Reactions of beta-propiolactone, beta-butyrolactone and gamma-butyrolactone with nucleic acids

Reactivity of beta-propiolactone, beta-butyrolactone and gamma-butyrolactone with guanosine, RNA, DNA and 4-(p-nitrobenzyl)pyridine was studied. beta-Propiolactone was 50--100 times more reactive with all the nucleophiles than beta-butyrolactone whereas gamma-butyrolactone was completely inactive. The rate of alkylation by the lactones was guanosine greater than RNA = denatured DNA greater than double-stranded DNA. The type of the adducts formed were characterized by fluorescence and ultraviolet spectroscopy. Similar alkylation products were formed by the two lactones. The main sites alkylated were N-1 at adenosine, N-3 at cytidine and N-7 at guanosine. The results suggest that the carcinogenic potency of the lactones correlates with their reactivity rather than with specificity of the adducts formed.     

Stabilization of alpha-chymotrypsin by ionic liquids in transesterification reactions.

Five different ionic liquids, based on dialkylimidazolium and quaternary ammonium cations associated with perfluorinated and bis (trifluoromethyl) sulfonyl amide anions, were used as reaction media to synthesize N-acetyl-L-tyrosine propyl ester by transesterification with alpha-chymotrypsin at 2% (v/v) water content at 50 degrees C. The synthetic activity was reduced by the increase in alkyl chains length of cations and by increases in anion size, which was related to the decrease in polarity. Incubation of the enzyme (with and without substrate) in ionic liquids exhibited first-order deactivation kinetics at 50 degrees C, allowing determination of deactivation rate constants and half-life times (1-3 h). Ionic liquids showed a clear relative stabilization effect on the enzyme, which was improved by increased chain length of the alkyl substituents on the imidazolium ring cations and the anion size. This effect was 10-times enhanced by the presence of substrate. For example, 1-butyl-3-methylimidazolium hexafluorophosphate increased the alpha-chymotrypsin half-life by 200 times in the presence of substrate with respect to the 1-propanol medium. These results show that ionic liquids are excellent enzyme-stabilizing agents and reaction media for clean biocatalysis in non-conventional conditions.     

Modeling of lipase catalyzed ring-opening polymerization of epsilon-caprolactone

Enzymatic ring-opening polymerization of epsilon-caprolactone by various lipases was investigated in toluene at various temperatures. The determination of molecular weight and structural identification was carried out with gel permeation chromatography and proton NMR, respectively. Among the various lipases employed, an immobilized lipase from Candida antartica B (Novozym 435) showed the highest catalytic activity. The polymerization of epsilon-caprolactone by Novozym 435 showed an optimal temperature of 65 degrees C and an optimum toluene content of 50/50 v/v of toluene and epsilon-caprolactone. As lipases can degrade polyesters, a maximum in the molecular weight with time was obtained due to the competition of ring opening polymerization and degradation by specific chain end scission. The optimum temperature, toluene content, and the variation of molecular weight with time are consistent with earlier observations. A comprehensive model based on continuous distribution kinetics was developed to model these phenomena. The model accounts for simultaneous polymerization, degradation and enzyme deactivation and provides a technique to determine the rate coefficients for these processes. The dependence of these rate coefficients with temperature and monomer concentration is also discussed.     

Cocrystallization of random copolymers of omega-pentadecalactone and epsilon-caprolactone synthesized by lipase catalysis

