Acyl migration and kinetics study of 1(3)-positional specific lipase of Rhizopus oryzae-catalyzed methanolysis of triglyceride for biodiesel production

The lipase of Rhizopus oryzae (R. oryzae) was reported to have 1(3)-positional specificity, but in the process of R. oryzae-catalyzed biodiesel production, the yield of biodiesel (methyl esters) could reach over 80%. Although during 1(3)-positional specific lipase-catalyzed methanolysis of triglycerides, acyl migration was thought as one of the major reasons for higher methyl ester yield, there was no further study on the mechanism exploration regarding to acyl migration. In this paper, acyl migration and the related kinetics of R. oryzae-mediated methanolysis of triolein was studied systematically. Through our study, it was revealed that during the methanolysis process, acyl migration between 2-MG and 1-MG as well as acyl migration between 1,2-DG and 1,3-DG could take place independent of enzymatic catalysis. The kinetic study showed that the acyl migration was first-order reversible reaction. Based on this finding, a two-step mechanic model including acyl migration was developed for the enzyme-mediated methanolysis for biodiesel production and it was found that the reaction included consecutive hydrolysis and esterification. Further investigation on kinetics showed that R. oryzae lipase was not restrict selectivity of 1(3)-position acyl group, but a preference of 1(3)-position over 2-position, which also contributed to the higher yield of methyl esters.     

Study on acyl migration in immobilized lipozyme TL-catalyzed transesterification of soybean oil for biodiesel production

During enzymatic transesterification of soybean oils with methanol for biodiesel production, it was supposed that the maximum biodiesel yield was only 66% since lipozyme TL was a typical lipase with a strict 1,3-positional specificity. However, it has been observed that over 90% biodiesel yield could be obtained. It was therefore assumed, and subsequently demonstrated, that acyl migration occurred during the reaction process. Different factors which may influence the acyl migration were explored further and it has been found that the silica gel acting as the immobilized material contributes significantly to the promotion of acyl migration in the transesterification process. The final biodiesel yield was only 66% when 4% lipozyme TL used, while about 90% biodiesel yield could be achieved when combining 6% silica gel with 4% lipozyme TL, almost as high as that of 10% immobilized lipase used for the reaction.     

Hydrogenation,isomerization and migration of unsaturated acyl moieties in natural triacylglycerols

Triacylglycerols of soybean were partially hydrogenated with a copper chromite catalyst, which reduced the octadecadienoyl and octadecatrienoyl moieties selectively to octadecenoyl moieties. Composition of the acyl moieties, including the distribution of positional isomers of cis- and trans-octadecenoyl moieties, both in 1,3- and 2-positions of triacylglycerols, was determined after hydrolysis with pancreatic lipase. The results show that the octadecadienoyl, but not the octadecatrienoyl moieties are reduced at a faster rate in the 1,3-positions than in the 2-position, whereas the accumulation of trans-octadecenoyl moieties is much higher in the 2-position than in the 1,3-positions. On the other hand, the distribution of positional isomers, both in cis- and trans-octadecenoyl moieties, is essentially the same in 1,3- and 2-positions. Practically no acyl migration occurs during hydrogenation under the conditions described.     

Solvent-free enzymatic synthesis of 1,3-diconjugated linoleoyl glycerol optimized by response surface methodology

An operation mode with N(2) bubbling under vacuum was employed for the solvent-free synthesis of 1,3-diconjugated linoleoyl glycerol (1,3-dCLG) from conjugated linoleic acid (CLA) catalyzed by Novozym 435. The response surface methodology (RSM) was adopted for the optimization of the reaction conditions with five major factors (incubation time, temperature, enzyme load, substrate mole ratio, and system vacuum) and three responses (CLA conversion, 1,3-dCLG yield, and acyl migration). Two sets of optimal conditions were recommended. Validation of the RSM model was verified by the good agreement between the experimental and the predicted values of 1,3-dCLG yield. Under the optimal conditions, the yield of 1,3-dCLG up to 93% was obtained. The reaction was scaled up to a production level of 100 g of 1,3-dCLG at a yield of 90.7%, indicating a promising feature of the technology in industrial applications.     

