Enzymatic Transacylation Research Articles

Light‐Controlled Radical Polymerization of Functional Methacrylates Prepared by Enzymatic Transacylation

Well‐defined homo and block copolymers are prepared with methyl methacrylate, pentyl methacrylate, 3‐phenylpropyl methacrylate, and 6‐thiobenzoylhexyl methacrylate using Ir(ppy)3 as a catalyst. It is shown that the advantages of the light‐controlled radical polymerization can be exploited also for these special monomers as prepared by enzymatic transacylation without time‐consuming labor‐intensive removal of side products. Furthermore, it is shown that the polymerization is significantly affected even by small variations in the irradiation and therefore also by choice of the light source. In contrast to our recent report we therefore recommend removal of the heterogeneous supported enzyme by filtration before polymerization. Because of the sensitivity of the polymerization process to the irradiation, turbidity caused by the bead‐supported enzyme can affect the reproducibility of the polymer preparation. Results obtained with methyl methacrylate as monomer could only be transferred to the above‐mentioned functional monomers by proper choice of the light source. On the basis of kinetic investigations, the effect of the irradiation source, the catalyst concentration, and the alcohol residue on the methacrylate used are discussed. Finally, the optimized experimental conditions are applied for the preparation of different block copolymers. image

Combining Enzymatic Monomer Transformation with Photoinduced Electron Transfer - Reversible Addition-Fragmentation Chain Transfer for the Synthesis of Complex Multiblock Copolymers.

A novel and facile method, involving enzymatic monomer synthesis and a photocontrolled polymerization technique, has been successfully employed for the preparation of high-order multiblock copolymers. New acrylate monomers were synthesized via enzymatic transacylation between an activated monomer, i.e., 2,2,2-trifluoroethyl acrylate (TFEA), and various functional alcohols. These synthesized monomers were successfully polymerized without further purification via photoinduced electron transfer-reversible addition-fragmentation chain transfer (PET-RAFT) polymerization under low energy blue LED light (4.8 W) in the presence of an iridium-based photoredox catalyst (fac-[Ir(ppy)3]). In this condition, PET-RAFT allows us to achieve high monomer conversion (∼100%) with excellent integrity of the end group (>80%). Different multiblock (co)polymers, including poly(hexyl acrylate) pentablock homopolymer, poly(methyl acylate-b-ethyl acrylate-b-n-propyl acrylate-b-n-butyl acrylate-b-n-pentyl acrylate) pentablock copolymer, and poly(3-oxobutyl acrylate-b-methyl acrylate-b-3-(trimethylsilyl)prop-2-yn-1-yl acrylate) triblock copolymer containing functional groups were rapidly prepared via sequential addition of monomers without purification steps.

One‐Pot Synthesis of Multifunctional Polymers by Light‐Controlled Radical Polymerization and Enzymatic Catalysis with Candida antarctica Lipase B

The preparation of multifunctional polymers and block copolymers by a straightforward one-pot reaction process that combines enzymatic transacylation with light-controlled polymerization is described. Functional methacrylate monomers are synthesized by enzymatic transacylation and used in situ for light-controlled polymerization, leading to multifunctional methacrylate-based polymers with well-defined microstructure.

Synthesis of thiol functionalized poly(meth)acrylates through enzymatic catalysis and a subsequent one pot reaction process

The synthesis and modification of thiol functionalized poly(meth)acrylates using a straightforward reaction concept that consists of an enzymatically catalyzed monomer synthesis, free radical polymerization and post-polymerization modification is presented. The well-known enzymatic transacylation of methyl acrylate and methyl methacrylate that runs under mild and environmentally friendly conditions was used to synthesize thiol protected acrylic and methacrylic monomers. Upon free radical polymerization and subsequent removal of protection groups, polymers with pendant thiol groups are obtained, which, in turn, can react in situ with Michael acceptors to form thiol-ene reaction products. The exceptional advantage of the proposed method is that upon removal of the enzyme from the monomer mixture, the polymerization, deprotection of thiols and subsequent Thio-Michael-type addition reaction can be conducted in one pot without purification of the intermediate products. Furthermore, the different reactivities of acetyl and benzoyl protection groups in lipase catalyzed reactions are discussed.

