general categories of diatom assemblages related to their habitats: (1) A planktonic assemblage composed of Chaetoceros muelleri var. subsalsum, Cyclotella choctawhatcheeana, Cyclotella sp., Cylindrotheca closterium, Pleurosigma ambrosianum, Thalassionema sp.; (2) a benthic assemblage with diatoms that live on the bottom (e.g. genera Caloneis, Diploneis, Entomoneis, Gyrosigma, Plagiotropis, Pleurosigma, Surirella and Tryblionella), or in algal mats (Proschkinia bulnheimii, several species of Navicula and Seminavis gracilenta); (3) an epiphytic community attached to the macroscopic green algae which grow on the rocks and other hard surfaces near shore (e.g. Achnanthes brevipes, Licmophora ehrenbergii, Tabularia parva); and (4) a freshwater assemblage composed of species that get washed in by the rivers and other inflows discharging into the Sea (e.g. Achnanthidium minutissimum, Cocconeis pediculus, Cyclotella atomus, C. scaldensis, Nitzschia elegantula, T. weissflogii). The most striking feature of the phytoplankton is the abundance of species formerly known only from marine environments; this is not surprising given the high salinity and the peculiar history of the lake.     

Novel diterpenoids and hydrocarbons in the Dufour gland of the ectoparasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae)

Chemical constituents contained in the Dufour gland of the ectoparasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae) were characterized. Three terpenes, beta-springene, a homo-beta-springene, and a homo-geranyllinalool constitute approximately 37% of the gland components, with the remaining 63% all being hydrocarbons. The hydrocarbons consist of a homologous series of n-alkanes (n-C21 to n-C31), a trace amount of 3-methyl C23, a homologous series of internally methyl-branched alkanes (11-methyl C23 to 13-methyl C35), one dimethylalkane (13,17-dimethyl C33), a homologous series of monoenes (C(25:1) to C(37:1)) with the double bonds located at Delta9, Delta13 and Delta15 for alkenes of carbon number 25 to 31 and at Delta13 and Delta15 for carbon numbers 33 to 37 and three homologous dienes in very low amounts with carbon numbers of 31, 32, and 33. The terpenoid and hydrocarbon composition of the Dufour gland was similar in virgin and mated females. However, in contrast to the hydrocarbons, the amount of beta-springene and homo-geranyllinalool increased significantly with time after adult emergence from the cocoon. Although many hydrocarbons in the Dufour gland are the same as those on the cuticle of this species [Howard and Baker, Arch. Insect Biochem. Physiol. 53:1-18 (2003)], substantial differences also occur. Of particular note is the chain length of alkenes and location of the double bonds: cuticular alkenes have a chain length of C23 to C29 and double bond locations at Delta5, Delta7, and Delta9, whereas the Dufour gland alkenes contains a greater range of carbon numbers and have no Delta5 or Delta7 alkenes. The Dufour gland contains only one of the long-chain dimethylalkanes found on the cuticle. Also, no terpenoids are found on the cuticle, and the Dufour gland contains none of the secondary wax esters that are major components on the cuticle. GC-MS analysis of lipids carried in the hemolymph of H. hebetor indicated that all hydrocarbons found on both the cuticle and in the Dufour gland are present, as are some of the wax esters. However, none of the terpenoids were detected in the hemolymph. This suggests that the hydrocarbons are synthesized in other tissues or cells, probably by oenocytes, and differentially partitioned between the cuticle and the Dufour gland. The terpenoids are most likely synthesized within the Dufour gland. Analysis of surface lipids from eggs laid within 18 h indicated that no diterpenoids were present. Rather, the lipids present on the eggs were n-alkanes, monomethylalkanes, alkenes, and secondary alcohol wax esters. This composition did not reflect that of the Dufour gland, hence eggs are not being coated with Dufour gland components during oviposition.     

An exception among diatoms: unique organization of genes involved in isoprenoid biosynthesis in Rhizosolenia setigera CCMP 1694

The marine diatom Rhizosolenia setigera is unique among this group of microalgae given that it is only one of a handful of diatom species that can produce highly branched isoprenoid (HBI) hydrocarbons. In our efforts to determine distinguishing molecular characteristics in R. setigera CCMP 1694 that could help elucidate the underlying mechanisms for its ability to biosynthesize HBIs, we discovered the occurrence of independent genes encoding for two isopentenyl diphosphate isomerases (RsIDI1 and RsIDI2) and one squalene synthase (RsSQS), enzymes that catalyze non‐consecutive steps in isoprenoid biosynthesis. These genes are peculiarly fused in all other genome‐sequenced diatoms to date, making their organization in R. setigera CCMP 1694 a clear distinguishing molecular feature. Phylogenetic and sequence analysis of RsIDI1, RsIDI2, and RsSQS revealed that such an arrangement of individually transcribed genes involved in isoprenoid biosynthesis could have arisen through a secondary gene fission event. We further demonstrate that inhibition of squalene synthase (SQS) shifts the flux of exogenous isoprenoid precursors towards HBI biosynthesis suggesting the competition for isoprenoid substrates in the form of farnesyl diphosphate between the sterol and HBI biosynthetic pathways in this diatom.     

