Synthesis of Ribose - Oleic Acid Esters in the Presence- and Absence of Candida antarctica Lipase B.

D-ribose-oleic acid esters were produced with or without a biocatalyst, using in the same organic media, dimethyl sulfoxide (DMSO): tert-butanol (TBU) or 2-methyl-2-butanol (2M2B). The yield of the ester product was above 90% in both of the reactions. The biocatalyst used was lipase B of Candida antarctica. Molecular characterization was performed by using all the analytical methods available: IR, 1H-NMR and 13C-NMR, HSQC, and ESI-MS.

Synthesis of 2-ethylhexyl oleate catalyzed by Candida antarctica lipase immobilized on a magnetic polymer support in continuous flow.

This study investigated the synthesis of 2-ethylhexyl oleate catalyzed by Candida antarctica lipase immobilized on magnetic poly(styrene-co-divinylbenzene) particles in a continuous packed-bed bioreactor. Runs were carried out in a solvent-free system at 50 °C. The performance of the reactor was evaluated for substrates composed by oleic acid and 2-ethylhexanol at five molar ratios (1:4-4:1), determining its operation limits in terms of substrate flow rate. The system performance was quantified for three different flow rates corresponding to space-time between 3 and 12 h. For each condition, the influence of the space-time in the ester formation, esterification yield and productivity was determined. The molar ratio of acid-to-alcohol interfered, in a remarkable way, in the formation of 2-ethylhexyl oleate and the best performance was attained for substrate at equimolar ratio running at 12 h space-time. Under this condition, average 2-ethylhexyl oleate concentration was 471.65 ± 2.98 g L-1 which corresponded to ester productivity of 23.16 ± 0.49 mmol g-1 L-1 h-1. This strategy also gave high biocatalyst operational stability, revealing a half-life time of 2063 h. A model based on the ping-pong Bi-Bi mechanism was developed to describe the kinetics of the esterification reaction and validated using experimental data. The goodness of fit of the model was satisfactory (R2 = 0.9310-0.9952).

Enhancing nitrobenzene biodegradation in aquatic systems: Feasibility of using plain soil as an inoculant and effects of adding ascorbic acid and peptone.

Nitrobenzene (NB) is recalcitrant to microbial biodegradation due to the electron-deficient character of the nitro group (NO2-). Prior work has found that the reductant could enhance NB biodegradation by providing excess electron donors. However, the existing theory couldn't explain the increase-and-decrease pattern of the NB biodegradation rate with an increase in a reductant concentration. Our results suggest that the reductant affects NB biodegradation by two mechanisms: the available electron donors and the stimulation or inhibition of biomass growth, which are linked by a pseudo-first-order reaction kinetics. In addition, the results showed that directly inoculating the plain soil into the aquatic system and then allowing the synergistic effect of the organic reductant (ascorbic acid) and the substrate (peptone) enhance NB biodegradation. Employing the new method, 200 mg L-1 NB was transformed in 72 h. GC-MS analysis detected two novel intermediate metabolites, indicating that NB was degraded into aniline and further transformed into acetanilide and 9-octadecenamide before its mineralization. This study sheds light on how to exploit the synergistic effects of the availability of excess electron donors and biomass growth by controlling the reductant and a substrate in the right concentration range (e.g., ascorbic acid < 0.8 mgL-1 + peptone).

Cellular uptake of self-assembled phytantriol-based hexosomes is independent of major endocytic machineries.

Despite increasing interests in non-lamellar liquid crystalline dispersions, such as hexosomes, for drug delivery, little is known about their interactions with cells and mechanism of cell entry. Here we examine the cellular uptake of hexosomes based on phytantriol and mannide monooleate by HeLa cells using live cell microscopy in comparison to conventional liposomes. To investigate the importance of specific endocytosis pathways upon particle internalization, we silenced regulatory proteins of major endocytosis pathways using short interfering RNA. While endocytosis plays a significant role in liposome internalization, hexosomes are not taken up via endocytosis but through a mechanism that is dependent on cell membrane tension. Biophysical studies using biomembrane models highlighted that hexosomes have a high affinity for membranes and an ability to disrupt lipid layers. Our data suggest that direct biomechanical interactions of hexosomes with membrane lipids play a crucial role and that the unique morphology of hexosomes is vital for their membrane activity. Based on these results, we propose a mechanism, where hexosomes destabilize the bilayer, allowing them to "phase through" the membrane. Understanding parameters that influence the uptake of hexosomes is critical to establish them as carrier systems that can potentially deliver therapeutics efficiently to intracellular sites of action.