Random copolymers were prepared by Candida antarctica lipase B (Novozyme-435) catalyzed copolymerization of omega-pentadecalactone (PDL) with epsilon-caprolactone (CL). Over the whole composition range PDL-CL copolymers are highly crystalline (melting enthalpy by differential scanning calorimetry, above 100 J/g; crystallinity degree by wide-angle X-ray scattering, WAXS, 60-70%). The copolymers melt at temperatures that linearly decrease with composition from that of poly(omega-pentadecalactone) (PPDL; 97 degrees C) to that of poly(epsilon-caprolactone) (PCL; 59 degrees C). The WAXS profiles of PCL and PPDL homopolymers are very similar, except for the presence in PPDL of the (001) reflection at 2theta = 4.58 degrees that corresponds to a 19.3 angstroms periodicity in the chain direction. In PDL-CL copolymers the intensity of this reflection decreases with increasing content of CL units and vanishes at 50 mol % CL, as a result of randomization of the ester group alignment and loss of chain periodicity. PDL-CL copolymers crystallize in a lattice that gradually changes from that of one homopolymer to that of the other, owing to comonomer isomorphous substitution. Cocrystallization of comonomer units is also shown by a random PDL-CL copolymer obtained in a polymerization/transesterification reaction catalyzed by C. antarctica lipase B (Novozyme-435) starting from preformed PCL and PDL monomer.     

Correlation between structure of the lactones and substrate specificity in enzyme-catalyzed polymerization for the synthesis of polyesters

Small-size (4-membered) and medium-size (5-, 6-, and 7-membered) unsubstituted lactones as well as unsubstituted macrolides (12 and 13 membered) were subjected to the ring-opening polymerization using the extracellular PHB depolymerase from Alcaligenes faecalis T1 (PhaZ(Afa)). The characteristic reactivities of the lactones were discussed based on a tertiary structure model of the active site of the PhaZ(Afa). With respect to the ring-size of the lactones, the 4-membered beta-propiolactone and 6-membered delta-valerolactone (delta-VL) showed the highest polymerization activity, and delta-VL seemed to be the upper size limit for the molecular recognition of the narrow active site cleft of PhaZ(Afa). On the other hand, epsilon-caprolactone, 11-undecanolide, and 12-dodecanolide, which showed excellent polymerization activities by lipases, were scarcely polymerized by PhaZ(Afa). This was ascribed to the difference in the recognition sites between PhaZ(Afa) and lipase. In addition, the effect of the substrate-binding domain of PhaZ(Afa) and the enantioselective ring-opening polymerization of (R,S)-beta-butyrolactone ((R,S)-beta-BL) were studied. The substrate-binding domain lacking PhaZ(Afa) showed higher reactivities than PhaZ(Afa) for the polymerization of the lactones and that a significant enantioselectivity was observed at the early stage of the polymerization of (R,S)-beta-BL to produce the (R)-enriched optically active poly(3-hydroxybutyrate).     

Over-stabilization of Candida antarctica lipase B by ionic liquids in ester synthesis

Four different ionic liquids, based on dialkylimidazolium cations associated with perfluorinated and bis(trifluoromethyl)sulfonyl amide anions were used as reaction media for butyl butyrate synthesis catalyzed by free Candida antarctica lipase B at 2% (v/v) water content and 50 °C. Lipase had enhanced synthetic activity in all ionic liquids in comparison with two organic solvents (hexane, and 1-butanol), the enhanced activity being related to the increase in polarity of ionic liquids. The continuous operation of lipase with all the assayed ionic liquids showed over-stabilization of the enzyme. The reuse of free lipase in 1-butyl-3-methylimidazolium hexafluorophosphate in continuous operation cycles showed a half-life time 2300 times greater than that observed when the enzyme was incubated in the absence of substrate (3.2 h), and a selectivity higher than 90%.     

Enzymatic preparation of novel thermoplastic di-block copolyesters containing poly[(R)-3-hydroxybutyrate] and poly(epsilon-caprolactone) blocks via ring-opening polymerization