Functional and reactive polymethacrylates suitable for preparation of peptide/protein–polymer conjugates

Three reactive polymers, poly(para-nitrophenoxycarbonyloxyethyl methacrylate- co-methyl methacrylate) P( p-NPCEMA- co-MMA) [ rCoP1], poly(methyl methacrylate- co-butyl methacrylate- co-allyl methacrylate- co-phenoxycarbonyloxyethyl methacrylate) P(MMA- co-BMA- co-AMA- co-PCEMA) [ rCoP2] and poly(methyl methacrylate- co-dimethylaminopropyl methacrylate- co-dodecyl methacrylate- co-phenoxycarbonyloxyethyl methacrylate) P(MMA- co-DMAPMA- co-DMA- co-PCEMA) [ rCoP3], respectively, were prepared following two synthetic concepts. Following the first concept, p-NPCEMA and PCEMA were prepared starting with commercially available HEMA and were copolymerized via ATRP with MMA, BMA and AMA. According to the second concept phenoxycarbonyloxy decorated polymethacrylates were obtained via polymer analogous reaction of a HEMA containing functional polymethacrylate, P(MMA- co-DMAPMA- co-DMA- co-HEMA) [ fCoP], obtained via a cascade reaction of enzymatic transacylation and free radical polymerization. The highly reactive poly(methacrylate)s and silk peptides, hen egg-white lysozyme and Candida antarctica lipase B were used for the preparation of protein/peptide-poly(methacrylate) bioconjugates.

Enzymatic Preparation of Structured Oils Containing Short-Chain Fatty Acids

Structured oils prepared by enzymatic transacylation with triacylglycerols (TAGs) and various fatty acids (FAs) were characterized. Transacylation with trilaurin and saturated FAs (C4:0-C16:0) was performed using Lipozyme RM-IM under standard reaction conditions. The structured oils thus produced had transacylation ratios of 25-37%, as medium-chain FAs > long-chain FAs > short-chain FAs. This result confirmed that short-chain FAs have little reactivity in enzymatic transacylation. All prepared oils shared the same composition of TAG molecular species, as demonstrated by HPLC analysis, and contained a mixture of mono-substituted, di-substituted, and non-substituted TAGs. The reaction conditions for transacylation with TAGs and short-chain FAs were optimized to improve transacylation ratios. The introduction ratios of C4:0, C5:0, and C6:0 into trilaurin were increased to 52.4, 42.5, and 34.1%, respectively, by extending the reaction time. Transacylation between TAGs and short-chain FAs was further examined by using Lipase PL. C4:0 was introduced at 51.1%, the same ratio as for Lipozyme RM-IM. When C5:0 and C6:0 were used as the FA substrate, the transacylation ratios obtained were 47.7 and 43.4%, respectively, higher than those for Lipase RM-IM. Lipase PL is therefore useful for introducing short-chain FAs into TAGs.

Thermoresponsive polyacrylates obtained via a cascade of enzymatic transacylation and FRP or NMP

Following a cascade reaction comprising enzymatic monomer synthesis and radical polymerization, different functional poly(acrylate)s were obtained. Transacylation of methyl acrylate (MA) with diethylene glycol monoethyl ether, 1,2-propanediol or 1,2,6-hexanetriol in the presence of Novozyme 435 led to functional acrylate monomers. In a subsequent step, after filtration of the enzyme, removal of MA by distillation and eventual addition of a hydrophilic co-monomer, (2-hydroxyethyl acrylate, HEA), the mixture was copolymerized via free radical and nitroxide mediated polymerization resulting in poly(acrylate)s with predefined functionalities. Furthermore the thermoresponsive properties of the resulting copolymers are discussed with respect to the polymer composition and the molecular weight distribution.