Tetra-unsaturated sesterterpenoids (Haslenes) from Haslea ostrearia and related species

The structures and distributions of C25 highly branched isoprenoid (HBI) alkenes (Haslenes) have been determined following isolation from cultures of the diatoms Haslea ostrearia, Haslea crucigera, Haslea pseudostrearia and Haslea saltstonica. The distributions of the HBIs change between Haslea species and also between different cultures of the same species. Large scale culturing of H. ostrearia and H. pseudostrearia has enabled the structures of three new tetra-unsaturated alkenes to be determined by NMR spectroscopy. The structural relationships between different Haslenes together with the potential significance of the biosynthesis of HBIs by the Haslea genus is discussed.     

Chemical basis for inter-colonial aggression in the stingless bee Scaptotrigona bipunctata (Hymenoptera: Apidae)

Inter-colonial aggression was tested using three colonies of Scaptotrigona bipunctata in a natural setting when their nests were moved and by artificial contact between individuals. Examination of the cuticular lipids of individuals from two colonies kept under identical conditions showed clear differences in their cuticular hydrocarbon profiles. The cuticular lipids were a mixture of hydrocarbons (saturated and unsaturated alkanes and alkenes) within the range of C23-C29. The use of multivariate analysis (PCA and discriminant analysis) showed that seven of the identified surface compounds are enough to separate workers from colonies A and B from each other.     

Xanthones from Garcinia smeathmannii (Oliver) and their antimicrobial activity

Two new xanthones, smeathxanthone A (1) (2-(3,7-dimethyl-2,6-octadienyl)-1,3,5,8-tetrahydroxyxanthone) and smeathxanthone B (2) (5,7,10-trihydroxy-2-methyl-2-(4-methylpent-3-enyl)[2H, 6H]pyrano[3,2-b]xanthen-6-one), have been isolated from the stem bark of Garcinia smeathmannii, and their structures elucidated on the basis of 1D and 2D NMR experiments. 1,3,5-Trihydroxyxanthone and 1,5-dihydroxyxanthone were also obtained. The compounds showed only modest activity against a range of bacteria and yeasts.     

The evolution of tachinid pollination in Neotinea ustulata is related to floral cuticular composition and the combined high relative production of (Z)-11-C23/C25enes

Among terrestrial orchids, and particularly among the subtribe Orchidinae, flies are underrepresented as pollinators. The European Neotinea ustulata, which developed specialized pollination by tachinid flies, is known to produce high relative concentrations of the floral cuticular alkenes (Z)-11-tricosene and (Z)-11-pentacosene (referred to as (Z)-11-C23/C25enes), which seem to be uncommon among orchid flowers. If the evolution of tachinid pollination is related to that of (Z)-11-C23/C25enes, we can expect that closely related species have a different floral chemical pattern and significantly small or no production of (Z)-11-C23/C25enes, independently of their pollinator guild identity (e.g., bees, flies, moths). We chemically compared the floral cuticular composition among Neotinea species, performed electrophysiological analyses, reconstructed the phylogenetic Orchidinae tree, and identified the evolutionary history of pollinator guild and (Z)-11-C23/C25enes production within the Orchidinae. Neotinea ustulata has evolved a markedly different floral cuticular composition compared to other Neotinea and produces both compounds ((Z)-11-C23/C25enes) in high relative quantities (i.e., above 8% in combination), which are detectable by tachinid antennae. Moreover, most Orchidinae taxa have minimal or no production of these alkenes, independently of the identity of their pollinator guild. Our ancestral reconstruction suggested that (Z)-11-C23/C25enes production was an evolutionary exaptation in Neotinea, whereas tachinid pollination was a unique evolutionary innovation for N. ustulata. Floral cuticular composition and, in particular, the combined production of (Z)-11-C23/C25enes at relatively high concentrations is intimately linked to the evolution of tachinid pollination within the Orchidinae.