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Novel functional groups can be introduced into vegetable oils using enzymes, resulting in value-added products. The transesterification kinetics of ethyl ferulate with MAG, DAG, and TAG were examined. Transesterification was catalyzed by immobilized Candida antarctica lipase B in solventless batch and packed-bed reactors. Initial reaction rates with TAG were slightly sensitive to water activity, whereas rates with MAG and DAG were water activity independent. Transesterification was also three-to sixfold faster with MAG and DAG. These observations indicate that the reaction is rate limited by the acyl acceptor, and that oils with free hydroxyl groups are preferred acyl acceptors in comparison with TAG, which must undergo partial hydrolysis before becoming reactive.

Cytochrome c immobilized on alkylthiol self-assembled monolayers exhibits a characteristic Fe(II)/Fe(III) redox signal that is lost when exposed to ionic liquids composed of a butylimidazolium cation combined with either hexafluorophosphate or bis(trifluoromethylsulfonyl)imide anion. In this study it was shown that exposure to the aqueous solubilized ionic liquid components, butyl-, hexyl-, and octyl-imidazolium cations and hexafluorophosphate, tetrafluoroborate, and bis(trifluoromethylsulfonyl)imide anions, resulted in partial electrochemical signal loss. Absorbance and fluorescence measurements showed that signal loss due to the cationic ionic liquid component followed a different mechanism than that of the anionic component. Although a portion of the signal was recoverable, irreversible signal loss also occurred in both cases. The source of the irreversible component is suggested to be the loss of protein secondary structure through complexation between the ionic liquid components and the protein surface residues. The reversible electrochemical signal loss is likely due to interfacial interactions imposed between the electrode and the cytochrome heme group. The influence of the amount of exposed surface residues was explored with a simplified model protein, microperoxidase-11.

There is growing interest in recovering ferulic acid from plant sources for use as feedstock for several high-value applications. Jojoba meal was examined as a potential source of ferulic acid. The feruloyl esterase domain of the Clostridium thermocellum cellulosomal xylanase was employed to hydrolyze ferulic acid from defatted jojoba meal. Esterase treatment produced 6.7 g of ferulic acid/kg of jojoba meal. The predominant source (86%) of the ferulate was found to originate from the meal's water-soluble simmondsin fraction. Seven feruloyl simmondsin species from jojoba meal were identified by liquid chromatography–mass spectroscopy. Only one species, a didemethylsimmondsin ferulate, displayed an enzymatic hydrolysis rate distinctly faster than the other feruloyl simmondsins. Complete hydrolysis of all feruloyl simmondsin species was achieved in 24–48 h at 60 °C with a 100:1 meal:enzyme weight ratio. Ferulic acid was efficiently recovered from the medium by ethyl acetate extraction. The recovered ferulic acid was readily converted to ethyl ferulate, demonstrating a facile procedure for producing a valuable product from defatted jojoba meal.

Lesquerella and castor oils were esterified with cinnamic acid (CA) and 4-methoxycinnamic acid (MCA). Esterification of the hydroxy oils reached 85% completion with CA and 50% conversion with MCA. The hydroxy oils were esterified at 200°C under a nitrogen atmosphere within a sealed system. Unreacted CA and MCA were removed from the reaction mixtures by sublimation at 100°C under vacuum. The resultant methoxycinnamic oils possessed a broader, more blue-shifted UV absorbance, 250 to 345 nm with a λmax of 305 nm, compared with the cinnamic oils, which absorbed from 260 to 315 nm, λmax of 270 nm. The methoxycinnamic oils provide better UV-B absorption and thus are better candidates to be used as sunscreen active ingredients. Esterifications of the hydroxy oils with MCA at 200°C resulted in conversion of 40% of the MCA to undesirable by-products. Esterifications with MCA performed at 175°C in the presence of a tin catalyst resulted in similar percent conversions to product without degradation of MCA. Esterifications of lesquerella oil with MCA at 175°C resulted in higher conversions, 43%, than analogous esterifications with castor oil, 29%. The hydroxyl groups of the lesquerella and castor oils provide their excellent emolliency, lubricity, and noncomedogenicity in skin and personal-care products. Therefore, reactions that convert only 50% of the available hydroxyl groups of the lesquerella oil to cinnamoyl-esters are preferred.