Enzymatic modification of a microbial polyester was achieved by the ring-opening polymerization of epsilon-caprolactone (CL) with low-molecular weight telechelic hydroxylated poly[( R)-3-hydroxybutyrate] (PHB-diol) as initiator and Novozym 435 (immobilized Candida antarctica Lipase B) as catalyst in anhydrous 1,4-dioxane or toluene. The ring-opening polymerization was investigated at different conditions with two different types of PHB-diols: PHB-diol(P), containing a primary OH and a secondary OH end groups, and PHB-diol(M), consisting of 91% PHB-diol(P) and 9% PHB-diol containing two secondary OH end groups. The reactions were followed by GPC analyses of the resulting polymers at different time points, and the optimal conditions were established to be 70 degrees C at a weight ratio of CL/enzyme/solvent of 8:1:24. The ring-opening polymerization of CL with PHB-diol(M) (Mn of 2380, NMR) at the molar ratio of 50:1 under the optimal conditions in 1,4-dioxane gave the corresponding poly[HB(56 wt %)-co-CL(44 wt %)] with Mn (NMR) of 3900 in 66% yield. Polymerization of CL and PHB-diol(P) ( Mn of 2010, NMR) at the same condition in toluene gave the corresponding poly[HB(28 wt %)-co-CL(72 wt %)] with Mn (NMR) of 7100 in 86% yield. Both polymers were characterized by (1)H and (13)C NMR and IR analyses as di-block copolyesters containing a PHB block with a secondary OH end group and a poly(epsilon-caprolactone) (PCL) block with a primary OH end group. NMR analyses and control experiments suggested no formation of random copolymers and no change of the PHB block during the reaction. The enzymatic ring-opening polymerization was selectively initiated by the primary OH group of PHB-diol, whereas the secondary OH group remained as an end group in the final polymers. The thermal properties of the di-block poly(HB-co-CL)s were analyzed by DSC, with excellent T g values for the elastomer domain: poly[HB(56 wt %)- co-CL(44 wt %)] with M n (NMR) of 3900 demonstrated a T g of -57 degrees C, Tm of 145, 123, and 53 degrees C; and poly[HB(28wt%)-co-CL(72wt%)] with Mn (NMR) of 7100 gave a Tg of -60 degrees C, Tm of 147 and 50 degrees C. Thus, the selective enzymatic ring-opening polymerization with PHB-diol as macro-initiator provides a new method for the preparation of PHB-based block copolymers as biomaterials with good thermoplastic properties and novel structures containing functional end groups.     

Syntheses, characterization, and in vitro degradation of ethyl cellulose-graft-poly(epsilon-caprolactone)-block-poly(L-lactide) copolymers by sequential ring-opening polymerization

Well-defined ethyl cellulose-graft-poly(epsilon-caprolactone) (EC-g-PCL) graft copolymers were successfully synthesized via ring-opening polymerization (ROP) of epsilon-caprolactone (CL) with an ethyl cellulose (EC) initiator and a tin 2-ethylhexanoate (Sn(Oct)2) catalyst in xylene at 120 degrees C. Then, novel ethyl cellulose-graft-poly(epsilon-caprolactone)-block-poly(L-lactide) (EC-g-PCL-b-PLLA) graft-block copolymers were prepared by ROP of L-lactide (L-LA) with a hydroxyl-terminated EC-g-PCL macroinitiator and Sn(Oct)2 catalyst in bulk at 120 degrees C. Various graft and block lengths of EC-g-PCL and EC-g-PCL-b-PLLA copolymers were obtained by adjusting the molar ratios of CL monomer to EC and the L-LA monomer to CL. The thermal properties and crystalline morphologies of EC-g-PCL and EC-g-PCL-b-PLLA copolymers were different from those of linear PCL. The in vitro degradation rate of EC-g-PCL-b-PLLA was faster than those of linear PCL and EC-g-PCL due to the presence of PLLA blocks.     

Ring-opening bulk polymerization of epsilon-caprolactone and trimethylene carbonate catalyzed by lipase Novozym 435.

