Osmoprotective effect of ubiquinone in lipid vesicles modelling the E. coli plasma membrane

Bacteria need to be able to adapt to sudden changes in their environment, including drastic changes in the surrounding osmolarity. As part of this adaptation, the cells adjust the composition of their cytoplasmic membrane. Recent studies have shown that ubiquinones, lipid soluble molecules involved in cell respiration, are overproduced by bacteria grown in hyperosmotic conditions and it is thus believed that these molecules can provide with osmoprotection. Hereby we explore the mechanisms behind these observations. Liposomes with a lipid headgroup composition mimicking that of the cytoplasmic membrane of E. coli are used as suitable models. The effect of ubiquinone-10 (Q10) on water transport across the membranes is characterized using a custom developed fluorescence-based experimental approach to simultaneously determine the membrane permeability coefficient and estimate the elastic resistance of the membrane towards deformation. It is shown that both parameters are affected by the presence of ubiquinone-10. Solanesol, a molecule similar to Q10 but lacking the quinone headgroup, also provides with osmoprotection although it only improves the resistance of the membrane against deformation. The fluorescence experiments are complemented by cryogenic transmission electron microscopy studies showing that the E. coli membrane mimics tend to flatten into spheroid oblate structures when osmotically stressed, suggesting the possibility of lipid segregation. In agreement with its proposed osmoprotective role, the flattening process is hindered by the presence of Q10.     

Discovery of phenyl acetamides as potent and selective GPR119 agonists

The paper describes the SAR/SPR studies that led to the discovery of phenoxy cyclopropyl phenyl acetamide derivatives as potent and selective GPR119 agonists. Based on a cis cyclopropane scaffold discovered previously, phenyl acetamides such as compound 17 were found to have excellent GPR119 potency and improved physicochemical properties. Pharmacokinetic data of compound 17 in rat, dog and rhesus will be described. Compound 17 was suitable for QD dosing based on its predicted human half-life, and its projected human dose was much lower than that of the recently reported structurally-related benzyloxy compound 2. Compound 17 was selected as a tool compound candidate for NHP (Non-Human Primate) efficacy studies.     

Effects of osmotic pretreatments on oxidative stress, antioxidant profiles and cryopreservation of olive somatic embryos

A three-day pretreatment of olive somatic embryos (SE) with 0.75 M sucrose, combined with cryoprotection (0.5 M DMSO, 1 M sucrose, 0.5 M glycerol and 0.009 M proline) and controlled rate cooling, supported regrowth (as 34.6% fresh weight gain) and resumption of embryo development after cryopreservation. Pretreatment with mannitol or sorbitol did not support regrowth. Profiles of sugars, proline, antioxidant enzymes, Reactive oxygen species (ROS), secondary oxidation products and ethylene were constructed for the most successful (0.75 M) pretreatment series. Sucrose was the optimal pretreatment for supporting recovery, it also elevated glutathione reductase (GR) activity compared to controls, whereas superoxide dismutase (SOD), catalase and guaiacol peroxidase activities remained relatively unchanged. Superoxide dismutase activity was higher in SE pretreated with sucrose, compared with those pretreated with polyols; H₂O₂ was enhanced in SE pretreated with sorbitol and sucrose compared to mannitol. The overall trend for ethylene and OH production revealed their levels were highest in SE pretreated with polyols albeit, for individual treatments this was not always the case. Generally, pretreatments did not significantly change embryo secondary oxidation profiles of ThioBarbituric Acid Reactive Substances (TBARS) and Schiff's bases. In combination these studies suggest oxidative processes may influence regrowth of cryopreserved olive SE and that optimal pretreatments could, in part, increase tolerance by an overall enhancement of endogenous antioxidants (particularly GR), proline and sugars.     

Synchronous bond molecular dynamics of conjugated chlorocyclopropyl alk-yn-enes revealed by ECD and UV–vis

Chlorocyclopropanes (CCPs) conjugated to alk-yn-enes occur in a unique family of polyketide natural products from marine sponges. Synthesis of several optically enriched analogs of CCPs and measurement of their UV–vis spectra and electronic circular dichroism (ECD) spectra reveal unusually strong hyperconjugation that constrains and aligns the cyclopropyl C-C bond with the π-plane of the distal ene-bond. This alignment imposes a barrier to rotation of at least 5.0 kcal·mol⁻¹. Comparison of red-shifted Cotton effects in chiral CCPs show the barrier is independent of alkene substituent and establishes an empirical rule for assignment of other CCP-containing natural products.     

Quadranguloside,a cycloartane triterpene glycoside from Passiflora quadrangularis

Quadranguloside, a new cyclopropane triterpene glycoside was isolated from the methanolic extract of the leaves of Passiflora quadrangularis. Its structure has been established as 9,19-cyclolanost-24Z-en-3β,21,26-triol-3,26-di-O-gentiobioside on the basis of hydrolysis and spectral evidence.     