The electrochemical behavior of cytochrome c (cyt- c ) adsorbed to gold electrodes through mercaptoalkanoic acid/mercaptoalcohol monolayers was investigated in the 1-butyl-3-methylimidazolium salts of bis(trifluoromethylsulfonyl)imide and hexafluorophosphate. Cyt- c does not retain its Fe(III)/Fe(II) redox activity in dry ionic liquids (ILs); however, the redox signal of the modified electrodes can be reconstituted upon subsequent treatment with aqueous buffer. Water saturated ILs, 1.4 wt.%, failed to support the redox activity of the adsorbed cyt- c ; therefore, the ILs’ deleterious effects on the redox behavior of the cyt- c modified electrodes cannot be attributed solely to the stripping of essential water from the enzyme's polypeptide lattice.

A biocatalytic process for covalent incorporation of ferulic acid onto the glycerol backbone of vegetable oil proceeds efficiently, although rather slowly, with Candida antarctica lipase B in a packed-bed reactor. The bioreactor shows considerable long-term stability. Product yield is influenced by the water content of the fluid phase and enzyme support. The enzyme support modulates substrate concentrations through adsorption and subsequent release of reactants over the course of the reaction. The resulting product has excellent UVA/UVB absorbing properties, making it a potential substitute for conventional petroleum-based sunscreen active agents.

Octadecyl ferulate was prepared using solid acid catalyst, monitored using Supercritical Fluid Chromatography and purified to a 42% yield. Differential scanning calorimetry measurements determined octadecyl ferulate to have melting/solidification phase transitions at 67 and 39°C, respectively. AFM imaging shows that 5-mol% present in a lipid bilayer induced domains to form. Phase behavior measurements confirmed that octadecyl ferulate increased transition temperature of phospholipids. Fluorescence measurements demonstrated that octadecyl ferulate stabilized liposomes against leakage, maintained antioxidant capacity within liposomes, and oriented such that the feruloyl moiety remained in the hydrophilic region of the bilayer. Molecular modeling calculation indicated that antioxidant activity was mostly influenced by interactions within the bilayer.

The ability of Fe(III)protoporphyrin(IX) chloride (hemin), microperoxidase-11 (MP-11), and cytochrome c (cyt-c) to oxidize 2-methoxyphenol (guaiacol) was examined in the room-temperature ionic liquids (IL) 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([bmim][Tf2N]) and the hexafluorophosphates of 1-butyl- and 1-octyl-3-methylimidazolium, ([bmim][PF6] and [omim][PF6]), respectively. All three biocatalysts displayed peroxidase activity when activated by an electron acceptor, tert-butyl hydroperoxide for hemin and hydrogen peroxide for MP-11 and cyt-c. Hemin required the addition of a coordinating base, pyridine or N-methylimidazole (NMI), to produce an active complex. Cyt-c did not require exogenous ligands for activity in IL, although their addition increased peroxidase activity. MP-11 could not be solubilized without an exogenous ligand, therefore, whether MP-11 was active in the absence of such ligands was not determined. Pyridine provided higher activities than NMI for the three catalysts. Hemin and MP-11 peroxidase activities were markedly higher in IL compared to molecular solvents of similar polarity, as characterized by probe solvatochromic behavior, while cyt-c activity was comparable between both types of solvents. There was no consistent preference by the catalysts for a particular IL. These observations indicate that IL are suitable media for bioelectrocatalysis.