The affects of lipase concentration on ring-opening bulk polymerizations of epsilon-caprolactone and trimethylene carbonate were studied by using Novozym 435 (immobilized form of lipase B from Candida antarctica) as biocatalyst. The polymerization of epsilon-caprolactone was carried out in bulk at 70 degrees C. Three lipase concentrations of 9.77, 1.80 and 0.50 mg/mmol epsilon-CL were used in the experiment. The results showed that increasing the lipase concentration used in the polymerization system resulted in an increased rate of monomer consumption. For an enzyme concentration of 9.8 mg lipase per mmol monomer, an 80% monomer conversion was achieved in a 4-h time period, while for the lower enzyme concentration of 1.8 mg lipase per mmol monomer, 48 h were needed to reach monomer conversion. Linear relationships between Mn and monomer conversions were observed in all three enzyme concentrations, suggesting that the product molecular weight may be controlled by the stoichiometry of the reactants for these systems. At the same monomer conversion level, however, Mn decreased with increasing enzyme concentration. After correcting for the amount of monomer consumed in initiation, the plot of ln[([M]o - [M]i)/([Mt] - [M]i)] versus reaction time was found to be linear, suggesting that the monomer consumption followed a first-order rate law and no chain termination occurred. For the TMC systems, the polymerization was carried out in bulk at 55 degrees C. Similar to the epsilon-CL systems, increasing the Novozym 435 concentration from 8.3 to 23.6 mg/mmol TMC increased the rate of monomer conversion. Unlike the epsilon-CL systems, however, nonlinear relationships were obtained between Mn and monomer conversion, indicating that possible chain transfer and/or slow initiation had taken place in these systems. Consistent with the above result, nonlinear behavior was observed for the plot of ln[[M]o/[M]t] versus reaction time.     

Tyrosinase activity in ionic liquids

Activity of mushroom tyrosinase was studied in three ionic liquids, 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF₆]), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF₄]) and 1-butyl-3-methylimidazolium methylsulfate ([BMIm][MeSO₄]), and was compared to that in chloroform. Kinetic parameters of the enzyme were determined and the results indicate that the enzyme in ionic liquids basically follows the same catalytic mechanism as in water, and that the ionic liquids may affect the enzyme activity by direct interacting with the enzyme and thus hindering the E–S binding due to their high hydrophilicity and polarity.     

Thiol-dependent changes in the properties of rat liver sulphotransferases

1. Two enzymes (A and B) which catalyse the sulphation of p-nitrophenol and l-tyrosine methyl ester have been isolated from female rat livers. One of these enzymes (A) also catalyses the sulphation of dehydroepiandrosterone. 2. The K(m) values for the sulphation of p-nitrophenol and l-tyrosine methyl ester by enzyme B at pH7.5 are 1.5mum and 2.9mm respectively. 3. Enzyme B is oxidized on keeping at 0 degrees C when the K(m) and V(max.) values for the sulphation of p-nitrophenol are increased approx. 200-fold and fourfold respectively. This oxidized preparation of enzyme B fails to catalyse the sulphation of l-tyrosine methyl ester. 4. When the oxidized form of enzyme B is kept at 0 degrees C and low ionic strength then further forms of p-nitrophenol sulphotransferase are produced having even lower affinities for the sulphate acceptor. 5. The K(m) value for adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate is not affected during storage of the enzyme under these conditions. 6. Prolonged storage of enzyme B at low ionic strength leads to a considerable degree of polymerization of p-nitrophenol sulphotransferase and l-tyrosine methyl ester sulphotransferase. 7. The changes in the kinetic properties and molecular size of enzyme B during storage are reversed by dithiothreitol.     

Synthesis of poly(epsilon-caprolactone)-b-poly(gamma-benzyl-L-glutamic acid) block copolymer using amino organic calcium catalyst

A biodegradable two block copolymer, poly(epsilon-caprolactone)-b- poly(gamma-benzyl-L-glutamic acid) (PCL-PBLG) was synthesized successfully by ring-opening polymerization of N-carboxyanhydride of gamma-benzyl-L-glutamate (BLG-NCA) with aminophenyl-terminated PCL as a macroinitiator. The aminophenethoxyl-terminated PCL was prepared via hydrogenation of a 4-nitrophenethoxyl-terminated PCL, which was novelly obtained from the polymerization of epsilon-caprolactone (CL) initiated by amino calcium 4-nitrobenzoxide. The structures of the block copolymer and its precursors from the initial step of PCL were confirmed and investigated by 1H NMR, FT-IR, GPC, and FT-ICRMS analyses and DSC measurements.     