Acyl Group Migrations in 2-Monoolein

Acyl migration in 2-monoolein dissolved in solvents under conditions common in lipid modification reactions has been studied. The effects on acyl migration of solvent, incubation temperature, water activity, polar additives and solid additives have been investigated. Extensive acyl migration occured in aliphatic hydrocarbons and water-miscible alcohols under dry conditions. The acyl migration rate could be decreased in several nonpolar solvents by adding a small amount of water or an alcohol. Increasing water activity had no effect in isooctane, but decreased the acyl migration rate dramatically in methyl tert-butyl ether and methyl isobutyl ketone. Several commonly used enzyme supports catalysed acyl migration, showing that supports with surface charges could catalyse acyl migration.     

Lipase-catalyzed synthesis of monoacylglycerol in a homogeneous system

The 1,3-regiospecifique lipase, Lipozyme IM, catalyzed the esterification of lauric acid and glycerol in a homogeneous system. To overcome the drawback of the insolubility of glycerol in hexane, which is extensively used in enzymatic synthesis, a mixture of n-hexane/tert-butanol (1:1, v/v) was used leading to a monophasic system. The conversion of lauric acid into monolaurin was 65% in 8 h, when a molar ratio of glycerol to fatty acid (5:1) was used with the fatty acid at 0.1 M, and the phenomenon of acyl migration was minimized.     

Biotransformations of organosilicon compounds: enantioselective reduction of acyl silanes by means of baker's yeast

The enantioselective reduction of acyl silanes has been performed employing baker's yeast (BY) in fermenting conditions: a series of substrates of different structure was investigated, showing that the reactivity as well as the level of enentioselectivity depends on the steric bulk of the substituents on the acyl silane. The products α-hydroxy silanes were obtained with chemical and optical yields over 90% in the most favourable cases.     

Kinetic study of the lipase-catalyzed synthesis of triolein

The kinetics of the synthesis of triolein catalyzed by immobilized Mucor miehei lipase were studied. Equilibrium constants for the synthesis of mono-, di-, and triolein were calculated from the equilibrium compositions for different initial ratios of glycerol and oleic acid by means of multiresponse regression. The 1,3-specific lipase can catalyze the synthesis of triolein because the ester enzymatically formed with the primary alcohol isomerizes, through acyl migration, to an ester on the secondary hydroxyl. The freed primary hydroxyl may then undergo further enzymatic conversion. The rates of isomerization depend on the concentration of oleic acid. (c) 1993 Wiley & Sons, Inc.     

Efficient kinetic resolution of dl-menthol by lipase-catalyzed enantioselective esterification with acid anhydride in fed-batch reactor

Acid anhydrides were used as highly reactive and non-water-producing acyl donors for hydrolase-catalyzed enantioselective esterification. Efficient kinetic resolution of dl-menthol has been achieved via lipase-catalyzed enantioselective esterification in cyclohexane when propionic anhydride as an acyl donor was continuously fed into a reactor containing dl-menthol and Candida cylindracea lipase OF 360, while a high concentration of the acid anhydride in a batch reaction system with a dehydrated organic solvent did not facilitate the reaction, because water necessary for the enzyme function was consumed by the competing hydrolysis of the anhydride catalyzed by the same enzyme. The efficiency of this fed-batch reaction system using acid anhydride was higher and the enzyme stability in repeated use was much better than those of conventional batch and fed-batch reaction systems using propionic acid as an acyl donor. The optical purity (more than 98% e.e.) of the l-menthyl ester produced in the fed-batch system using the anhydride was comparable to that in the system using the corresponding acid. *** DIRECT SUPPORT *** AG903062 00002     

Acyl migration during deacylation of phospholipids rich in docosahexaenoic acid (DHA): an enzymatic approach for evidence and study

