New deuterated oligo(ethylene glycol) building blocks and their use in the preparation of surface active lipids possessing labeled hydrophilic tethers

For the introduction of additional analysis protocols of tethered molecules, a method is presented to prepare functionalized, deuterated oligo(ethylene glycols) from ethylene glycol-d4. Partial oligomerization of ethylene glycol-d4 and conversion to ditosylates is accompanied by coupling reactions to prepare doubly benzyl protected oligo(ethylene glycols) with two to five repeating units. The tetramer bearing 16 deuteria was elaborated at both ends to eventually prepare 2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-d,l-alpha-lipoic acid ester (DPTL), which bears a fully deuterated tetra(ethylene glycol) spacer group. Through linking of functionalized components, an analogue of DPTL possessing an octa(ethylene glycol) spacer group was prepared, both in deuterated and unlabeled form.     

The Impact of an Anchoring Layer on the Formation of Tethered Bilayer Lipid Membranes on Silver Substrates

Tethered bilayer lipid membranes (tBLMs) have been known as stable and versatile experimental platforms for protein–membrane interaction studies. In this work, the assembly of functional tBLMs on silver substrates and the effect of the molecular chain-length of backfiller molecules on their properties were investigated. The following backfillers 3-mercapto-1-propanol (3M1P), 4-mercapto-1-butanol (4M1B), 6-mercapto-1-hexanol (6M1H), and 9-mercapto-1-nonanol (9M1N) mixed with the molecular anchor WC14 (20-tetradecyloxy-3,6,9,12,15,18,22 heptaoxahexatricontane-1-thiol) were used to form self-assembled monolayers (SAMs) on silver, which influenced a fusion of multilamellar vesicles and the formation of tBLMs. Spectroscopic analysis by SERS and RAIRS has shown that by using different-length backfiller molecules, it is possible to control WC14 anchor molecules orientation on the surface. An introduction of increasingly longer surface backfillers in the mixed SAM may be related to the increasing SAMs molecular order and more vertical orientation of WC14 at both the hydrophilic ethylenoxide segment and the hydrophobic lipid bilayer anchoring alkane chains. Since no clustering of WC14 alkane chains, which is deleterious for tBLM integrity, was observed on dry samples, the suitability of mixed-component SAMs for subsequent tBLM formation was further interrogated by electrochemical impedance spectroscopy (EIS). EIS showed the arrangement of well-insulating tBLMs if 3M1P was used as a backfiller. An increase in the length of the backfiller led to increased defectiveness of tBLMs. Despite variable defectiveness, all tBLMs responded to the pore-forming cholesterol-dependent cytolysin, vaginolysin in a manner consistent with the functional reconstitution of the toxin into phospholipid bilayer. This experiment demonstrates the biological relevance of tBLMs assembled on silver surfaces and indicates their utility as biosensing elements for the detection of pore-forming toxins in liquid samples.     

Enzyme activity to augment the characterization of tethered bilayer membranes

The rate of Ca2+ -triggered phospholipase A2 (PLA2) degradation of tethered bilayer membranes (tBLMs), composed of a synthetic lipid, beta-mercaptoethanol, and palmitoyloleoylphosphatidylcholine (POPC), is approximately 80 times greater than for those prepared with diphytanoylphosphatidylcholine (DPhyPC). Electrochemical impedance spectroscopy (EIS) and neutron reflectivity (NR) data indicate complete, water-free tBLMs that exhibit near ideal capacitive behavior and the presence of a water reservoir in the bilayer subspace proximal to the substrate (Au) surface for both tBLMs. Together these data indicate that the POPC and the DPhyPC tBLMs are structurally similar along the surface normal but markedly different at the outer leaflet/solution interface and that PLA2 is a sensitive probe of short length scale structural differences not revealed by EIS and NR.     