Enzymatic catalysis of formation of Z-aspartame in ionic liquid - An alternative to enzymatic catalysis in organic solvents

We present the first report of enzymatic catalysis in an ionic liquid. The virtually nonexistent vapor pressure makes ionic liquids an exciting new alternative for enzyme-catalyzed syntheses in environmentally friendly environments. Z-aspartame was synthesized in a thermolysin-catalyzed reaction of carbobenzoxy-L-aspartate and L-phenylalanine methyl ester hydrochloride in 1-butyl-3-methylimidazolium hexafluorophosphate (BP6). Ionic liquids such as BP6 are thermally stable and have a remarkable range of temperatures over which they remain liquid (300 degrees C). With an initial rate of 1.2 +/- 0.1 nmol min(-)(1) mg(-)(1), we observed a competitive rate in comparison to that of enzymatic synthesis in organic solvent. Additionally, the enzyme exhibits outstanding stability, which would normally require immobilization.     

Alpha-chymotrypsin catalysis in imidazolium-based ionic liquids

The transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with 1-propanol catalyzed by alpha-chymotrypsin was examined in the ionic liquids 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF(6)]) and 1-octyl-3-methylimidazolium hexafluorophosphate ([omim][PF(6)]), and in combination with supercritical carbon dioxide (SC-CO(2)). The activity of alpha-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. alpha-Chymotrypsin freeze-dried with K(2)HPO(4), KCl, or poly(ethylene glycol) demonstrated no activity in [bmim][PF(6)] or [omim][PF(6)] 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][PF(6)] than [bmim][PF(6)]. Added water was not necessary for enzyme activity when ionic liquids were combined with SC-CO(2). These results indicate that [bmim][PF(6)] and [omim][PF(6)] provide a relatively polar environment, which can be modified with nonpolar SC-CO(2) to optimize enzyme activity.     

Toward advanced ionic liquids. Polar,enzyme-friendly solvents for biocatalysis

Ionic liquids, also called molten salts, are mixtures of cations and anions that melt below 100°C. Typical ionic liquids are dialkylimidazolium cations with weakly coordinating anions such as (MeOSO₃) or (PF₆). Advanced ionic liquids such as choline citrate have biodegradable, less expensive, and less toxic anions and cations. Deep eutectic solvents are also included in the advanced ionic liquids. Deep eutectic solvents are mixtures of salts such as choline chloride and uncharged hydrogen bond donors such as urea, oxalic acid, or glycerol. For example, a mixture of choline chloride and urea in 1:2 molar ratio liquefies to form a deep eutectic solvent. Their properties are similar to those of ionic liquids. Water-miscible ionic liquids as cosolvents with water enhance the solubility of substrates or products. Although traditional water-miscible organic solvents also enhance solubility, they often inactivate enzymes, while ionic liquids do not. The enhanced solubility of substrates can increase the rate of reaction and often increases the regioor enantioselectivity. Ionic liquids can also be solvents for non-aqueous reactions. In these cases, they are especially suited to dissolve polar substrates. Polar organic solvent alternatives inactivate enzymes, but ionic liquids do not even when they have similar polarities. Besides their solubility properties, ionic liquids and deep eutectic solvents may be greener than organic solvents because ionic liquids are nonvolatile, and can be made from nontoxic components. This review covers selected examples of enzyme catalyzed reaction in ionic liquids that demonstrate their advantages and unique properties, and point out opportunities for new applications. Most examples involve hydrolases, but oxidoreductases and even whole cell reactions have been reported in ionic liquids.     