Non-enzymatic acyl migration could be counter-productive for the preparation of structured phospholipids with docosahexaenoic acid (DHA) at a designated position. Therefore enzymatic approaches have been developed to investigate acyl migration. First, acyl migration from sn-2 to sn-1 position has been set into relief by a three step enzymatic method using a typo-selective lipase, a phospholipase A2 and a non-selective lipase. The effect of reaction temperature on acyl migration from sn-2 to sn-1 was monitored: lowering the reaction temperature from 40 to 30°C allowed a reduction of DHA migration rate of 40%. Secondly, acyl migration from sn-1 to sn-2 position was negligible. This last result was obtained through the study of structured phosphatidylcholine selective deacylation using a phospholipase A2.     

Acyl migration in 1,2-dipalmitoyl-sn-glycerol

The acyl migration of 1,2-dipalmitoyl-sn-glycerol (1,2-DPG) to 1,3-dipalmitoylglycerol (1,3-DPG) in different states, neat, in the presence of egg yolk lecithin (sonicated and unsonicated) and on silica gel was studied. The isomerization was quantitated by scanning densitometry of charred TLC plates, at different temperatures and for varying periods of time. At equilibrium the amount of 1,3-DPG was found to be 56%. The rates of initial isomerization, and the time required to isomerize to half the equilibrium quantity (i.e., t1/2 eq. = 1,3-DPG 28%) under the above conditions was estimated. In the case of neat melt at 74 degrees C and in an organic solvent the time required to t1/2 eq. is 18 h and a few days, respectively. However, at 62 degrees C in the presence of a polar solvent (sodium phosphate buffer at pH 7.0) the t1/2 eq. is 1-2 h. On dry silica gel (TLC plate) at 24 degrees C the t1/2 eq. is reached in less than 1 h.     

Enzymatic synthesis of lysophosphatidic acid and phosphatidic acid

Immobilised 1,3-specific lipase from Rhizopus arrhizus was used as catalyst for the esterification of -glycero-3-phosphate and fatty acid or fatty acid vinyl ester in a solvent-free system. With lauric acid vinyl ester as acyl donor, a_w<0.53 favored the synthesis of lysophosphatidic acid (1-acyl-rac-glycero-3-phosphate, LPA1) and the spontaneous acyl migration of the fatty acid on the molecule. Subsequent acylation by the enzyme resulted in high phosphatidic acid (1,2-diacyl-rac-glycero-3-phosphate, PA) formation and high total conversions (>95%). With oleic acid, maximum conversions of 55% were obtained at low water activities. Temperatures below melting point of the product favored precipitation and resulted in high final conversion and high product ratio [LPA/(PA+LPA)]. Thus, LPA was the only product with lauric acid vinyl ester as acyl donor at 25°C. Increased substrate ratio ( -glycero-3-phosphate/fatty acid) from 0.05 to 1 resulted in a higher ratio of LPA to PA formed, but a lower total conversion of -glycero-3-phosphate. Increased amounts of enzyme preparation did not result in higher esterification rates, probably due to high mass-transfer limitations.     

O-N intramolecular acyl migration reaction in the development of prodrugs and the synthesis of difficult sequence-containing bioactive peptides