Increased activity of enzymatic transacylation of acrylates through rational design of lipases

A rational design approach was used to create the mutant Candida antarctica lipase B (CALB, also known as Pseudozyma antarctica lipase B) V190A having a k cat three times higher compared to that of the wild type (wt) enzyme for the transacylation of the industrially important compound methyl methacrylate. The enzymatic contribution to the transacylation of various acrylates and corresponding saturated esters was evaluated by comparing the reaction catalysed by CALB wt with the acid (H 2SO 4) catalysed reaction. The performances of CALB wt and mutants were compared to two other hydrolases, Humicola insolens cutinase and Rhizomucor mihei lipase. The low reaction rates of enzyme catalysed transacylation of acrylates were found to be caused mainly by electronic effects due to the double bond present in this class of molecules. The reduction in rate of enzyme catalysed transacylation of acrylates compared to that of the saturated ester methyl propionate was however less than what could be predicted from the energetic cost of breaking the π-system of acrylates solely. The nature and concentration of the acyl acceptor was found to have a profound effect on the reaction rate.

Hydroxy functional acrylate and methacrylate monomers prepared via lipase—catalyzed transacylation reactions

Candida antarctica lipase B (CAL-B, Novozyme 435) catalyzes the transacylation of methyl acrylate and methyl methacrylate with diols and triols in 2-methyl-2-butanol at 50 °C. Under the experimental conditions, up to 70 mol% of the acyl donor methyl acrylate was converted. Methyl methacrylate is the less efficient acyl donor (up to 60 mol%) due to the higher sterical hindrance in the enzymatic transacylation. Under the reaction conditions high yields of the mono-acylated products are obtained, which contain minor amounts of bis(meth)acrylates. In addition it was observed that Novozyme 435 catalyzes regioselectively the acylation of the primary hydroxyl groups. In comparison with the chemical catalyzed route no selectivity was observed for unsubstituted diols. For substituted diols more mono-acylated product was formed in the lipase-catalyzed reaction than in the chemical catalyzed reaction.

Enzymatic synthesis of capsaicin analogs and their effect on the T-type Ca 2+ channels

Capsaicin (Cap) and its analogs (CAPanalogs) have diverse effects in sensory neurons including analgesia, implying they modulate other cellular targets besides the TRPV1 Cap receptor. Since Cap and CAPanalogs are not largely available and their chemical synthesis is cumbersome, they have been obtained through a direct lipase-catalyzed reaction. Capsiate, the ester CAPanalog, was synthesized using a novel enzymatic transacylation one-pot strategy. Five different CAPanalogs were synthesized by amidation in 2-methyl-2-butanol with higher yields than previously reported. Voltage-dependent Ca 2+ channels (Ca vs) are among the main Ca 2+ entry paths into cells. They are classified as high-voltage-activated Ca 2+ channels (HVA) and low-voltage-activated Ca 2+ channels (LVA) constituted only by T-type channels. Though HVA Ca vs are Cap sensitive, it is not known if capsaicinoids inhibit LVA Ca vs which participate in the primary sensory neuron pain pathway. Here we first report that Cap, dihydrocapsaicin, N-VAMC 8, N-VAMC 9, and N-VAMC 10 can directly and partially reversibly inhibit T-type Ca vs, whereas olvanil, capsiate, and vanillylamine cannot. The Cap inhibition of T-type Ca vs was independent of TRPV1 activation.

Chemical and enzymatic transacylation of amide-linked FA of buttermilk gangliosides.

