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).     

Enzymatic synthesis of poly(hydroxyalkanoates) in ionic liquids

Ring-opening polymerization of five lactones catalyzed by Candida antarctica lipase B in ionic liquids yielded poly(hydroxyalkanoates) of moderate molecular weights up to Mn=13,000. In the ionic liquid 1-butyl-3-methylimidazolium bis(trifluoromethane)-sulfonimide and with a low weight ratio of enzyme to lactone (1:100) we obtained polymers from beta-propiolactone, delta-valerolactone, and epsilon-caprolactone with degrees of polymerization as high as 170, 25, and 85, respectively; oligomers from beta-butyrolactone and gamma-butyrolactone with degrees of polymerization of 5; and a copolymer of beta-propiolactone and beta-butyrolactone with a degree of polymerization of 180. Water-immiscible ionic liquids were superior to water-miscible ionic liquids. Reducing the water content of the enzyme improved the degree of polymerization by as much as 50% for beta-propiolactone and epsilon-caprolactone.     

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.     

Enzymatic degradation of block copolymers prepared from epsilon-caprolactone and poly(ethylene glycol)

Block copolymers were prepared by ring-opening polymerization of epsilon-caprolactone in the presence of monohydroxyl or dihydroxyl poly(ethylene glycol) (PEG), using Zn powder as catalyst. The resulting poly(epsilon-caprolactone) (PCL)-PEG diblock and PCL-PEG-PCL triblock copolymers were characterized by various analytical techniques such as NMR, size-exclusion chromatography, differential scanning calorimetry, and X-ray diffraction. Both copolymers were semicrystalline polymers, the crystalline structure being of the PCL type. Films were prepared by casting dichloromethane solutions of the polymers on a glass plate. Square samples with dimensions of 10 x 10 mm were allowed to degrade in a pH = 7.0 phosphate buffer solution containing Pseudomonas lipase. Data showed that the introduction of PEG blocks did not decrease the degradation rate of poly(epsilon-caprolactone).     

Structural characterization of a lipase-catalyzed copolymerization of epsilon-caprolactone and D,L-lactide

The copolymerization of epsilon-caprolactone (epsilon-CL) and d,l-lactide catalyzed by Candida antarctica lipase B was studied. Copolymerizations with different epsilon-CL-to-lactide ratios were carried out, and the product was monitored and characterized by MALDI-TOF MS, GPC, and (1)H NMR. The polymerization of epsilon-CL, which is normally promoted by C. antarctica lipase B, is initially slowed by the presence of lactide. During this stage, lactide is consumed more rapidly than epsilon-CL, and the incorporation occurs dimer-wise with regard to the lactic acid (LA) units. As the reaction proceeds, the relative amount of CL units in the copolymer increases. The nonrandom copolymer structure disappears with time, probably due to a lipase-catalyzed transesterification reaction. In the copolymerizations with a low content of lactide, macrocycles of poly(epsilon-caprolactone) and copolymers having up to two LA units in the ring were detected.     

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.     

Porous scaffolds from high molecular weight polyesters synthesized via enzyme-catalyzed ring-opening polymerization

Several aliphatic polyesters have been synthesized until now using enzyme-catalyzed ring-opening polymerization (ROP) of different lactones, although their molecular weight, hence mechanical strength, was not sufficient enough to fabricate porous scaffolds from them. To achieve this target, 1,5-dioxepan-2-one (DXO) and epsilon-caprolactone (CL) were polymerized in bulk with Lipase CA as catalyst at 60 degrees C, and porous scaffolds were prepared from the polymers obtained thereof using a salt leaching technique. The CL/DXO molar feed ratio was varied from 1.5 to 10, and the reactivity ratios of CL and DXO were determined using the Kelen-Tudos method under such conditions of polymerization. NMR results showed a slightly lower CL/DXO molar ratio in the copolymers than in the feed due to high reactivity of DXO toward Lipase CA catalysis. The crystallinity of the PCL segment of the copolymers was affected by the presence of soft and amorphous DXO domains. The copolymers having high CL content were thermally more stable. The porosity of the scaffolds was in the range 82-88%, and the SEM analysis showed interconnected pores in the scaffolds. Of the two parameters which could affect the mechanical properties, viz., the copolymer composition and the scaffold pore size, the pore size showed a significant effect on the mechanical properties of the scaffolds. The porous scaffolds developed in this way for tissue engineering are free from toxic organometallic catalyst residues, and they are highly suitable for biomedical applications.     