Tethered bilayer lipid membranes on mixed self-assembled monolayers of a novel anchoring thiol: impact of the anchoring thiol density on bilayer formation

Tethered bilayer lipid membranes (tBLMs) are designed on mixed self-assembled monolayers (SAMs) of a novel synthetic anchoring thiol, 2,3-di-o-palmitoylglycerol-1-tetraethylene glycol mercaptopropanoic acid ester (TEG-DP), and a new short dilution thiol molecule, tetraethylene glycol mercaptopropanoic acid ester (TEG). tBLM formation was accomplished by self-directed fusion of small unilamellar vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. The influence of the dilution of the anchoring thiol molecule in the SAM on the vesicle fusion process and on the properties of the resulting tBLMs is studied. It is observed by quartz crystal microbalance that vesicle fusion is a one-step process for a pure TEG-DP SAM as well as for mixed SAMs containing a high concentration of the anchoring thiol. However, upon dilution of the anchoring thiol to moderate concentrations, this process is decelerated and possibly follows a pathway different from that observed on a pure TEG-DP SAM. Electrochemical impedance spectroscopy is used to qualitatively correlate the composition of the SAM to the electrical properties of the tBLM. In this paper we also delineate the necessity of a critical concentration of this anchoring TEG-DP thiol as a requisite for inducing the fusion of vesicles to form a tBLM.     

Formation and finite element analysis of tethered bilayer lipid structures

Rapid solvent exchange of an ethanolic solution of diphytanoyl phosphatidylcholine (DPhyPC) in the presence of a mixed self-assembled monolayer (SAM) [thiolipid/β-mercaptoethanol (βME) (3/7 mol/mol) on Au] shows a transition from densely packed tethered bilayer lipid membranes [(dp)tBLMs], to loosely packed tethered bilayer lipid membranes [(lp)tBLMs], and tethered bilayer liposome nanoparticles (tBLNs) with decreasing DPhyPC concentration. The tethered lipidic constructs in the aqueous medium were analyzed by atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). Finite element analysis (FEA) was applied to interpret spectral EIS features without referring to equivalent circuit modeling. Using structural data obtained earlier from neutron reflectometry and dielectric constants of lipid bilayers, we reproduced experimentally observed features of the electrochemical impedance (EI) spectra of complex surface constructs involving small pinhole defects, large membrane-free patches, and bound liposomes. We demonstrated by FEA that highly insulating (dp)tBLMs with low-defect density exhibit EI spectra in the shape of a perfect semicircle with or without low-frequency upward "tails" in the Cole-Cole representation. Such EI spectra were observed at DPhyPC concentrations of >5 × 10(-3) mol L(-1). While AFM was not able to visualize very small lateral defects in such films, EI spectra unambiguously signaled their presence by increased low frequency "tails". Using FEA we demonstrate that films with large diameter visible defects (>25 nm by AFM) produce EI spectral features consisting of two semicircles of comparable size. Such films were typically obtained at DPhyPC concentrations of <5 × 10(-3) mol L(-1). At DPhyPC concentrations of <1.0 × 10(-3) mol L(-1) the planar bilayer structures were replaced by ellipsoidal liposomes with diameters ranging from 50 to 500 nm as observed in AFM images. Despite the distinct surface morphology change, the EI curves exhibited two semicircle spectral features typical for the large size defects in planar tBLMs. FEA revealed that, to account for these EI features for bound liposome systems (50-500 nm diameter), one needs to assume much lower tBLM conductivities of the submembrane space, which separates the electrode surface and the phospholipid bilayer. Alternatively, FEA indicates that such features may also be observed on composite surfaces containing both bound liposomes and patches of planar bilayers. Triple semicircular features, observed in some of the experimental EI curves, were attributed to an increased complexity of the real tBLMs. The modeling demonstrated that such features are typical for heterogeneous tBLM surfaces containing large patches of different defectiveness levels. By integrating AFM, EIS, and FEA data, our work provides diagnostic criteria allowing the precise characterization of the properties and the morphology of surface supported bilayer systems.     

