Polycyclic Triterpenoids Research Articles

Terpenoids in surface soils from different ecosystems on the Tibetan Plateau

Realizing the potential of terpenoid biomarkers as an unequivocal land ecosystem and floral diversity indicator calls for work to establish a direct link between the terpenoids extracted from the plants and those found in the underlying soils. To this end, we have characterized the triterpenoid profile of soils and vegetation along two altitudinal transects representing very different ecosystems. The first transect, comprising 31 sample sites, was on the eastern slopes of Mt Gongga (eastern Tibetan Plateau) rising from 1230 m to 4500 m above sea level, an area with a temperate monsoonal climate. The second transect, sampling 29 sites, was on the northern slopes of Mt West Kunlun, a region characterized by montane desert, meadow and alpine steppe (located between 1300 m and 5050 m above sea level). The dominant terpenoid biomarkers (polycyclic triterpenoids of the taraxerane, oleanane, friedelane, ursane, lupane, glutinane and lanostane series) identified in the soils were found to be consistent with the overlying vegetation. However, the discovery of some unusual triterpenoids, including the pentacyclic triterpene 3-methyl ethers (PTMEs), D:A-friedo-2,3-secoolean-2,3-dioic acid, methyl 3β-acetoxylup-20(29)-en-28-oate, 24-methylenecycloartan-3-one, 9,19-cyclolanostan-3-yl 24-methylene acetate, 3α-methoxy-9β-lanosta-7,24-dien-26,23-olide and 3α-methoxy-9β-lanost-7-en-26,23-olide in the Gongga soils, does not match the current vegetation and may be indicative of a long vanished forest-type vegetation. Thus, soil biomarkers may be a useful tool in the reconstruction of palaeo-vegetation.

Chemical constituents and their cytotoxicities from mushroom Tricholoma imbricatum

Two undescribed triterpenes, tricholimbrins A and B, three undescribed steroids, tricholimbrins C‒E, one undescribed 4-chromanone derivative, along with 27 known compounds were isolated from fruiting bodies of the mushroom Tricholoma imbricatum. Tricholimbrins A and B are two polycyclic triterpenoids with a carbon degradation, while tricholimbrin C is a ring-rearranged steroid containing an aromatic moiety that might be derived from an ergosterol. Isocyathisterol, 3β,15α-dihydroxyl-(22E,24R)-ergosta-5,8(14),22-trien-7-one, demethylincisterol A3, and volemolide showed cytotoxicities to six human cancer cell lines. 3β-Hydroxyl-(22E,24R)-ergosta-5,8,22-trien-7,15-dione and 3β-hydroxyl-(22E,24R)-ergosta-5,8,22-trien-7-one showed preferable cytotoxicities against HL-60 while chaxine C and volemolide showed preferable cytotoxicities against A-549, with IC50 values less than 10 μM.

Leaf lipid degradation in soils and surface sediments: A litterbag experiment

The fate of leaf lipids upon early diagenesis was monitored in a two year litterbag experiment in a soil and at the water-sediment interface of an adjacent pond. The biomarker content of degrading leaves exhibited substantial variability among litterbags, even for a given time step within a given environmental condition, likely reflecting natural microenvironmental variability. Due to this variability and the oxic conditions in the pond, no substantial difference between the soil and the pond could be evidenced in the biomarker degradation pattern. An occasional increase in the abundance of several biomarkers (β- sitosterol, oleanolic acid, C16 phytyl ester, C27n-alkane) was also noted during the experiment, which was attributed to release of bound compounds and/or an external contribution. Nevertheless, absolute quantification showed that the concentration of all lipid constituents was reduced, but they exhibited different decay profiles: (i) rapid extensive degradation (phytyl ester), (ii) exponential-like decrease (fatty lipids) and (iii) variable degradation profile (polycyclic triterpenoids). However, all the main constituents initially present in the senescent leaves were still detected after two years of degradation in both environments. Fatty lipids abundances generally decreased to<10% of the initial content but the main distribution features (carbon number maximum and predominance) remained unchanged. The results thus tend to validate their use as proxy for source and environment in ancient organic matter. They also suggest that, on a mid-term basis, a plant biomarker signature is not substantially affected by differential degradation in soil and at the water-sediment interface, at least for a qualitative approach.

Characterization of the 2,3-Oxidosqualene Cyclase Gene from Antrodia cinnamomea and Enhancement of Cytotoxic Triterpenoid Compound Production.