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.     

Enzymatic polymerization catalyzed by surfactant-coated lipases in organic media

Structural ring-opening of lactones driven by enzymatic polymerization has been performed using low concentration dosages of surfactant-coated lipases in organic media. By comparison, enzymatic polymerization rate with coated lipase proceeded at a rate 100-fold better than native powder. Similarly a higher polymeric molecular weight (21,300), narrow dispersity (Mw/Mn=1.9) and better conversion (100%) were obtained following polyesterification tests with surfactant-coated lipase.     

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.     

Lipase-catalyzed biodegradation of poly(epsilon-caprolactone) blended with various polylactide-based polymers

Poly(epsilon-caprolactone) was blended with various polylactide-based polymers and processed to films by the solution casting method. Blends of 25/75, 50/50, 75/25, 90/10, and 95/5 (w/w) poly(epsilon-caprolactone)/poly(l-lactide), a 95/5 (w/w) blend of poly(epsilon-caprolactone) with a poly(d-lactide), a 50/50 (w/w) poly(l-lactide)-poly(d-lactide) mixture, and a poly(l-lactide-co-epsilon-caprolactone) copolymer were considered comparatively. The various phase-separated films were allowed to degrade in the presence of Pseudomonas lipase, biodegradation being monitored by proton nuclear magnetic resonance, size exclusion chromatography, differential scanning calorimetry, X-ray diffraction, and environmental scanning electron microscopy. The formation of separated phases during solvent evaporation and their morphologies are discussed. The introduction of poly(l-lactide) dramatically decreased the degradation rate of poly(epsilon-caprolactone)/poly(l-lactide) blends. The higher the percentage of poly(l-lactide), the slower the degradation, while the presence of cracks and increasing the lipase concentration acted in favor of the enzymatic degradation. Long-term enzymatic degradation of the various 95/5 blends was investigated over 480 h. The poly(epsilon-caprolactone) phase was enzymatically degraded by the lipase regardless of the blend type, the degradation rate depending on the nature of the co-components.     

Synthesis and micellization of amphiphilic brush-coil block copolymer based on poly(epsilon-caprolactone) and PEGylated polyphosphoester

A novel biodegradable amphiphilic brush-coil block copolymer consisting of poly(epsilon-caprolactone) and PEGylated polyphosphoester was synthesized by ring opening polymerization. The composition and structure of the copolymer were characterized by 1H NMR, 13C NMR, and FT-IR, and the molecular weight and molecular weight distribution were analyzed by gel permeation chromatograph (GPC) measurements to confirm the diblock structure. These amphiphilic copolymers formed micellar structures in water, and the critical micelle concentrations (CMCs) were around 10(-3) mg/mL, which was determined using pyrene as a fluorescence probe. Transmission electron microscopy (TEM) images showed that the micelles took an approximately spherical shape with core-shell structure, which was further demonstrated by laser light scattering (LLS) technique. The degradation behavior of the polymeric micelle was also investigated in the presence of Pseudomonas lipase and characterized by GPC measurement. Such polymer micelles from brush-coil block copolymers are expected to have wide utility in the field of drug delivery.     