The direct electrochemical reduction of hemin, protoporphyrin(IX) iron(III) chloride, ligated with strong or weak heterocyclic bases, was investigated in the ionic liquids (IL), 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF6]) and 1-octyl-3methylimidazolium hexafluorophosphate ([omim][PF6]), using cyclic voltammetry and chronocoulometry. Hemin complexed with N-methylimidazole (NMI) or with pyridine had E1/2 values slightly (4–59 mV) more positive in IL (without electrolyte) than in methanol (1.0 M electrolyte) using a gold electrode. NMI-ligated hemin had a lower E1/2 than pyridine-ligated hemin in either IL, consistent with the stronger electron donor characteristic of NMI. [Bmim][PF6] solutions consistently yielded E1/2 values 30 mV more negative than [omim][PF6] solutions. The diffusion coefficients Do of hemin in the IL ranged between 1.50 and 2.80 ×10 −7 cm 2 s −1 , while the heterogeneous electron-transfer rate constants ks ranged between 3.7 and 14.3 × 10 − 3 cm s − 1 . Cyclic voltammetry of hemin adsorbed to a gold surface through 4,4-bispyridyl disulfide (AT4) linkages showed a large positive shift in the oxidation wave, indicating that adsorption stabilizes the reduced hemin state. The surface concentration o of the adsorbed hemin was determined to be 1.21 ×10 − 10 mol cm − 2 , indicating the presence of one or more complete monolayers of hemin. These findings suggest that while hemin is electrochemically active in IL, its behavior is modified by the ligand field strength and surface adsorption phenomena. Published by Elsevier Science B.V.

The alkyl esters of plant-derived phenols may serve as slow-release sources for cutaneous delivery of antioxidants. The ability of skin esterases to hydrolyze phenolic esters was examined. Esters of tyrosol and hydroxytyrosol were prepared from decanoic and lipoic acids. Ferulic acid was esterified with octadecanol, glycerol, and dioleoylglycerol. These phenolic derivatives were treated in taurodeoxycholate microemulsion and unilamellar liposomes with ex vivo porcine skin and an aqueous extract of the skin. Extracted esterases hydrolyzed the microemulsions at rates in the order: tyrosyl lipoate > tyrosyl decanoate > hydroxytyrosyl lipoate > hydroxytyrosyl decanoate. The tyrosyl decanoate was subject to comparatively little hydrolysis (10–30% after 24 h) when incorporated into liposomes, while hydroxytyrosyl decanoate in liposomes was not hydrolyzed at all by the skin extract. Ferulate esters were not hydrolyzed by the extract in aqueous buffer, microemulsion, nor liposomes. Tyrosyl decanoate applied topically to skin explants in microemulsion were readily hydrolyzed within 4 h, while hydrolysis was minimal when applied in liposomes. These findings indicate that porcine skin displays a general esterase activity toward medium-chain esters of tyrosol and hydroxytyrosol, which can be moderated by the physiochemical properties of the lipid vehicle, but no feruloyl esterase activity.

The acylglycerides of lipoic and dihydrolipoic acids may serve as slow-release sources for cutaneous delivery of these antioxidants when formulated in a liposomal vehicle.Testing was conducted to determine the storage stability of lipoyl glycerides in phospholipid-based liposomes.Lipoyl glycerides prepared by transesterification of lipoic acid with high oleic sunflower oil were incorporated into unilamellar liposomes comprised of soy phosphatidylcholine (soyPC) or dioleoylphosphatidylcholine (DOPC).Lipoyl glycerides were stable in soyPC at 4 °C (90% remaining after five weeks) and decayed with a half-life (t(½)) of 14 d at 40 °C. In contrast, lipoyl glycerides embedded in DOPC were completely stable for four weeks at 40 °C. Dihydrolipoyl glycerides in soyPC converted to lipoyl glycerides at 4 °C (t(½) = 14 d) over four weeks, and much more rapidly so at 40 °C (t(½) = 1 d). A hydroperoxide accumulation analysis indicated that lipoyl glycerides and dihydrolipoyl glycerides were modified or degraded while suppressing autoxidation of the polyunsaturated fatty acids present in soyPC. Dynamic light scattering measurements found that liposomes containing lipoyl glycerides or dihydrolipoyl glycerides did not undergo significant size changes for at least 48 d, indicating that inclusion of the lipoic acid derivatives did not induce vesicle aggregation.Substitution of the soyPC with DOPC, which is not readily subject to autoxidation, provided a much more stable storage environment for lipoyl glycerides. These findings confirm the expectation that phospholipid liposomes need to be oxidatively stable vehicles for dermal delivery of lipoic acid derivatives.

The transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with 1-propanol catalyzed by α-chymotrypsin was examined in the ionic liquids 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF6]) and 1-octyl-3-methylimidazolium hexafluorophosphate ([omim][PF6]), and in combination with supercritical carbon dioxide (SC-CO2). The activity of α-chymotrypsin was studied to determine whether trends in solvent polarity, water activity, and enzyme support properties, observed with this enzyme in conventional organic solvents, hold for the novel environment provided by ionic liquids. α-Chymotrypsin freeze-dried with K2HPO4, KCl, or poly(ethylene glycol) demonstrated no activity in [bmim][PF6] or [omim][PF6] at very low water concentrations, but moderate transesterification rates were observed with the ionic liquids containing 0.25% water (v/v) and higher. However, the physical complexation of the enzyme with poly(ethylene glycol) or KCl did not substantially stimulate activity in the ionic liquids, unlike that observed in hexane or isooctane. Activities were considerably higher in [omim][PF6] than [bmim][PF6]. Added water was not necessary for enzyme activity when ionic liquids were combined with SC-CO2. These results indicate that [bmim][PF6] and [omim][PF6] provide a relatively polar environment, which can be modified with nonpolar SC-CO2 to optimize enzyme activity. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 181–186, 2001.

Novozym® 435-catalyzed transesterification of ethyl 4-hydroxy-3-methoxy cinnamate (ethyl ferulate, EF) with triolein to form the ultraviolet (UV)-absorbing lipids monoferuloylmonooleoyl-glycerol (FMO) and feruloyl-dioleoyl-glycerol (FDO) has been conducted using supercritical CO2 (SC−CO2) batch reactions. The alcoholysis of 0.1 M EF with 0.1 M 1-octanol in SC−CO2 to form octyl ferulate was used as a model reaction to optimize pressure and temperature conditions. Conditions ranging from 45 to 80°C and 10.3 to 34.5 MPa (1,500 to 5,000 psi) were tested with a maximal conversion of 53% of the EF being achieved at 13.8 MPa (2,000 psi) and 80°C after 24 h. These optimized conditions applied to the transesterification of EF with triolein effected a combined FMO and FDO yield of 69%. Triolein exhibits higher solubilities in SC−CO2 at higher pressures; therefore, the transesterification was performed at 80°C over a range of pressures from 13.8 to 34.5 MPa (2,000 to 5,000 psi). Results showed that a maximal yield of 74% of FMO and FDO was reached at 80°C and 24.1 MPa (3,500 psi) after 48 h. Compared to the FMO and FDO synthesis conducted neat or in toluene, the synthesis of the UV-absorbing lipids in SC−CO2 affords higher yields within a shorter amount of time. Therefore, the transesterification of EF with triolein in a SC−CO2 batch reaction is a viable route to UV-absorbing lipids that could be used as active ingredients in sunscreen formulations.

Four commercially available esterases were screened for their ability to esterify ferulic acid (4-hydroxy-3-methoxy cinnamate). Novozym® 435 was found to be the only one of those screened to convert ferulic acid to ethyl and octyl ferulate at 20 and 14% yields, respectively. The highest percentage conversion was obtained using a 1∶1 mole ratio of alcohol to ferulic acid in stirred batch reactions in anhydrous 2-methyl-2-propanol at 60°C using one equivalent (wt/wt based on ferulic acid) of Novozym 435. Increased water content and a higher alcohol/ethyl ferulate ratio had adverse effects on the lipase-catalyzed esterification. The Novozym 435 activity was tested in less polar solvents (anhydrous toluene and hexane) by monitoring the alcoholysis of ethyl ferulate with 1-octanol, which resulted in a 50% yield of octyl ferulate. The alcoholysis was improved to 83% by applying a 16 mm Hg vacuum for 5 min every 24 h to remove the ethanol co-product. The optimal alcoholysis parameters were applied to the alcoholysis of ethyl ferulate with monoolein and the transesterification with triolein. The transesterification of ethyl ferulate with triolein in anhydrous toluene produced a combined 44% yield of ferulyl monoolein and ferulyl diolein, a 20% greater yield than that obtained for alcoholysis using monoolein. The highest yield, 77%, of ferulyl monoolein and ferulyl diolein was achieved using a threefold excess of neat triolein. The lipase-catalyzed transesterification of ethyl ferulate with triolein appears to be a technically feasible route to ferulyl-substituted acylglycerols, which are potentially useful sunscreen ingredients.