Antrodia cinnamomea is a scarce, epiphyte, host-specific, brown-rot fungus that produces diverse bioactive compounds with potent biological activity. Natural wild-type fruiting bodies of A. cinnamomea are rare and highly valued, but their artificial culture poses challenges. Triterpenoids are a group of secondary metabolites that contribute to the bioactivities of A. cinnamomea. 2,3-Oxidosqualene cyclase (OSC) is a key enzyme in triterpenoid biosynthesis, which converts 2,3-oxidosqualene (OS) into polycyclic triterpenoids. In this study, we isolated a 2,3-oxidosqualene cyclase gene from A. cinnamomea with degenerate primers and designated it as AcOSC. The full length AcOSC cDNA was subcloned into a yeast expression vector, and AcOSC activity was confirmed. RT-PCR results showed that AcOSC expression was highest in the wild-type fruiting body and correlated with a higher concentration of triterpenoids. Agrobacterium-mediated gene transformation was conducted to enhance the triterpenoid synthesis capacity of the cultured mycelium. Metabolite profiling was conducted by LC-MS/MS and principal component analysis (PCA). The compositions and contents of metabolites in the AcOSC transgenic lines were different from those in the wild-type mycelium and vector control. The levels of two important triterpenoids, dehydrosulphurenic acid (DSA) and dehydroeburicoic acid (DEA), were increased in A. cinnamomea oxidosqualene cyclase overexpression strains compared to controls. In summary an Agrobacterium-mediated gene transformation procedure was established that successfully increased the level of transgene expression and enhanced the triterpenoid content in cultured A. cinnamomea.

Chemical Conversions of Betulone Produced by Biotransformation

The possibility of transforming betulone formed by bio-oxidation of betulin by intact R. rhodochrous IEGM 66 cells was studied. The corresponding mono- and diglutaryl derivatives were prepared from betulone and its oxime. Beckmann fragmentation of 28-benzoyloxy-3-hydroxyiminolup-20(29)-ene synthesized 3,4-seco- lupane derivatives. Polycyclic triterpenoids with an oxidized 3-hydroxyl are convenient starting platforms for preparing semi-synthetic A-seco-triterpenoids and derivatives with an annelated heterocyclic moiety, many of which exhibit antiviral or cytotoxic activity. The structure of the pentacyclic triterpenoid betulin, which is commonly used to develop new pharmacologically active derivatives, includes two hydroxyls, a primary one on C-28 and a secondary one on C-3 (1, 2). Chemical approaches enabling oxidation of the secondary 3-hydroxyl of betulin to form betulone were reported. However, as a rule, traditional oxidants formed mixtures of products (3) whereas a more selective three-step synthesis of betulone included additional introduction/removal steps for protecting the primary hydroxyl of betulin (4). Biocatalytic technology for one-step oxidation of structurally complicated terpene substrates is being actively pursued as an alternative to chemical methods (5). Thus, a one-step method for bioconversion of betulin into betulone using intact mycelial fungal cells was reported. Application of them for preparative purposes was limited by the low betulin concentration and its low conversion yields (6, 7). Recently, we demonstrated that actinobacteria of the genus Rhodococcus could be used as effective biocatalysts for biotransformation of betulin (1) into betulone (2) and proposed a biocatalytic method for preparing betulone using dormant IEGM 66 R. rhodochrous cells (8, 9). Herein the synthesis of new derivatives from betulone, obtained by a one-step regioselective biotransformation of betulin, is reported. A suspension of intact IEGM 66 R. rhodochrous cells in phosphate buffer was used for preparative biocatalytic oxidation of betulin over 24 h (9). The target compound 2 (36.8% yield) was isolated by flash chromatography from the obtained extract of the biotransformation products. Reaction of 2 with 3,3-dimethylglutaric acid anhydride produced ester 3 (58% yield). The PMR spectrum of 3 showed a weak-field shift of the doublets for the C-28 methylene protons (3.84 and 4.27 ppm) compared with those of starting 2 and characteristic resonances for the 3,3-dimethylglutaric acid. The 13 C NMR spectrum of 3 exhibited resonances at 173.76 and 183.25 ppm for the ester and carboxyl C atoms. Treatment of ester 3 or the mixture of biotransformation products with hydroxylamine hydrochloride in EtOH:Py produced oximes 4 or 5 in yields of 69 and 24%, respectively. IR spectra of oximes 4 and 5 showed strong bands at 1640-1642 cm -1 for hydroxylamine C=N stretching vibrations. The C-3 resonance in 13 C NMR spectra was observed at 167.13-168.61 ppm. Reaction of oxime 5 with 3,3-dimethylglutaric acid anhydride afforded diester 6. The doubled integrated intensity of the corresponding proton resonances at 2.42-2.46 ppm confirmed that 6 contained two 3,3-dimethylglutaric acids.