Lipase-mediated deacetylation and oligomerization of lactonic sophorolipids

The direct enzymatic polymerization of lactonic sophorolipids (SLs) was investigated with four lipases, including porcine pancreatic lipase (PPL), immobilized Mucor miehei lipase (MML), lyophilized Candida antarctica lipase (Fraction B, CAL-B), and lyophilized Pseudomonas sp. lipase (PSL). Several organic solvents, covering a wide range of polarity, were compared for suitability as the reaction medium. Isopropyl ether and toluene were found most effective. According to the quantification and structure identification by HPLC and LC-MS, the reaction proceeded with the formation of monoacetylated lactonic SLs and the subsequent conversion of the intermediates to oligomers and polymers, presumably through ring-opening polymerization. Temperature was found to have significant effects on the reaction. Both the conversion of reactant SLs and the subsequent formation of oligomers and polymers from the intermediates were faster at 60 degrees C than at 50 degrees C. The substrate selectivity among the three dominant reactant SLs also differed with the temperature. The conversion rate increased with the ring size of the lactones at 60 degrees C, but it decreased with the size at 50 degrees C.     

Lactones 29 . Enzymatic resolution of racemic γ-lactones

Studies on the application of commercially available enzymes to resolution of the racemic unsaturated γ-lactones: 5-(3-methylbutylidene)-4-methyl-tetrahydrofuran-2-one (1a) and 5-(3,3-dimethylbutylidene)-4-methyl-tetrahydrofuran-2-one (2a) are presented. Lipase PS, Rhizopus niveus lipase, Rhizopus arrhizus lipase, porcine pancreas lipase and hog liver esterase transformed substrates into their respective γ-keto acids with good efficiency (50-75%). Three of them hydrolysed the studied lactones with moderate enantioselectivity. Enantiomeric excesses determined by GC for the unreacted lactones were in the range of 20-60%. Lipase PS preferentially hydrolysed the (+) enantiomers of lactones 1a and 2a whereas R. niveus lipase hydrolysed the (-) enantiomers, respectively.     

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.     

Humicola insolens cutinase-catalyzed lactone ring-opening polymerizations: kinetic and mechanistic studies

This paper explores reaction kinetics and mechanism for immobilized Humicola insolenscutinase (HIC), an important new biocatalyst that efficiently catalyzes non-natural polyester synthetic reactions. HIC, immobilized on Lewatit, was used as catalyst for epsilon-caprolactone (CL) and omega-pentadecalactone (PDL) ring-opening polymerizations (ROPs). Plots of percent CL conversion vs time were obtained in the temperature range from 50 to 90 degrees C. The kinetic plot of ln([M]0/[M]t) vs time (r2 = 0.99) for HIC-catalyzed bulk ROP of CL was linear, indicating that chain termination did not occur and the propagation rate is first order with respect to monomer concentration. Furthermore, linearity to 90% conversion for M(n) vs fractional CL conversion is consistent with a chain-end propagation mechanism. Deviation from linearity above 90% conversion indicates that a competition between ring-opening chain-end propagation and chain growth by steplike polycondensations takes place at high monomer conversion. HIC was inactive for catalysis of L-lactide and (R,S)-beta-butyrolactone ROP. HIC-catalyzed ROP of epsilon-CL and PDL in toluene were successfully performed, giving high molecular weight poly(epsilon-caprolactone) and omega-poly(pentadecalactone). In addition, the relative activities of immobilized Candida antarctica lipase B (CALB) and HIC for epsilon-CL and PDL polymerizations are reported herein.     

Resistance to macrolides and lincosamides in Streptomyces lividans and to aminoglycosides in Micromonospora purpurea.

Ribosomal (r) resistance to gentamicin in clones containing DNA from the producing organism Micromonospora purpurea is determined by grmA, and not by kgmA as originally reported. The kgmA gene originated in Streptomyces tenebrarius and is identical to kgmB. Both grmA and kgm encode enzymes that methylate single specific sites within 16S rRNA, although the site of action of the grmA product has not yet been determined. In either case, the methylated nucleoside is 7-methyl G. Inducible resistance to lincomycin (Ln) and macrolides in Streptomyces lividans TK21 results from expression of two genes: lrm, encoding an rRNA methyltransferase and mgt, encoding a glycosyl transferase (MGT), that specifically inactivates macrolides. The lrm product monomethylates residue A2058 within 23S rRNA (Escherichia coli numbering scheme) and confers high-level resistance to Ln with much lower levels of resistance to macrolides. Substrates for MGT, which utilises UDP-glucose as cofactor, include macrolides with 12-, 14-, 15- or 16-atom cyclic polyketide lactones (as in methymycin, erythromycin, azithromycin or tylosin, respectively) although spiramycin and carbomycin are not apparently modified. The enzyme is specific for the 2'-OH group of saccharide moieties attached to C5 of the 16-atom lactone ring (corresponding to C5 or C3 in 14- or 12-atom lactones, respectively). The lrm and mgt genes have been cloned and sequenced. The deduced lrm product is a 26-kDa protein, similar to other rRNA methyltransferases, such as the carB, tlrA and ermE products, whereas the mgt product (deduced to be 42 kDa) resembles a glycosyl transferase from barley.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis, characterization, and degradation behavior of amphiphilic poly-alpha,beta-[N-(2-hydroxyethyl)-L-aspartamide]-g-poly(epsilon-caprolactone)