Described are two new ferrocenes, each containing two different straps linking the pair of cyclopentadienyl rings. The first, Fe(C5H3)2-1,1‘-S3-3,3‘-C2Me4, was characterized by single-crystal X-ray...

Polyferrocenylene persulfides, [(BuC{sub 5}H{sub 3})(C{sub 5}H{sub 4})FeS{sub 2}]n, have been prepared by desulfurization of butyltrithiaferrocenophane (BuC{sub 5}H{sub 3})(C{sub 5}H{sub 4})FeS{sub 3}. The molecular precursor was prepared by dilithiation of butylferrocene followed by treatment with elemental sulfur and isolated as a 7:1 mixture of the 1,3,1{prime} and 1,2,1{prime} isomers. The trisulfide was converted to polymer by treatment with the desulfurization agent PBu{sub 3}. The rate of this desulfurization and the nature of the polymer depends critically on the reaction solvent. Desulfurization in DMF and THF afforded insoluble but swellable rubbers. Desulfurization of BufcS{sub 3} in mixed CH{sub 2}Cl{sub 2}-THF solutions gave soluble polymers with M{sub n}, ranging from 12000 to 359000, with lower molecular weight products being favored at high [CH{sub 2}Cl{sub 2}]. The effect of high [CH{sub 2}- Cl{sub 2}] was modeled by the reaction of [fcS{sub 2}]n with a CH{sub 2}Cl{sub 2} solution of PBu{sub 3} to give fcS{sub 2}CH{sub 2}. Molecular weights for [BufcS{sub 2}]n were confirmed by light scattering measurements. Soluble [BufcS{sub 2}]n undergoes reversible electrochemical oxidation in two steps, centered at 780 V (vs Ag/AgCl) and separated by {approximately}300 mV. The first oxidation potential is similar to that for the trisulfide monomer and is thought tomore » correspond to oxidation of alternating Fe centers, followed by conversion of the remaining Fe{sup II} sites. The polymers photodegrade upon exposure to UV light in air. Deselenization of BufcSe{sub 3} gave red soluble [BufcSe{sub 2}]n with only moderate molecular weights and with high polydispersities. The microstructure of this polymer was established by {sup 77}Se NMR studies which support the presence of head-to-head, head-to-tail, and tail-to-tail dyads. 21 refs., 9 figs., 2 tabs.« less

Antioxidants have critical roles in maintaining cellular homeostasis and disease-state prevention. The multi-functional agent α-lipoic acid offers numerous beneficial effects to oxidatively stressed tissues. α-Lipoic acid was enzymatically incorporated into a triglyceride in conjunction with oleic acid, creating lipoyl dioleoylglycerol, and chemically reduced to form dihydrolipoyl dioleoylglycerol. The triglyceride forms of lipoic acid stabilized dioleoylphosphatidylcholine unilamellar liposomal vesicles, as judged by calcein–cobalt leakage. Stabilization resulted from increased packing density of phospholipid acyl chains. Scavenging activity against the 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) radical was monitored by oxidation of 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-Bodipy). Dihydrolipoyl dioleoylglycerol in vesicles demonstrated strong antioxidant capacity in comparison to the conventional Trolox standard. Fluorescence quenching measurements indicated the lipoyl moiety of dihydrolipoyl dioleoylglycerol is positioned near the vesicle aqueous/lipid boundary. Treatment of intact vesicles with a nonpenetrating sulfhydryl reagent indicated that 80% of the dihydrolipoyl dioleoylglycerol was available for reaction. Molecular modeling of lipoyl dioleoylglycerol and dihydrolipoyl dioleoylglycerol in a phospholipid layer confirmed the existence of an extended configuration for the molecules that accounts for the interfacial location of the lipoyl moiety, which may allow the antioxidant to readily react with radical species approaching membranes from the aqueous phase.