Asymmetrical, Symmetrical, Divalent, and Y-Shaped (Bola)amphiphiles: The Relationship between the Molecular Structure and Self-Assembly in Amino Derivatives of Sophorolipid Biosurfactants.

Conventional head-chain but also more exotic divalent, Gemini, or bolaform amphiphiles have in common well-defined hydrophilic and hydrophobic blocks with often a predictable self-assembly behavior. However, new categories of amphiphiles, such as microbial biosurfactants, challenge such conventional understanding because of the poorly defined boundaries between the hydrophilic and hydrophobic portions. Microbial glycolipids, such as sophorolipids, rhamnolipids, or cellobioselipids, interesting biodegradable, nontoxic, alternatives to synthetic surfactants, all represent interesting examples of atypical amphiphiles with partially predictable self-assembly properties. However, their limited molecular diversity strongly limits their application potential. For this reason, we used them as ready-made platform to prepare a whole class of new derivatives. In particular, a broad range of amino derivatives of sophorolipid biosurfactant was recently prepared with the goal of producing biobased antimicrobial and transfection agents, of which the efficiency strongly depends on their molecular structure and unpredictable self-assembly behavior. The new compounds contain a set of asymmetrical and symmetrical bolaamphiphiles, the latter with three or four hydrophilic centers, divalent amphiphiles with asymmetric polar headgroups and even Y-shaped amphiphiles, bearing two sophorose groups connected to one nitrogen atom. In this contribution, we employ small-angle X-ray scattering to establish a relationship between their peculiar molecular structures and the self-assembly properties in water. We find that all divalent and Y-shaped compounds form micelles, of which the hydrophilic shell is composed of a bulky sophorose-C x( x = 8,11)-amine moiety, with aggregation numbers between 30 and 100. On the contrary, most symmetrical and asymmetrical bolaamphiphiles display poor self-assembly properties, generally showing aggregation numbers below 20, especially in the presence of either short spacers or large spacers containing hydrophilic centers.

Optimization of Alkyldiethanolamides Synthesis from Terminalia catappa L. Kernel Oil through Enzymatic Reaction.

Alkyldiethanolamides (fatty acid diethanolamides) synthesis from Terminalia catappa L. kernel oil was optimized using lypozyme as a catalyst. The result showed that the optimal reaction conditions were 2 hours reaction time, with a ratio of oil mass (g) to diethanolamine (mmol) of 1:5, a ratio of oil mass to enzyme (g) of 1: 0.075, and a temperature of 40°C. The percentage of alkyldiethanolamides at optimum condition was 56-60%. The synthesis results were also analyzed by FTIR. FTIR spectra revealed specific absorption at several wave numbers (3434 cm-1, 1655 cm-1, 1280 cm-1), indicating that amide and alcohol bonds (C=O, C-N, and O-H) were formed. GC-MS was employed to identify the types of fatty acid diethanolamides that were successfully synthesized. The fatty acid diethanolamides formed were palmitoyldiethanolamide (Rt = 32.96 min) and oleyldiethanolamide (Rt = 35.57 min). The total nitrogen content of alkyldietanolamides was 0.26%, or 0.19 mmol of the amide group in 1 g of sample.

Fungal polysaccharides as a water-adsorbing material in esters production with the use of lipase from Rhizomucor variabilis.