Synthesis and HPLC enantioseparation of the cyclopropane analogue of valine (c3Val)

A new and efficient method is presented for the preparation of the N-Boc-protected cyclopropane analogue of valine, 1-(N-tert-butoxycarbonyl)amino-2,2-dimethylcyclopropanecarboxylic acid, both in racemic and enantiomerically pure forms. Cyclopropanation of the exocyclic double bond of 2-phenyl-4-isopropylidene-5(4H)-oxazolone with diazomethane followed by elaboration of the heterocyclic moiety provided multigram quantities of the racemic target compound. Subsequent HPLC resolution of a racemic precursor on a noncommercial chiral stationary phase has given access to enantiomerically pure products. Almost 1.5 g of the first-eluted enantiomer and 1.0 g of the second-eluted enantiomer have been isolated in optically pure form using a 150 x 20 mm ID column containing mixed 10-undecenoate/3,5-dimethylphenylcarbamate of cellulose covalently bonded to allylsilica gel with a mixture of hexanes/tert-butyl methyl ether/ethyl acetate as the mobile phase.     

Identification of bottlenecks in the accumulation of cyclic fatty acids in camelina seed oil

Modified fatty acids (mFA) have diverse uses; for example, cyclopropane fatty acids (CPA) are feedstocks for producing coatings, lubricants, plastics and cosmetics. The expression of mFA‐producing enzymes in crop and model plants generally results in lower levels of mFA accumulation than in their natural‐occurring source plants. Thus, to further our understanding of metabolic bottlenecks that limit mFA accumulation, we generated transgenic Camelina sativa lines co‐expressing Escherichia coli cyclopropane synthase (EcCPS) and Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT). In contrast to transgenic CPA‐accumulating Arabidopsis, CPA accumulation in camelina caused only minor changes in seed weight, germination rate, oil accumulation and seedling development. CPA accumulated to much higher levels in membrane than storage lipids, comprising more than 60% of total fatty acid in both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) versus 26% in diacylglycerol (DAG) and 12% in triacylglycerol (TAG) indicating bottlenecks in the transfer of CPA from PC to DAG and from DAG to TAG. Upon co‐expression of SfLPAT with EcCPS, di‐CPA‐PC increased by ~50% relative to lines expressing EcCPS alone with the di‐CPA‐PC primarily observed in the embryonic axis and mono‐CPA‐PC primarily in cotyledon tissue. EcCPS‐SfLPAT lines revealed a redistribution of CPA from the sn‐1 to sn‐2 positions within PC and PE that was associated with a doubling of CPA accumulation in both DAG and TAG. The identification of metabolic bottlenecks in acyl transfer between site of synthesis (phospholipids) and deposition in storage oils (TAGs) lays the foundation for the optimizing CPA accumulation through directed engineering of oil synthesis in target crops.     

Synthesis and properties of methyl 5-(1'R,2'S)-(2-octadecylcycloprop-1-yl)pentanoate and other omega-19 chiral cyclopropane fatty acids and esters related to mycobacterial mycolic acids

A 23-26-carbon chain length range of omega-19 (1'R,2'S) cyclopropane fatty acids, related to mycobacterial mycolic acids, has been prepared. The key cyclopropyl intermediate, (1'R,2'S)-(Z)-1-formyl-2-octadecylcyclopropane, underwent Wittig chemistry with various reagents to provide vinylic precursors, which were selectively reduced to the corresponding saturated omega-19 cyclopropane fatty acids or esters. The 24-carbon omega-19 cyclopropane ester was made by chain elongation of the 23-carbon ester. Saturated and unsaturated chiral cyclopropane acids and esters were assayed, using wall extracts of Mycobacterium smegmatis; the incorporation of 14C-acetate was used to measure inhibition or stimulation of mycolic acid synthesis. Minor inhibition (2-3%) was shown by the 23- and 24-carbon saturated esters; all the other compounds were stimulants. The most effective (38-55%) stimulators of mycolate synthesis were the unsaturated esters with 23- and 26-carbons and the saturated and unsaturated 25-carbon acids.     

Synthesis and Antitumor Activity of Cyclopropane Derivatives of Betulinic and Betulonic Acids

New cyclopropane derivatives of betulin were synthesized by attachment of dichlorocarbenes or dibromocarbenes to the double bond of betulin diacetate, followed by the deprotection of hydroxyl groups. The betulin cyclopropane derivative was obtained from 20,29-dihydro-20,29-dichloromethylenebetulin by treatment with lithium in tert-butanol. These compounds were converted into the corresponding derivatives of betulonic acid by oxidation with chromium trioxide. The reduction of oxo group with sodium borohydride led to the corresponding derivatives of betulinic acid. 20,29-Dihydro-20,29-dichloromethylenebetulinic acid proved to be the most cytotoxic toward human melanoma of the Colo 38 and Bro lines and human ovarian carcinoma of the CaOv line (IC₅₀ 10 μM). 20,29-Dihydro-20,29-dibromomethylenebetulinic acid inhibited the growth of the Bro melanoma cell line and the CaOv carcinoma cell line practically by 50% at a concentration of 10 μM. The other derivatives of betulinic and betulonic acids were active toward all of the three cancer cell lines at concentrations higher than 10 μM.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 320–325.Original Russian Text Copyright © 2005 by Symon, Veselova, Kaplun, Vlasenkova, Fedorova, Lyutik, Gerasimova, Shvets.