Hopanoids Play a Role in Membrane Integrity and pH Homeostasis in Rhodopseudomonas palustris TIE-1

Sedimentary hopanes are pentacyclic triterpenoids that serve as biomarker proxies for bacteria and certain bacterial metabolisms, such as oxygenic photosynthesis and aerobic methanotrophy. Their parent molecules, the bacteriohopanepolyols (BHPs), have been hypothesized to be the bacterial equivalent of sterols. However, the actual function of BHPs in bacterial cells is poorly understood. Here, we report the physiological study of a mutant in Rhodopseudomonas palustris TIE-1 that is unable to produce any hopanoids. The deletion of the gene encoding the squalene-hopene cyclase protein (Shc), which cyclizes squalene to the basic hopene structure, resulted in a strain that no longer produced any polycyclic triterpenoids. This strain was able to grow chemoheterotrophically, photoheterotrophically, and photoautotrophically, demonstrating that hopanoids are not required for growth under normal conditions. A severe growth defect, as well as significant morphological damage, was observed when cells were grown under acidic and alkaline conditions. Although minimal changes in shc transcript expression were observed under certain conditions of pH shock, the total amount of hopanoid production was unaffected; however, the abundance of methylated hopanoids significantly increased. This suggests that hopanoids may play an indirect role in pH homeostasis, with certain hopanoid derivatives being of particular importance.

Hypotheses for the origin and early evolution of triterpenoid cyclases.

Hopanes and steranes are found almost universally in the sedimentary rock record where they often are used as proxies for aerobic organisms, metabolisms, and environments. In order to interpret ancient lipid signatures confidently we require a complementary understanding of how these modern biochemical pathways evolved since their conception. For example, generally it has been assumed that hopanoid biosynthesis was an evolutionary predecessor to steroid biosynthesis. Here we re-evaluate this assumption. Using a combined phylogenetic and biochemical perspective, we address the evolution of polycyclic triterpenoid biosynthesis and suggest several constraints on using these molecules as aerobic biomarkers. Amino acid sequence data show that the enzymes responsible for polycyclic triterpenoid biosynthesis (i.e. squalene and 2,3-oxidosqualene cyclases) are homologous. Numerous conserved domains correspond to active sites in the enzymes that are required to complete the complex cyclization reaction. From these sites we develop an evolutionary analysis of three independent characters to explain the evolution of the major classes of polycyclic triterpenoids. These characters are: (i) the number of unfavourable anti-Markovnikov ring closures, (ii) all-chair (CCC) or chair-boat-chair (CBC) substrate conformation, and (iii) the choice between squalene and 2,3-oxidosqualene as the substrate. We use these characters to construct four competing phylogenies to describe the evolution of polycyclic triterpenoid biosynthesis. The analysis suggests that malabaricanoids would be the most ancient polycyclic triterpenoids. The two most parsimonious evolutionary trees are the ones in which hopanoid and steroid cyclases diverged from a common ancestor. The transition from a CCC- to CBC-fold marks the major divergence in the evolution of these pathways, and it is diagnosable in the geological record. However, this transition does not require the simultaneous adoption of the aerobic substrate, 2,3-oxidosqualene, because these characters are controlled by independent parts of the enzyme.

2,3‐oxidosqualene cyclase: From azasqualenes to new site‐directed inhibitors

2,3-Oxidosqualene cyclases (OSC) are enzymes which convert 2,3-oxidosqualene (OS) into polycyclic triterpenoids such as lanosterol, cycloartenol, and alpha- and beta-amyrin. Our interest in the study of OSC is the development of new OSC inhibitors for potential use as hypocholesterolemic, antifungal, or phytotoxic drugs. In particular, we describe the biological activity and the mechanism of a series of acyclic azasqualene derivatives mimicking the C-2, C-8, and C-20 carbonium ions formed during OS cyclization. Some of these carbonium ion analogues are very promising as specific hypocholesterolemic agents. The toxicity, the biodistribution, and the pharmacokinetics of different azasqualene derivatives in mice are also presented. In order to obtain new, site-directed irreversible inhibitors of OSC, a series of squalene derivatives containing functional groups that can link covalently to an active-site thiol group was designed. Among these compounds, squalene maleimide was the most active toward mammalian OSC, whereas squalene Ellman behaved as an irreversible inhibitor of OSC from yeast.

Analysis of extractables from one euphorbia

AbstractChemical analyses have been made of the heptane extractable material ofEuphorbia lathyris, a plant which has been proposed as an “energy farm” candidate. The heptane extract is 4–5% of the dry plant weight and has a heat value of ≈18×103 BTU/Ib. This reduced photosynthetic material consists almost entirely of polycyclic triterpenoids.

Biomimetic polyene cyclizations.

AbstractCertain polyenic substances having trans olefinic bonds in a 1,5 relationship can be induced to undergo stereospecific, non‐enzymic, cationic cyclization to give polycyclic products with all‐trans (“natural”) configuration. These transformations appear to mimic in principle the biogenetic conversion of squalene into polycyclic triterpenoids. Acetal and allylic alcohol functions are effective initiators for these cyclizations, and methylacetylenic end groups are particularly useful terminators since they lead to five‐membered rings. Thus a tetraenic acetal having no chiral centers has been converted in a single step into a tetracyclic product having seven such centers. The process is highly stereoselective, giving only two of 64 possible racemates. Systems have also been developed for effecting the total synthesis of the steroid nucleus in a single step starting from a substrate containing only one ring. The mechanism of these biomimetic cyclizations and also that of their enzymic counterparts is still unknown, but the balance of the evidence favors a synchronous process.