The extracellular crude Rhizomucor variabilis lipase was used for synthesis of flavor ester butyl caprylate and 1-butyl oleate often used as a diesel additive, a polyvinyl chloride plasticizer, a water-resisting agent, and an additive to hydraulic fluids. The influence of various reaction parameters such as the molar ratio, time, enzyme and substrate concentration, and effect of various fungal polysaccharides was estimated. The rate of catalyzed synthesis of esters largely depends on the solvent medium, and the maximum activity was found when n-hexane was used as a solvent. The maximum conversion yield of 58.2% and 59.3% was obtained for butyl caprylate and butyl oleate, respectively, under the following conditions: amount of free lipase 500 U; caprylic acid:butanol molar ratio 1:1; oleic acid:butanol molar ratio 2:1. The addition of naturally obtained fungal polysaccharides significantly enhanced the ester synthesis. The highest conversion rate of 95.2% was observed for butyl caprylate in the presence of AbEPS after 24 h with 500 U of free R. variabilis lipase. In the case of butyl oleate synthesis in the presence of LsPS, a maximum conversion yield of 91.2% was observed after the 24-h reaction.

Point mutation Arg153-His at surface of Bacillus lipase contributing towards increased thermostability and ester synthesis: insight into molecular network.

In order to design proteins with improved properties i.e. thermostability, catalytic efficiency and to understand the mechanisms underlying, a thermostable variant of Bacillus lipase was generated by site-directed mutagenesis with enhanced thermal (∆Tm = + 12 °C) and chemical (∆Cm denaturation for Gdmcl = + 1.75 M) stability as compared to WT. Arg153-His variant showed 72-fold increase in thermostability (t 1/2 = 6 h) at 60 °C as compared to WT (t 1/2 = 5 min). Increase in thermostability might be contributed by the formation of additional hydrogen bonds between His153/AO-Arg106/ANH2 as well as His153-Arg106/ANE. The variant demonstrated broad substrate specificity. A maximum conversion of 59 and 62% was obtained for methyl oleate and methyl butyrate, respectively, using immobilized variant lipase, whereas immobilized WT enzyme synthesizes 35% methyl oleate. WT enzyme was unable to synthesize methyl butyrate as it showed negligible activity with pNP-butyrate.

Lactose oleate as new biocompatible surfactant for pharmaceutical applications.

Sugar fatty acid esters are an interesting class of non-ionic, biocompatible and biodegradable sugar-based surfactants, recently emerged as a valid alternative to the traditional commonly employed (e.g. polysorbates and polyethylene glycol derivatives). By varying the polar head (carbohydrate moiety) and the hydrophobic tail (fatty acid), surfactants with different physico-chemical characteristics can be easily prepared. While many research papers have focused on sucrose derivatives, relatively few studies have been carried out on lactose-based surfactants. In this work, we present the synthesis and the physico-chemical characterization of lactose oleate. The new derivative was obtained by enzymatic mono-esterification of lactose with oleic acid. Thermal, surface, and aggregation properties of the surfactant were studied in detail and the cytotoxicity profile was investigated by MTS and LDH assays on intestinal Caco-2 monolayers. Transepithelial electrical resistance (TEER) measurements on Caco-2 cells showed a transient and reversible effect on the tight junctions opening, which correlates with the increased permeability of 4 kDa fluorescein-labelled dextran (as model for macromolecular drugs) in a concentration dependent manner. Moreover, lactose oleate displayed a satisfactory antimicrobial activity over a range of Gram-positive and Gram-negative bacteria. Overall, the obtained results are promising for a further development of lactose oleate as an intestinal absorption enhancer and/or an alternative biodegradable preservative for pharmaceutical and food applications.

Stereoselective synthesis of 17,18-epoxy derivative of EPA and stereoisomers of isoleukotoxin diol by ring opening of TMS-substituted epoxide with dimsyl sodium.

The reaction of TMS-substituted epoxy alcohols (and derivatives) with dimsyl sodium (NaDMSO) to give 1-alkene-3,4-diols was used for the synthesis of enantiomerically enriched 17(R),18(S)-EpETE and two diastereoisomers of isoleukotoxin diol. In the synthesis of 17(R),18(S)-EpETE, the α-ethoxyethyl ether (EE) of the epoxy alcohol derived from (R)-1-TMS-1-penten-3-ol underwent reaction with NaDMSO to give the mono EE-protected 1-hexene-3,4-diol. The aldehyde obtained by hydroboration/oxidation was subjected to Wittig reaction to afford a mono EE-protected diol. The corresponding mono mesylate was prepared and subjected to epoxide ring formation to afford 17(R),18(S)-EpETE stereoselectively. Similarly, a reaction of the anti epoxy alcohol derived from (R)-1-TMS-1-octen-3-ol with NaDMSO gave the anti form of 1-nonene-3,4-diol, which was converted to 12(S),13(R)-isoleukotoxin diol through Wittig reaction. 12(R),13(R)-Isoleukotoxin diol was synthesized in a similar manner.