Soyscreen oil was studied as a formulation ingredient to protect Beauveria bassiana (Balsamo) Vuillemin conidia from UV degradation. Feruloylated soy glycerides, referred to as Soyscreen oil, are biobased UV-absorbing molecules made by combining molecules of soybean oil with ferulic acid. Conidia stored in Soyscreen oil for 28 wk at 25, 30, and 35 degrees C retained viability as well as conidia stored in sunflower oil, demonstrating that Soyscreen did not adversely affect viability with prolonged storage. For samples applied to glass and exposed to simulated sunlight (xenon light), conidia in sunflower oil with or without sunscreens (Soyscreen or oxyl methoxycinnimate) had similar conidia viability after exposure. These oil formulations retained conidia viability better than conidia applied as an aqueous treatment. However, the 10% Soyscreen oil formulation applied to field grown cabbage (Brassica oleracea L.) and bean (Phaseolus vulgaris L.) plants, did not improve residual insecticidal activity compared with aqueous applications of unformulated conidia or two commercial formulations when assayed against Trichoplusia ni (Hübner) larvae. Our results suggest that the oil applications lose UV protection because the oil was absorbed by the leaf. This conclusion was supported in subsequent laboratory exposures of conidia in oil-based formulations with UV screens applied to cabbage leaves or balsa wood, which lost protection as measured by decreased viability of conidia when exposed to simulated sunlight. As a result, additional formulation techniques such as encapsulation to prevent separation of the protective oil from the conidia may be required to extend protection when oil formulations are applied in the field.

Redox inactivation, but not removal, of electrostatically adsorbed cytochrome c (cyt-c) on an alkanethiol modified gold surface was observed after exposure of the electrode to 1.0 M aqueous NaCl, NaClO4, KCl, or KClO4 solutions.

As environmentally conscious consumers push for environmentally friendly pest control, interest in controlling insects with microbial-based biopesticides increases. Specific bacteria, fungi, and viruses can provide effective pest control when sprayed onto plants. However, these microbes are rapidly degraded by sunlight, which limits their field efficacy. One strategy to combat this degradation is to develop formulations to protect the beneficial microbe from sunlight energy, specifically the ultraviolet (UV) wavelengths. Formulation technologies (ingredients and processes) continue to be developed and adapted to provide UV protection. We have been conducting research on soyscreen and lignin as formulation ingredients to provide UV protection to a variety of insect pathogens. Soyscreen consists of feruloylated soy glycerides (FSG) specifically developed to absorb UV energy. Previous research demonstrated that FSG in oil-based formulations protected the viability of hydrophobic fungal conidia of Beauveria bassiana exposed to simulated sunlight, but not when applied in the field to oil absorbing substrates, including cabbage and tree bark. To prevent the FSG from being absorbed, the oil was encapsulated in starch using a jet cooking process forming a product with improved UV absorption. These starch-FSG composites may be useful to formulate aqueous-based systems without synthetic surfactants to deliver and protect microbial biopesticides. Lignin has been used with a spray-drying process to encapsulate insecticidal viruses, which are extremely sensitive to degradation by sunlight. The insoluble particles remain intact during application and can extend insecticidal activity by several days. Techniques are currently being developed to produce an adjuvant formulation with water-soluble lignin in an effort to provide UV protection for a broader range of commercial pesticides. These technologies are considered to be environmentally benign and are intended to fit in the biopesticide concept. The versatility of combining various products and processes provides variable formulations, some of which are suitable for specific biopesticide applications.