N-Ointramolecular acyl migration in Ser- or Thr-containing peptides is a well-known side reaction in peptide chemistry. It results in the mutual conversion of ester and amide bonds. Our medicinal chemistry study focused on the fact that the O-acyl product can be readily converted to the original N-acyl form under neutral or slightly basic conditions in an aqueous buffer and the liberated ionized amino group enhances the water solubility of O-acyl products. Because of this, we have developed a novel class of "O-N intramolecular acyl migration"-type water-soluble prodrugs of HIV-1 protease inhibitors. These prodrugs released the parent drugs via a simple chemical mechanism with no side reaction. In this study, we applied this strategy to important cancer chemotherapeutic agents, paclitaxel and its derivatives, to develop water-soluble taxoid prodrugs, and found that these prodrugs, 2'-O-isoform of taxoids, showed promising results with higher water solubility and proper kinetics in their parent drug formation by a simple pH-dependent chemical mechanism with O-N intramolecular acyl migration. These results suggest that this strategy would be useful in toxicology and medical economics. After the successful application of O-N intramolecular acyl migration in medicinal chemistry, this concept was recently used in peptide chemistry for the synthesis of "difficult sequence-containing peptides." The strategy was based on hydrophilic O-acyl isopeptide synthesis followed by the O-N intramolecular acyl migration reaction, leading to the desired peptide. In a model study with small, difficult sequence-containing peptides, synthesized "O-acyl isopeptides" not only improved the solubility in various media and efficiently performed the high performance liquid chromatography purification, but also altered the nature of the difficult sequence during SPPS, resulting in the efficient synthesis of O-acyl isopeptides with no complications. The subsequent O-N intramolecular acyl migration of purified O-acyl isopeptides afforded the desired peptides as precipitates with high yield and purity. Further study of the synthesis of a larger difficult sequence-containing peptide, Alzheimer's disease-related peptide (A beta 1-42), surprisingly showed that only one insertion of the O-acyl group drastically improved the unfavorable nature of the difficult sequence in A beta 1-42, and achieved efficient synthesis of 26-O-acyl isoA beta 1-42 and subsequent complete conversion to A beta 1-42 via the O-N intramolecular acyl migration reaction of 26-O-acyl isoA beta 1-42. This suggests that our new method based on O-N intramolecular acyl migration is an important method for the synthesis of difficult sequence-containing bioactive peptides.     

Lipase-catalyzed diacylation of 1,3-butanediol

Summary A kinetic resolution of 1,3-butanediol was accomplished by lipase-catalyzed enantioselective diacylations in organic solvent. Diacylation of 1,3-butanediol was carried out using immobilized lipase SP382 (from Candida sp.) to produce (R) -1,3-diacetoxybutane with 85.8% e.e.. And then, this optically active product was chemically hydrolyzed to diol, and re-acylated with lipase SP382 to (R) -1,3-diacetoxybutane with over 98% e.e..     

Enzymes in stereoselective pharmacokinetics of endogenous substances.

The use of enzymes to assay individual components of the L-carnitine family in pharmaceuticals, foodstuffs, and biological fluids with various forms of detection is reviewed. The most useful enzyme in the assay of compounds of the L-carnitine family is carnitine acetyl transferase (CAT), which catalyses the reversible interconversion of L-carnitine and its short-chain acyl esters. CAT can be used in one or more coupled reactions combined with U.V., or radiolabelled detection, or combined with HPLC, allowing, enantioselective, structurally specific, and, in the case of radiolabelled tracing, highly sensitive assays to be carried out. When compared with chromatographic separation of enantiomers or diastereoisomers, enantioselective enzyme mediated assays may be cheaper, more sensitive, and simpler, but they do not allow the nonpreferred isomer to be assayed. Consequently, they are appropriate for the specific assay of endogenous enantiomeric substrates of the enzyme concerned, in biological samples. The analysis of the other enantiomer in raw materials or in pharmaceuticals must be more properly approached by enantioselective chromatographic methods.     

Pseudomonas cepacia lipase-catalysed resolution of racemic alcohols in ionic liquid using succinic anhydride: role of triethylamine in enhancement of catalytic activity

Racemic secondary alcohols were resolved via enantioselective acylation using succinic anhydride as acyl donor catalysed by lipase from Pseudomonas cepacia supported on celite (PS-C) in ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate [bmim]PF₆. Organic base, namely triethylamine as an additive in ionic liquid has been found to enhance the rate of the reaction.     