The goal of this work was to alter the composition of amide-linked FA of bovine buttermilk gangliosides, particularly the disialoganglioside GD3, to adjust lipid sources to special food specifications and pharmacological or cosmetic applications. The chemical transacylation of amide-linked FA of buttermilk gangliosides with free arachidic acid (20:0) by a combination of basic hydrolysis and diethylphosphorylcyanide/triethylamine-catalyzed reacylation was compared to an enzymatic sphingolipid ceramide N-deacylase (EC 3.5.1.23)-catalyzed FA exchange by GC analysis and nano electrospray ionization-MS. The buttermilk predominantly contained the disialoganglioside GD3 and the monosialoganglioside GM3. The heterogeneity of FA that are incorporated into gangliosides, mainly palmitic acid (29.4 wt%), stearic acid (16.9 wt%), oleic acid (17.8 wt%), and myristic acid (8.5 wt%), was effectively altered by both transesterification techniques. Arachidic acid, which was not integrated into the initial buttermilk gangliosides, was transacylated to total gangliosides with 23.2 wt% (GD3, 6.7 wt%) by the chemical process and with 8.7 wt% (GD3, 13.8 wt%) when catalyzed enzymatically. Mainly behenic acid and lignoceric acid of GD3 were exchanged chemically, and stearic acid was exchanged by the enzymatic process. This observation might depend on hydrolytic sensitivities of amide-linked very long chain saturated FA or specific enzyme subtrate affinities, respectively. Results of chemical hydrolysis indicated there was a risk of sialic acid decomposition and unspecific degradations. Regarding specificity and avoidance of critical agents, the enzymatic transesterification is recommended for industrial-scale production of consumer goods.

Modification of Soybean Phospholipids by Enzymatic Transacylation.

Modified glycerophospholipids with expected novel physiological functions were prepared by introducing different acyl groups into soybean phospholipids. The method was based on the enzymatic transesterification of phospholipids with monoacylglycerol (MG) or diacylglycerol (DG). The transacylation ratios reached 46-59% and nearly quantitative recovery of phospholipids was obtained in the case of transacylation of phospholipids with MGs having C12 to C18 saturated and monounsaturated acyl groups. With C18:2-MG or C18:3-MG, however, transacylation ratios were less than ca. 20% MGs with C4 to C8 saturated acyl groups also gave very low transacylation ratios, but the ratios were moderately improved by using DGs with C6 and C8 acyl groups. It is considered from a series of the results that higher transacylated phospholipids might be obtained y repeating transacylations owing to be able to recover the phospholipids quantitatively after the reaction because MG with longer acyl groups than C10 or DG with C8 acyl groups acted as acyl donors and replacement for essential water, to suppress the simultaneous hydrolysis, in an enzymatic reaction.

Production of Recombinant Salmon Calcitonin by Amidation of Precursor Peptide Using Enzymatic Transacylation and Photolysis in Vitro

The C terminal amidation is required for full biological activity of salmon calcitonin (sCT). We constructed BL21(DE3)/pGEX-sCT-Ala, an engineering Escherichia coli strain. The soluble fusion protein of GST-sCT-Ala expressed from BL21(DE3)/pGEX-sCT-Ala was purified by affinity chromatography after high density, high expression culture and sonication of bacteria. Following S-sulfonation of the fusion protein, the 33 alanine-extended peptides were released from the fusion protein by cyanogen bromide. The S-sulfonated precursor peptide was transacylated by CPD-Y, o-PNGA as a nucleophile, to produce photosensitive SO−3-sCT-o-PNGA. After photolysis and folding, the biological activity of sCT was assayed as standard.

ChemInform Abstract: Peptide Amidation by Enzymatic Transacylation and Photolysis.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Peptide amidation by enzymatic transacylation and photolysis.

A series of model peptides with a C-terminal protected amide group were prepared by enzymatic transacylation. The protection groups were removed by photolysis to give the warranted peptide amides in high yields. Furthermore, fragments of human calcitonin were prepared. Various protective groups were employed, and the pH, solvent and concentration dependency of the enzymatic transcylation were examined. The photo-cleavage reaction was examined for wavelength, concentration and pH dependency. It was shown that the optimal yields required addition of a chemical scavenger for the photolysis byproducts.

Effects of substrate fatty acids on products of lecithin hydrolysis and acyl-CoA-independent transacylation with cholesterol by aortic enzyme preparations.