One-step Preparation of Carbon-based Solid Acid Catalyst from Water Hyacinth Leaves for Esterification of Oleic Acid and Dehydration of Xylose.

Carbon-based solid acid catalysts were successfully obtained via one-step hydrothermal carbonization (HTC) of water hyacinth (WH) in the presence of p-toluenesulfonic acid (PTSA). Increasing the HTC temperature from 180 to 240 °C resulted in carbonaceous materials with increased sulfur content and less adsorbed water. The material obtained at 220 °C (WH-PTSA-220) contains the highest amount of acid sites and promotes the highest initial rate of two transformations, that is, methanolysis of oleic acid and dehydration of xylose to furfural. While all PSTA-treated WH catalysts gave comparable fatty acid conversions (≈97 %) and furfural yields (≈60 %) after prolonged reaction times, the WH-PTSA-240 system bearing a relatively low acid density maintains the most favorable reusability profile. Higher HTC temperatures (220-240 °C) improved the catalyst reusability profiles due to graphitization and hydrophobicity of the carbon surface. The catalyst systems derived herein from biomass may have potential applications in biorefining platforms, utilizing the conversion of waste biomass to chemicals.

Synthesis and biological evaluation of novel lipoamino acid derivatives.

Seven novel lipoamino acid conjugates were synthesized from methyl oleate and amino acids. Methyl oleate was grafted to different amino acids using thioglycolic acid as a spacer group. Seven derivatives (3a-g) were prepared and characterized by spectral data (NMR, IR and MS spectral studies). All the derivatives were studied for their antimicrobial, anti-biofilm and anticancer activities. Among all the derivatives, it was found that compound 3b was the most potent antibacterial compound which showed good activity against four Gram positive bacterial strains and also exhibited excellent antifungal activity against a fungal strain. In the anti-biofilm assay, compound 3b showed promising activity with IC50 value of 2.8µM against Bacillus subtilis MTCC 121. All the compounds showed anticancer activities with 3c showing promising anticancer activity (IC50=15.3-22.4µM) against the four cell lines tested.

Quantitative Structure-Cytotoxicity Relationship of Oleoylamides.

Eighteen oleoylamides were subjected to quantitative structure-activity relationship analysis based on their cytotoxicity, tumor selectivity and anti-HIV activity, in order to assess their biological activities. Cytotoxicity against four human oral squamous cell carcinoma (OSCC) cell lines and five human oral normal cells (gingival fibroblast, periodontal ligament fibroblast, pulp cell, oral keratinocyte, primary gingival epithelial cells) was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Tumor-selectivity (TS) was evaluated by the ratio of the mean 50% cytotoxic concentration (CC50) against normal human oral cells to that against OSCC cell lines. Potency-selectivity expression (PSE) was determined by the ratio of TS to CC50 against OSCC. Anti-HIV activity was evaluated by the ratio of CC50 to the concentration leading to 50% cytoprotection from HIV infection (EC50). Physicochemical, structural and quantum-chemical parameters were calculated based on the conformations optimized by the LowModeMD method. Among 18 derivatives, compounds 8: with a catechol group) and 18: with a (2-pyridyl)amino group) had the highest TS. On the other hand, doxorubicin and 5-fluorouracil (5-FU) were more highly cytotoxic to normal epithelial cells, displaying unexpectedly lower TS and PSE values. None of the compounds had anti-HIV activity. Among 330 chemical descriptors, 75, 73 and 19 descriptors significantly correlated to the cytotoxicity to normal and tumor cells, and TS, respectively. Multivariate statistics with chemical descriptors for molecular polarization and hydrophobicity may be useful for the evaluation of cytotoxicity and TS of oleoylamides.