Tethered bilayer lipid membranes studied by simultaneous attenuated total reflectance infrared spectroscopy and electrochemical impedance spectroscopy

The formation of tethered lipid bilayer membranes (tBLMs) from unilamelar vesicles of egg yolk phosphatidylcholine (EggPC) on mixed self-assembled monolayers (SAMs) from varying ratios of 6-mercaptohexanol and EO(3)Cholesteryl on gold has been monitored by simultaneous attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS). The influence of the lipid orientation (and hence the anisotropy) of lipids on a gold film on the dichroic ratio was studied by simulations of spectra with a matrix method for anisotropic layers. It is shown that for certain tilt angles of the dielectric tensor of the adsorbed anisotropic layer dispersive and negative absorption bands are possible. The experimental data indicate that the structure of the assemblies obtained varies with varying SAM composition. On SAMs with a high content of EO(3)Cholesteryl, tBLMs with reduced fluidity are formed. For SAMs with a high content of 6-mercaptohexanol, the results are consistent with the adsorption of flattened vesicles, and spherical vesicles have been found in a small range of surface compositions. The kinetics of the adsorption process is consistent with the assumption of spherical vesicles as long-living intermediates for surfaces of a high 6-mercaptohexanol content. No long-living spherical vesicles have been detected for surfaces with a large fraction of EO(3)Cholesteryl tethers. The observed differences between the surfaces suggest that for the formation of tBLMs (unlike supported BLMs) no critical surface coverage of vesicles is needed prior to lipid bilayer formation.     

Electrochemical impedance spectroscopy of tethered bilayer membranes

The electrochemical impedance spectra (EIS) of tethered bilayer membranes (tBLMs) were analyzed, and the analytical solution for the spectral response of membranes containing natural or artificially introduced defects was derived. The analysis carried out in this work shows that the EIS features of an individual membrane defect cannot be modeled by conventional electrical elements. The primary reason for this is the complex nature of impedance of the submembrane ionic reservoir separating the phospholipid layer and the solid support. We demonstrate that its EIS response, in the case of radially symmetric defects, is described by the Hankel functions of a complex variable. Therefore, neither the impedance of the submembrane reservoir nor the total impedance of tBLMs can be modeled using the conventional elements of the equivalent electrical circuits of interfaces. There are, however, some limiting cases in which the complexity of the EIS response of the submembrane space reduces. In the high frequency limit, the EIS response of a submembrane space that surrounds the defect transforms into a response of a constant phase element (CPE) with the exponent (α) value of 0.5. The onset of this transformation is, beside other parameters, dependent on the defect size. Large-sized defects push the frequency limit lower, therefore, the EIS spectra exhibiting CPE behavior with α ≈ 0.5, can serve as a diagnostic criterion for the presence of such defects. In the low frequency limit, the response is dependent on the density of the defects, and it transforms into the capacitive impedance if the area occupied by a defect is finite. The higher the defect density, the higher the frequency edge at which the onset of the capacitive behavior is observed. Consequently, the presented analysis provides practical tools to evaluate the defect density in tBLMs, which could be utilized in tBLM-based biosensor applications. Alternatively, if the parameters of the defects, e.g., ion channels, such as the diameter and the conductance are known, the EIS data analysis provides a possibility to estimate other physical parameters of the system, such as thickness of the submembrane reservoir and its conductance. Finally, current analysis demonstrates a possibility to discriminate between the situations, in which the membrane defects are evenly distributed or clustered on the surface of tBLMs. Such sensitivity of EIS could be used for elucidation of the mechanisms of interaction between the proteins and the membranes.     

Redox enzymes in tethered membranes

An electrode surface is presented that enables the characterization of redox-active membrane enzymes in a native-like environment. An ubiquinol oxidase from Escherichia coli, cytochrome bo(3) (cbo(3)), has been co-immobilized into tethered bilayer lipid membranes (tBLMs). The tBLM is formed on gold surfaces functionalized with cholesterol tethers which insert into the lower leaflet of the membrane. The planar membrane architecture is formed by self-assembly of proteoliposomes, and its structure is characterized by surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS), and tapping-mode atomic force microscopy (TM-AFM). The functionality of cbo(3) is investigated by cyclic voltammetry (CV) and is confirmed by the catalytic reduction of oxygen. Interfacial electron transfer to cbo(3) is mediated by the membrane-localized ubiquinol-8, the physiological electron donor of cbo(3). Enzyme coverages observed with TM-AFM and CV coincide (2-8.5 fmol.cm(-)(2)), indicating that most-if not all-cbo(3) on the surface is catalytically active and thus retains its integrity during immobilization.     

Saponins from leaves of Acanthopanax hypoleucus Makino.