A series of biodegradable amphiphilic graft polymers were successfully synthesized by grafting poly(epsilon-caprolactone) (PCL) sequences onto a water-soluble poly-alpha,beta-[N-(2-hydroxyethyl)-L-aspartamide] (PHEA) backbone. The graft copolymers were prepared through the ring-opening polymerization of epsilon-caprolactone (CL) initiated by the macroinitiator PHEA with pendant hydroxyl groups without adding any catalyst. By controlling the feed ratio of the macroinitiator to the monomer, the copolymers with different branch lengths and properties can be obtained. The successful grafting of PCL sequences onto the PHEA backbone was verified by FTIR, 1H NMR, and combined size-exclusion chromatography and multiangle laser light scattering (SEC-MALLS) analysis. The hydrolytic degradation and enzymatic degradation of these graft copolymers were investigated. The results show the hydrolytic degradation rate increases with increasing content of hydrophilic PHEA backbone. While the enzymatic degradation rate is affected by two competitive factors, the catalytic effect of Pseudomonas cepacia lipase on the degradation of PCL branches and the hydrophilicity which depends on the copolymer composition. In situ observation of the degradation under polarizing light microscope (PLM) demonstrates the different degradation rates of different regions in the polymer samples.     

Lactones 29. Enzymatic resolution of racemic γ-lactones

Studies on the application of commercially available enzymes to resolution of the racemic unsaturated γ-lactones: 5-(3-methylbutylidene)-4-methyl-tetrahydrofuran-2-one (1a) and 5-(3,3-dimethylbutylidene)-4-methyl-tetrahydrofuran-2-one (2a) are presented. Lipase PS, Rhizopus niveus lipase, Rhizopus arrhizus lipase, porcine pancreas lipase and hog liver esterase transformed substrates into their respective γ-keto acids with good efficiency (50–75%). Three of them hydrolysed the studied lactones with moderate enantioselectivity. Enantiomeric excesses determined by GC for the unreacted lactones were in the range of 20–60%. Lipase PS preferentially hydrolysed the (+) enantiomers of lactones 1a and 2a whereas R. niveus lipase hydrolysed the (?) enantiomers, respectively.     

Genetic engineering of aminodeoxyhexose biosynthesis in Streptomyces fradiae

The antibacterial properties of macrolide antibiotics (such as erythromycin, tylosin, and narbomycin) depend ultimately on the glycosylation of otherwise inactive polyketide lactones. Among the sugars commonly found in such macrolides are various 6-deoxyhexoses including the 3-dimethylamino sugars mycaminose and desosamine (4-deoxymycaminose). Some macrolides (such as tylosin) possess multiple sugar moieties, whereas others (such as narbomycin) have only single sugar substituents. As patterns of glycosylation markedly influence a macrolide's drug activity, there is considerable interest in the possibility of using combinatorial biosynthesis to generate new pairings of polyketide lactones with sugars, especially 6-deoxyhexoses. Here, we report a successful attempt to alter the aminodeoxyhexose-biosynthetic capacity of Streptomyces fradiae (a producer of tylosin) by importing genes from the narbomycin producer Streptomyces narbonensis. This engineered S. fradiae produced substantial amounts of two potentially useful macrolides that had not previously been obtained by fermentation.