Lipase-catalyzed synthesis of ascorbyl oleate in acetone: optimization of reaction conditions and lipase reusability.

Lipase-catalyzed ascorbyl oleate synthesis is eco-friendly and selective way of production of liposoluble biocompatible antioxidants, but still not present on an industrial level due to the high biocatalyst costs. In this study, response surface methodology was applied in order to estimate influence of individual experimental factors, identify interactions among them, and to determine optimum conditions for enzymatic synthesis of ascorbyl oleate in acetone, in terms of limiting substrate conversion, product yield, and yield per mass of consumed enzyme. As a biocatalyst, commercial immobilized preparation of lipase B from Candida antarctica, Novozym 435, was used. In order to develop cost-effective process, at reaction conditions at which maximum amount of product per mass of biocatalyst was produced (60°C, 0.018 % (v/v) of water, 0.135 M of vitamin C, substrates molar ratio 1:8, and 0.2 % (w/v) of lipase), possibilities for further increase of ester yield were investigated. Addition of molecular sieves at 4(th) hour of reaction enabled increase of yield from 16.7 mmol g⁻¹ to 19.3 mmol g⁻¹. Operational stability study revealed that after ten reaction cycles enzyme retained 48 % of its initial activity. Optimized synthesis with well-timed molecular sieves addition and repeated use of lipase provided production of 153 mmol per gram of enzyme. Further improvement of productivity was achieved using procedure for the enzyme reactivation.

Novel acylethanolamide derivatives that modulate body weight through enhancement of hypothalamic pro-opiomelanocortin (POMC) and/or decreased neuropeptide Y (NPY).

Newly synthesized acylethanolamide derivatives oleoyl-L-valinolamide (1), oleoyl-D-valinolamide (2), elaidoyl-L-valinolamide (3), elaidoyl-D-valinolamide (4) stearoyl-L-valinolamide (5), and palmitoyl-L-valinolamide (6) were investigated in mice as antiobesity compounds. Compounds 1, 2, 5, 6 significantly decreased body weight by 6.57% following eight injections of 1 mg/kg i.p. during 39 days, while 3 and 4 showed no such activity. Receptor binding indicated that no compound activated CB1, CB2, PPARα, or TRPV1 receptors. Hypothalamic RT-PCR showed that mRNA expression of the anorexigenic genes POMC and CART was up-regulated by 1, 2, 5 and 1, 2, respectively, while that of the orexigenic genes NPY and CaMKK2 was down-regulated by the respective compounds 1, 5, 6 and 1, 2, 5. Oleoyl-L-valinolamide enhances anorectic pathways and lead to decreased glucose levels, enhanced locomotor activity, and improved cognition. Effects of oleoyl-L-valinolamide on weight were dose-dependent, and it could be given orally. 1, 2, 4, 5 down-regulated FAAH mRNA expression.

Fabrication of microspheres via solvent volatization induced aggregation of self-assembled nanomicellar structures and their use as a pH-dependent drug release system.

A series of oleamide derivatives, (C(18)H(34)NO)(2)(CH(2))(n) [n = 2 (1a), 3 (1b), 4 (1c), or 6 (1d); C(18)H(34)NO = oleic amide fragment] and (C(18)H(34)NO)(CH(2))(6)NH(2) (2), have been synthesized and their self-assembly is investigated in ethanol/water media. Self-assembly of 1a and 1b in ethanol/water (1/0.1 v/v) solution (5 mg mL(-1)) yields microspheres (MSs) with the average diameter ∼10 µm via a gradual temperature reduction and solvent volatilization process. Under the same self-assembly conditions, microrods (average diameter ∼6 µm and several tens of micrometers in length), micronecklace-like, and shape-irregular microparticles are formed from the self-assembly of 1c, 1d, and 2, respectively. The kinetics of evolution for their self-assemblies by dynamic light scattering technique and in situ observation by optical microscopy reveals that the microstructures formation is from a well-behaved aggregation of nanoscale micelles induced by solvent volatilization. The FT-IR and temperature-dependent (1)H-NMR spectra demonstrate the hydrogen bonding force and π-π stacking, which drove the self-assembly of all oleamide derivatives in ethanol/water. Among the fabricated microstructures, the MSs from 1a exhibit the best dispersity, which thus have been used as a scaffold for the in vitro release of doxorubicin. The results demonstrate a pH-sensitive release process, enhanced release specifically at low pH 5.2.