From the leaves of Acanthopanax hypoleucus Makino (Araliaceae), five triterpenoidal saponins, having oleanolic acid and hederagenin as sapogenins, were isolated. On the basis of chemical and spectral data, the structures of two new saponins, named hypoleucosides A (1), and B (5) were elucidated as follows: 1; 3-O-beta-D-glucopyranosyl 11 alpha-methoxy-oleanolic acid 28-O-beta-D-glucopyranosyl ester, 5; 3-O-beta-D-glucopyranosyl-(1----2)-alpha-L-arabinopyranosyl-(1---- 4)-beta-D-glucopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl-(1----6)-beta-D-glucopyranosyl ester.     

Study on the Chemical Constituent from the Seed of Litchi chinensis Sonn.

Seed of Litchi chinensis Sonn. is a traditional Chinese medicine, having curative effects on gastric disease, diabetes,and relieving pain.[1] Two new compounds 4-O-p-coumaroylquinic acid butyl ester (1) and 3-O-p-coumaroylquinic acid butyl ester (2), along with five known compounds: 5-O-p-coumaroylquinic acid butyl ester (3),[2] 3-O-p-coumaroylquinic acid methyl ester (4),[3] n-butyl-1-β-D-fructopyranoside (5),[4] D-1-O-methylmyo-inositol (6)[5] and 1H-imidazole-2,3-dihydro-2-oxo-4-carboxylic acid methyl ester (7)[5] were isolated and purified from the ethanol extracts of the seed of L.chinensis Sonn. Their structures, shown in Figure 1, were elucidated by ESI-MS, 1H NMR and 13C NMR, including 2DNMR spectroscopy.     

新型锂离子电池电解液添加剂的合成与应用

设计并合成了一系列基于苯环和环状碳酸酯的有机分子双(2,3-环碳酸甘油酯)对苯二甲酸酯、三(2,3-环碳酸甘油酯)均苯三甲酸酯和四(2,3-环碳酸甘油酯)均苯四甲酸酯,采用倍率测试、恒流充放电测试、交流阻抗测试和扫描电子显微镜测试等手段研究了这些添加剂对锂离子电池性能的影响.通过对循环20周前后球化石墨电极形貌的对比,发现含均苯四甲酸酯和均苯三甲酸酯的电解液球化石墨电极表面相对于空白电解液可形成一层致密而稳定的固体电解质中间相膜(SEI),从而优化电极-电解液的界面性能,且电池电阻增加较小;在测试电池的倍率性能时发现,均苯四甲酸酯的加入可以改善电池的倍率性能,而对苯二甲酸酯的加入则未能改善电池的循环性能.     

Studies on the metabolism of 4-methylpiperazine-1-carbodithioc acid 3-cyano-3,3- diphenylpropyl ester hydrochloride in rat bile by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

4-Methylpiperazine-1-carbodithioc acid 3-cyano-3,3-diphenylpropyl ester hydrochloride (TM208) is a newly synthesized compound which has shown excellent in vivo and in vitro anticancer activity and low toxicity. In the present study, the major metabolites of TM208 in rat bile were studied by high-performance liquid chromatography/tandem mass spectrometry with an electrospray ionization (ESI) interface. It was demonstrated that TM208 was extensively metabolized in rat bile and nine metabolites (M1-M9) were definitely or tentatively identified: (2-aminoethyl)dithiocarbamic acid 3-cyano-3,3-diphenylpropyl ester (M1), (2-methylaminoethyl)dithiocarbamic acid 3-cyano-3,3-diphenylpropyl ester (M2), 4-[(4-methylpiperazin-1-yl)thioxomethanesulfinyl]-2,2-diphenylbutyronitrile (M3), 4-methylpiperazine-1-carbodithioic acid 3-cyano-3-(4-hydroxyphenyl)-3-phenylpropyl ester(M4), the sulfine of (4-methylpiperazine-1-carbodithioc acid 3-cyano-3,3-diphenylpropyl ester) (M5), 4-methylpiperazine-1-carbothioic acid S-(3-cyano-3,3-diphenylpropyl) ester (M6), piperazine-1-carbodithioic acid 3-cyano-3,3-diphenylpropyl ester (M7), 4-hydroxymethylpiperazine-1-carbothioic acid S-(3-cyano-3,3-diphenylpropyl) ester (M8) and the sulfine of [4-methylpiperazine-1-carbodithioic acid 3-cyano-3-(4-hydroxyphenyl)-3-phenylpropyl ester] (M9).     