Buffer-mediated activation of Candida antarctica lipase B dissolved in hydroxyl-functionalized ionic liquids

Ionic-liquid buffer having phosphate anion was synthesized for the development of buffered enzymatic ionic liquid systems. Both the conformation and transesterification activity of Candida antarctica lipase B (CALB) dissolved in the hydroxyl-functionalized ionic liquids were buffer dependent. Intrinsic fluorescence studies indicated that the CALB possessed a more compact conformation in the medium consisted of ionic liquid buffer having phosphate anion and hydroxyl-functionalized ionic liquids like 1-(1-hydroxyethyl)-3-methyl-imidazolium tetrafluoroborate and 1-(1-hydroxyethyl)-3-methyl-imidazolium nitrate. High activity and outstanding stability could be obtained with the CALB enzyme in the buffered ionic liquids for the transesterification.     

Superior solubility of polysaccharides in low viscosity, polar, and halogen-free 1,3-dialkylimidazolium formates

We successfully prepared low viscosity, polar, and halogen-free ionic liquids as potential solvents for a wide range of polysaccharides. A series of 1,3-dialkylimidazolium formates were produced as liquids having strong hydrogen bond acceptability. These formates had significantly lower viscosity than previously reported polar ionic liquids, in particular 1-allyl-3-methylimidazolium formate (viscosity 66cP at 25 degrees C) and 1-allyl-3-ethylimidazolium formate (67cP at 25 degrees C). Because of their strong hydrogen bond ability, various polysaccharides including amylose and (scarcely soluble) cellulose were dissolved in high concentrations under mild condition.     

Polyester coating of cellulose fiber surfaces catalyzed by a cellulose-binding module-Candida antarctica lipase B fusion protein

A new approach to introduce polymers to cellulosic materials was developed by using the ability of a cellulose-binding module-Candida antarctica lipase B conjugate to catalyze ring-opening polymerization of epsilon-caprolactone in close proximity to cellulose fiber surfaces. The epsilon-caprolactone was introduced to the cellulose surfaces either by simple addition of liquid monomer or through gas phase. The effects of water activity and temperature on the lipase-catalyzed polymerization process were investigated. Analysis showed that the water content in the system primarily regulated the obtained polymer molecular weight, whereas the temperature influenced the reaction rate. The hydrophobicity of the obtained surfaces did not arise from covalent attachment of the poly(epsilon-caprolactone) to the surface hydroxyl groups but rather from surface-deposited polymers which could be readily extracted. The degree of lipase-catalyzed hydrolysis through introduction of water to the polymer-coated cellulose fiber surfaces was also investigated and shown to be significant.     

Dissolution of cellulose in phosphate-based ionic liquids

Two kinds of alkylimidazolium salts containing dimethyl phosphate or diethyl phosphate were obtained as room temperature ionic liquids synthesized by one step, and both of them have the ability to dissolve untreated cellulose. Especially, 1-ethyl-3-methylimidazolium diethylphosphonate ([EMIM]DEP) could obtain 4 wt% cellulose solution within 10 min under 90. The effects of dissolution temperature on cellulose dissolution time and degree of polymerization were investigated. As dissolution temperature increased, dissolution time was greatly reduced. Both the original and regenerated cellulose samples were characterized with wide-angle X-ray diffraction, thermogravimetric analysis and scanning electron micrograph. The results showed that the crystalline structure of cellulose was converted to cellulose II from cellulose I in native cellulose. It was also found that the regenerated cellulose had good thermal stability with [EMIM]DEP ionic liquid.     

Poly(ethylene glycol)-lipase complex that is catalytically active for alcoholysis reactions in ionic liquids

Lipase-catalyzed alcoholysis was investigated in three different ionic liquids. Lyophilized native lipase had a low activity in all the ionic liquids but a poly(ethylene glycol) (PEG)-lipase complex (with a molar ratio of the polymer/enzyme of 10:1) had an increased activity of over 14-fold. Of several lipases tested, PEG-lipase PS (from Pseudomonas cepacia) exhibited the highest activity (1.07 mmol/(h g^–1 protein)) in 1-octyl-3-methylimidazolium hexafluorophosphate.