Mitochondria targeting of non-peroxidizable triphenylphosphonium conjugated oleic acid protects mouse embryonic cells against apoptosis: role of cardiolipin remodeling.

Peroxidation of cardiolipin in mitochondria is essential for the execution of apoptosis. We suggested that integration of oleic acid into cardiolipin generates non-oxidizable cardiolipin species hence protects cells against apoptosis. We synthesized mitochondria-targeted triphenylphosphonium oleic acid ester. Using lipidomics analysis we found that pretreatment of mouse embryonic cells with triphenylphosphonium oleic acid ester resulted in decreased contents of polyunsaturated cardiolipins and elevation of its species containing oleic acid residues. This caused suppression of apoptosis induced by actinomycin D. Triacsin C, an inhibitor of acyl-CoA synthase, blocked integration of oleic acid into cardiolipin and restored cell sensitivity to apoptosis.

Synthesis of oleoylethanolamide using lipase.

An effective process for the enzymatic synthesis of oleoylethanolamide is described in this study. The process included purification of a commercial oleic acid product and then optimization of the reaction between the purified oleic acid and ethanolamine in the presence of hexane and a lipase. Under the optimal amidation reaction conditions identified, oleoylethanolamide was obtained with 96.6% purity. The synthesis was also conducted on a large scale (50 mmol of each of the reactants), and oleoylethanolamide purity and yield after crystallization purification were 96.1 and 73.5%, respectively. Compared to the previous studies, the current method of preparing high-purity oleoylethanolamide is more effective and economically feasible. The scalability and ease for such synthesis make it possible to study the biological and nutritional functions of the cannabinoid-like oleoylethanolamide in animal or human subjects.

Enzymatic synthesizing of phytosterol oleic esters.

A method of synthesizing the phytosterol esters from oleic acid and sterols was studied, using immobilized lipase Candida sp. 99-125 as catalyst. Molar ratio (oleic acid/phytosterols), temperature, reaction period, organic solvents, catalyst, and silica-gel drier were optimized, and the result showed that 93.4% of the sterols had been esterified under the optimal synthetic condition: the molar ratio of oleic acid/phytosterol is 1:1 in 10 mL iso-octane, immobilized lipase (w, 140% of the sterols), incubated in an orbital shaker (200 rpm) at a temperature of 45 °C for 24 h. The immobilized lipase could be reused for at least 13 times with limited loss of esterification activity. The conversion still maintained up to 86.6%. Hence, this developed process for synthesizing phytosterol esters could be considered as simple and low-energy consumption compared to existing chemical processes.

Nucleolipid nanovectors as molecular carriers for potential applications in drug delivery.

Novel thymidine- or uridine-based nucleolipids, containing one hydrophilic oligo(ethylene glycol) chain and one or two oleic acid residues (called ToThy, HoThy and DoHu), have been synthesized with the aim to develop bio-compatible nanocarriers for drug delivery and/or produce pro-drugs. Microstructural characterization of their aggregates has been determined in pure water and in pseudo-physiological conditions through DLS and SANS experiments. In all cases stable vesicles, with mean hydrodynamic radii ranging between 120 nm and 250 nm have been revealed. Biological validation of the nucleolipidic nanocarriers was ensured by evaluation of their toxicological profiles, performed by administration of the nanoaggregates to a panel of different cell lines. ToThy exhibited a weak cytotoxicity and, at high concentration, some ability to interfere with cell viability and/or proliferation. In contrast, DoHu and HoThy exhibited no toxicological relevance, behaving similarly to POPC-based liposomes, widely used for systemic drug delivery. Taken together, these results show nucleolipid-based nanocarriers as finely tunable, multi-functional self-assembling materials of interest for the in vivo transport of biomolecules or drugs.