Saponins from Amaranthus hypochondriacus.

Four triterpenoid saponins were isolated from Amaranthus hypochondriacus which are grain crops in the Nepal, Mexico and South America. Their structures were elucidated based on spectral evidence to be: (1) 3-O-alpha-L-rhamnopyranosyl(1----3)-beta-D-glucuronopyranosyl-2 beta,3 beta-dihydroxyolean-12-en-28-oic acid 28-O-beta-D-glucopyranosyl ester. (2) 3-O-alpha-L-rhamnopyranosyl(1----3)-beta-D-glucuronopyranosyl-2 beta,3 beta- dihydroxyolean-12-en-23-al-28-oic acid 28-O-beta-D-glucopyranosyl ester; (3) 3-O-alpha-L-rhamnopyranosyl(1----3)-beta-D-glucuronopyranosyl-2 beta, 3 beta-dihydroxy-30-norolean-12,20(29)-dien-28-oic acid 28-O-beta-D-glucopyranosyl ester. (4) 3-O-alpha-L-rhamnopyranosyl (1----3)-beta-D-glucuronopyranosyl-2 beta, 3 beta-dihydroxy-30-norolean-12, 20(29)-dien-23-al-28-oic acid 28-O-beta-D-glucopyranosyl ester.     

Studies on the constituents of Aster scaber Thunb. III. Structures of scaberosides B7, B8 and B9, minor oleanolic acid glycosides isolated from the root.

Three new oleanolic acid 3,28-O-bisdesmosides, scaberosides B7, B8 and B9, were isolated as minor saponins from the root of Aster scaber THUNB. (Compositae), and their structures were determined based on spectral and chemical evidence as follows. Scaberoside B7 is 3-O-beta-D-glucopyranosyluronic acid oleanolic acid 28-[O-beta-D-apiofuranosyl-(1----3)-[O-beta-D-xylopyranosyl-(1---- 4)-O-alpha-L-rhamnopyranosyl-(1----2)-alpha-L-arabinopyranosyl] ester, scaberoside B8, 3-O-beta-D-glucopyranosyl oleanolic acid 28-[O-beta-D-xylopyranosyl-(1----4)-O-alpha-L-rhamnopyranosyl-(1----2)-a lpha-L-arabinopyranosyl] ester, and scaberoside B9, 3-O-beta-D-glucopyranosyluronic acid oleanolic acid 28-[O-alpha-L-rhamnopyranosyl-(1----2)-[O-beta-D-xylopyranosyl-(1----6)] -beta-D-glucopyranosyl] ester. Scaberosides B7 and B9 were obtained as their methyl esters.     

Antioxidant secondary metabolites from Geranium lasiopus Boiss. & Heldr

Chromatographic studies on the EtOAc soluble portion of the MeOH extract of Geranium lasiopus led to the isolation of eight flavonoids (kaempferol (1), quercetin (2), quercetin 3-O-β-glucopyranoside (3), quercetin 3-O-β-galactopyranoside (4), kaempferol 3-O-α-rhamnopyranosyl-(1?→?6)-β-glucopyranoside (5), quercetin 3-O-α-rhamnopyranosyl-(1?→?6)-β-glucopyranoside (6), kaempferol 3-O-α-rhamnopyranosyl-(1?→?2)-β-glucopyranoside (7) and quercetin 3-O-α-rhamnopyranosyl-(1?→?2)-β-glucopyranoside (8)), two simple phenolic compounds (gallic acid (9) and its methyl ester (10)) and a hydrolysable tannin (pusilagin (11)). The structures of the compounds were elucidated by 1- and 2-dimensional NMR techniques ((1)H, (13)C, COSY, HMBC, HMQC) and ESI-TOF-MS spectrometry. Inhibitory effects on H(2)O(2)-induced lipid peroxidation in human red blood cells of the different extracts of G. lasiopus, as well as isolated compounds, were investigated. All tested compounds showed comparable or higher activity than that of ascorbic acid and trolox.     