In vitro stereoselective degradation of carprofen glucuronide by human serum albumin. Characterization of sites and reactive amino acids

Acyl glucuronides formed from carboxylic acids can undergo hydrolysis, acyl migration, and covalent binding to proteins. In buffers at physiological pH, the degradation of acylglucuronide of a chiral NSAID, carprofen, consisted mainly of acyl migration. Acidic pH reduced hydrolysis and acyl migration, thus stabilizing the carprofen acyl glucuronides. Addition of human serum albumin (HSA) led to an increased hydrolysis of the conjugates of both enantiomers. This protein protected R-carprofen glucuronide from migration and therefore improved its overall stability. Hydrolysis was stereoselective in favor of the S conjugate. The protein domains and the amino acid residues likely to be responsible for the hydrolytic activity of HSA were deduced from the results of various investigations: competition with probes specific of binding sites, effects of pH and of chemical modifications of albumin. Dansylsarcosine (DS), a specific ligand of site II of HSA, impaired the hydrolysis, whereas dansylamide (DNSA) and digoxin, which are specific ligands of sites I and III, respectively, had no effect. The extent of hydrolysis by HSA strongly increased with pH, indicating the participation of basic amino acids in this process. The results obtained with chemically modified HSA suggest the major involvement of Tyr and Lys residues in the hydrolysis of glucuronide of S-carprofen, and of other Lys residues for that of its diastereoisomer.     

Wax esters synthesis from heavy fraction of sheep milk fat and cetyl alcohol by immobilised lipases

Wax esters were obtained from lipase-catalysed alcoholysis of triglycerides with cetyl alcohol, using n-hexane as solvent. The heavy triglyceride fraction (HTF), obtained by fractionation of sheep milk fat, was used as raw material. In the natural fat mixture GC analysis showed that palmitic, myristic, stearic and oleic acids are the most abundant fatty acids which are useful to produce wax esters. Reactions were tested for different amounts of Lipozyme RMIM catalyst, and the optimum concentration of 10 mg catalyst/ml solution has been determined. The formation of the four main products, i.e. cetyl myristate, cetyl palmitate, cetyl oleate and cetyl stearate, was determined by HPLC/ELSD quantitative analysis. The optimum water activity in the reaction medium a_w=0.35 in the case of Lipozyme RMIM, and a_w=0.53 for Novozym 435 was found. Lipozyme RMIM (immobilised sn-1,3-specific lipase from Rhizomucor miehei) was more active than Novozym 435 (immobilised nonspecific lipase-B from Candida antarctica) towards wax esters production. The acyl migration of 2-monoglycerides was suggested as a crucial step to explain the higher yields produced by the 1,3-specific lipase.     

Water-soluble prodrugs of dipeptide HIV protease inhibitors based on O-->N intramolecular acyl migration: Design, synthesis and kinetic study

To improve the low water-solubility of HIV protease inhibitors, we synthesized water-soluble prodrugs of KNI-727, a potent small-sized dipeptide-type HIV-1 protease inhibitor consisting of an Apns-Dmt core (Apns; allophenylnorstatine, Dmt; (R)-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid) as inhibitory machinery. These prodrugs contained an O-acyl peptidomimetic structure with an ionized amino group leading to an increase in water-solubility, and were designed to regenerate the corresponding parent drugs based on the O-->N intramolecular acyl migration reaction via a five-membered ring intermediate at the alpha-hydroxy-beta-amino acid residue, that is Apns. The synthetic prodrug 3a improved the water-solubility (13 mg/mL) more than 8000-fold in comparison with the parent compound, which is the practically acceptable value as water-soluble drug. Furthermore, to understand the structural effects of the O-acyl moiety on the migration rate, we evaluated several phenylacetyl-type and benzoyl-type prodrugs. These prodrugs were stable as an HCl salt and in a strongly acidic solution corresponding to gastric juice (pH 2.0), and could be converted to the parent compounds promptly under aqueous conditions from slightly acidic to basic pH at 37 degrees C.     

Improving lipase-catalyzed enantioselective ammonolysis of phenylglycine methyl ester in organic solvent by in situ racemization

Lipase-catalyzed enantioselective ammonolysis of phenylglycine methyl ester was processed by in situ racemization with ammonium carbamate as the acyl acceptor. Using 1 mM benzaldehyde or 0.6 mM chloropyridoxal as the racemizing catalyst, 80% substrate conversion with an enantiomeric excess of the product of 95% were achieved at 20 °C after 7 h reaction.