The acyl composition of substrates and products of enzymatic hydrolysis and transacylation of lecithin with cholesterol in the arterial wall was investigated. Saturated acyl residues predominated in lysolecithin and unsaturated ones in acids released by hydrolysis of egg lecithin. In the reaction system with cholesterol, saturated acyls predominated in both lysolecithin and acids released whereas unsaturated ones were more abundant in newly formed acylcholesterols. Mainly unsaturated acyls were present in the hydrolysis products from soybean lecithin in the reaction systems with and without cholesterol. For acylcholesterols formed in the presence of either lecithin, the percent values are in the numerical order of C18:2 greater than C18:1 greater than C16:0 greater than or equal to C18:0. It It is concluded that acyl preferences and interactions in the enzyme-catalyzed reactions studied may contribute to the different accumulation and removal of the compounds involved from the artery.

Enzymatic degradation of polar lipids in deep-frozen parsley.

Under the frozen storage at usual storage temperatures of leafy tissues not pretreated by heat, enzymatic lipid degradation reactions take place, which lead already after a few weeks to a considerable or complete loss of the native polar lipids. These degradation processes being accompanied by a deterioration of the flavour have been studied in greater detail in parsley leaves. Among the reaction products we found large amounts of 6-acylmonogalactosyl diglycerides (formed from monogalactosyl diglycerides by enzymatic transacylation) and phosphatidic acid (formed from phospholipids through phospholipase-D action). The generally assumed reaction sequence: formation of free fatty acids by acyl hydrolases followed by hydroperoxidation through lipoxygenase and degradation of the hydroperoxidation through lipoxygenase and degradation of the hydroperoxides into off-flavour compounds may hence take place, if at all, only to a limited extent. Considerable phospholipase D as well as minor acyl transferase activities are detected at --24 degrees C, whereas at --32 degrees C the lipid loss is very low. Deterioration processes can be avoided by blanching, a treatment not leading to any substantial quality loss.

Phospholipid modifications and adenylate cyclase in rat liver plasma membranes.

Rat liver plasma membrane phospholipid headgroups or fatty acids were modified by enzymatic transmethylation or transacylation. To evaluate the effect of phospholipid modification on adenylate cyclase (AC), one of the enzymes of phospholipid metabolism as well as AC were measured in the same incubation medium. Methyl transfer from S-adenosyl-L-methionine (SAM) to phosphatidylcholine (PC) was the highest at pH 9.2. At low concentrations of SAM, synthesis of PC as well as synthesis of the monomethyl derivative were considerably decreased and Mg2+ had no effect at pH 9.2 or at pH 6.5. When phospholipid methylation was increased in relation to SAM concentration, there was no change in basal, NaF- and glucagon-stimulated AC. Methylation was not modified when AC was stimulated. On the other hand, there was an increase in the basal, NaF- and glucagon-stimulated AC when linoleate was incorporated into the membrane phospholipids. Other unsaturated fatty acids had no effect. The synthesis of linoleoyl-PC from added lysophosphatidylcholine (LPC) also stimulated AC and this in turn partly prevented the inhibitory effect of LPC. Thus in isolated plasma membranes transmethylation has no direct effect on AC, whereas synthesis of linoleoyl molecular species can modulate AC in a way which does not depend on the membrane fluidity.

Galactolipid transformations and photochemical activities of spinach chloroplasts

1. When spinach chloroplasts are isolated in media containing high concentrations of NaCl, monogalactolipase activity can be observed. In media containing sucrose or mannitol, there is also an enzymatic transacylation which results in a decrease in digalactolipid and the formation of acylated derivatives of the mono- and digalactolipids. The rate of release of free fatty acids is slower under the latter conditions. 2. In order to establish a relation between these phenomena and the photochemical capacities of the chloroplasts, several light reactions have been measured in fresh and aged chloroplasts. 3. Hill reactions in Photosystem II and NADP + reduction in Photosystem I are not directly influenced by the lipid transformations. 4. Photophosphorylation and Hill reaction at limiting light intensities are very labile under the experimental conditions. Lipid transformations may contribute to this effect. 5. Serum albumin has no effect on the transformations of chloroplast galactolipids. It has a stimulating and stabilizing effect on photophosphorylation and improves the efficiency of photophosphorylation. Only a part of this effect can be explained by the known capacity of serum albumin to bind free fatty acids.