Anchor-lipid monolayers at the air-water interface; prearranging of model membrane systems

Model membrane systems are gaining more and more interest both for basic studies of membrane-related processes as well as for biotechnological applications. Several different model systems have been reported among which the tethered bilayer lipid membranes (tBLMs) form a very attractive and powerful architecture. In all the proposed architectures, a control of the lateral organization of the structures at a molecular level is of great importance for an optimized preparation. For tBLMs, a homogeneous and not too dense monolayer is required to allow for the functional incorporation of complex membrane proteins. We present here an alternative approach to the commonly used self-assembly preparation. Lipids are spread on the air-water interface of a Langmuir film balance and form a monomolecular film. This allows for a better control of the lateral pressure and distribution for subsequent transfer to solid substrates. In this paper, we describe the properties of the surface monolayer, in terms of surface pressure, structure of the lipid molecule, content of lipid mixtures, temperature, and relaxations features. It is shown that a complete mixing of anchor-lipids and free lipids can be achieved. Furthermore, an increase of the spacer lengths and a decrease of the temperature lead to more compact films. This approach is a first step toward the fully controlled assembly of a model membrane system.     

Antithrombotic lipids from Semen Persicae

Chemical investigation of Semen Persicae has led to the isolation of decane (1), triolein (2), nonacosanoic acid (3), oleic acid ethyl ester (4), palmitic acid (5), oleic acid (6) and 15,16-dihydroxy-9Z,12Z-octadecadienoic acid 2,3-dihydroxypropyl ester (7). Amongst these, compound 7 is a new lipid. Their structures were elucidated by chemical and extensive spectral analysis. Their anticoagulative activities were also evaluated in vitro, which showed that petroleum ether extract and compounds 5-6 could significantly prolong thrombin time while methanol extract could obviously inhibit platelet aggregation.     

6063铝合金三价铬化学转化膜的制备与电化学性能

以硫酸铬钾及磷酸为原料在6063铝合金上制备了三价铬化学转化膜. 采用极化曲线及交流阻抗技术研究了不同条件下三价铬转化膜的电化学性能. 结果表明, 温度为30-40 ℃、沉积时间为9 min、pH值为2.0-3.0、KCr(SO4)2为15-25 g·L-1及H3PO4的浓度为10-20 g·L-1的条件为最优条件. Tafel极化曲线结果表明化学转化膜比基体铝合金具有更正的腐蚀电位(Ecorr)、小孔腐蚀电位(Epit)和更低的腐蚀电流(icorr), 说明化学转化膜具有良好的耐腐蚀性能. 利用交流阻抗谱的数据建立了等效电路模型, 并拟合出了腐蚀参数, 如表面电阻(Rcoat)及电容(Ccoat), 电荷转移电阻(Rct)及双电层电容(Cdl)等. 三价铬化学转化膜的交流阻抗谱结果与极化曲线的电化学测试结果相吻合.     

New Exo-Functional Cyclophane Hosts. Synthesis and Crystal Structures of Inclusion Compounds

The new cyclophane type host compounds 1–3, containing rigid aromatic units and two exo-topic carboxylic acid functions, have been synthesized. Crystalline solvent inclusions, involving the dicarboxylic acid hosts and their corresponding ester intermediates 6 (a–c), namely 1·DMSO (1:4), 2·pyridine (1:3), 3·pyridine (1:4), 6a·pyridine (1:2) and 6a·benzene (1:2), have been prepared and studied using X-ray diffraction on single-crystals. Moreover, X-ray structure analyses of the solvent-free crystals of the 6 (a, b) intermediates were also carried out for comparison. Co-crystals of the carboxylic hosts 1–3 contained H-bonded 1:2 host-guest associates as building blocks, together with additional space-filling guests, whereas only loosely bounded space-filling solvent molecules were found in the two solid inclusion compounds of the 6a cyclophane ester host. In addition to the mentioned conventional H-bond interactions between carboxylic hosts and their guests, the crystal structures proved to be held together by relatively weak C–H…O bonds besides the ordinary van der Waals' interactions. Packing relations, and the effects of structural variations, guest molecules and anisotropic packing forces on the conformation of the semi-rigid cylcophane ring have been discussed and compared in seven crystal structures.