Enzyme-catalyzed Ring-opening Polymerization Research Articles

Recent Advances in the Enzymatic Synthesis of Polyester.

Polyester is a kind of polymer composed of ester bond-linked polybasic acids and polyol. This type of polymer has a wide range of applications in various industries, such as automotive, furniture, coatings, packaging, and biomedical. The traditional process of synthesizing polyester mainly uses metal catalyst polymerization under high-temperature. This condition may have problems with metal residue and undesired side reactions. As an alternative, enzyme-catalyzed polymerization is evolving rapidly due to the metal-free residue, satisfactory biocompatibility, and mild reaction conditions. This article presented the reaction modes of enzyme-catalyzed ring-opening polymerization and enzyme-catalyzed polycondensation and their combinations, respectively. In addition, the article also summarized how lipase-catalyzed the polymerization of polyester, which includes (i) the distinctive features of lipase, (ii) the lipase-catalyzed polymerization and its mechanism, and (iii) the lipase stability under organic solvent and high-temperature conditions. In addition, this article also focused on the advantages and disadvantages of enzyme-catalyzed polyester synthesis under different solvent systems, including organic solvent systems, solvent-free systems, and green solvent systems. The challenges of enzyme optimization and process equipment innovation for further industrialization of enzyme-catalyzed polyester synthesis were also discussed in this article.

Beyond nylon 6: polyamides via ring opening polymerization of designer lactam monomers for biomedical applications.

Ring opening polymerization (ROP) of lactams is a highly efficient and versatile method to synthesize polyamides. Within the last ten years, significant advances in polymerization methodology and monomer diversity are ushering in a new era of polyamide chemistry. We begin with a discussion of polymerization techniques including the most widely used anionic ring opening polymerization (AROP), and less prevalent cationic ROP and enzyme-catalyzed ROP. Next, we describe new monomers being explored for ROP with increased functionality and stereochemistry. We emphasize the relationships between composition, structure, and properties, and how chemists can control composition and structure to dictate a desired property or performance. Finally, we discuss biomedical applications of the synthesized polyamides, specifically as biomaterials and pharmaceuticals, with examples to include as antimicrobial agents, cell adhesion substrates, and drug delivery scaffolds.

Comparison of Candida antarctica Lipase B Variants for Conversion of ε-Caprolactone in Aqueous Medium-Part 2.

Enzyme-catalyzed ring-opening polymerization of lactones is a method of increasing interest for the synthesis of polyesters. In the present work, we investigated which changes in the structure of Candida antarctica lipase B (CaLB) shift the catalytic equilibrium between esterification and hydrolysis towards polymerization. Therefore, we present two concepts: (i) removing the glycosylation of CaLB to increase the surface hydrophobicity; and (ii) introducing a hydrophobic lid adapted from Pseudomonas cepacia lipase (PsCL) to enhance the interaction of a growing polymer chain to the elongated lid helix. The deglycosylated CaLB (CaLB-degl) was successfully generated by site-saturation mutagenesis of asparagine 74. Furthermore, computational modeling showed that the introduction of a lid helix at position Ala148 was structurally feasible and the geometry of the active site remained intact. Via overlap extension PCR the lid was successfully inserted, and the variant was produced in large scale in Pichia pastoris with glycosylation (CaLB-lid) and without (CaLB-degl-lid). While the lid variants show a minor positive effect on the polymerization activity, CaLB-degl showed a clearly reduced hydrolytic and enhanced polymerization activity. Immobilization in a hydrophobic polyglycidol-based microgel intensified this effect such that a higher polymerization activity was achieved, compared to the “gold standard” Novozym® 435.

Enzymatic Ring-Opening Polymerization (ROP) of Polylactones: Roles of Non-Aqueous Solvents.

Aliphatic polyesters such as polylactides (PLAs) and other polylactones are thermoplastic, renewable and biocompatible polymers with high potentials to replace petro-chemical-based synthetic polymers. A benign route for synthesizing these polyesters is through the enzyme-catalyzed ring-opening polymerization (ROP) reaction; this type of enzymatic process is very sensitive to reaction conditions such as solvents, water content and temperature. This review systematically discusses the crucial roles of different solvents (such as solvent-free or in bulk, organic solvents, supercritical fluids, ionic liquids, and aqueous biphasic systems) on the degree of polymerization and polydispersity. In general, many studies suggest that hydrophobic organic solvents with minimum water contents lead to efficient enzymatic polymerization and subsequently high molecular weights of polyesters; the selection of solvents is also limited by the reaction temperature, e.g. the ROP of lactide is often conducted at above 100 °C, therefore, the solvent typically needs to have its boiling point above this temperature. The use of supercritical fluids could be limited by its scaling-up potential, while ionic liquids have exhibited many advantages include their low-volatility, high thermal stability, controllable enzyme-compatibility, and a wide range of choices. However, the fundamental and mechanistic understanding of the specific roles of ionic liquids in enzymatic ROP reactions is still lacking. Furthermore, the lipase specificity towards l- and d-lactide is also surveyed, followed by the discussion of engineered lipases with improved enantioselectivity and thermal stability. In addition, the preparation of polyester-derived materials such as polyester-grafted cellulose by the enzymatic ROP method is briefly reviewed.

Characterization of Polyesters Obtained by Enzyme-Catalyzed Ring-Opening Polymerization of Pentadecanolide at high Temperature

ABSTRACTDifferences on physicochemical properties of poly(pentadecanolide), PPDL, synthesized by enzymatic ring opening polymerization at two different temperatures, 70 C and 90 C, using Novozyme-430 were assessed. PPDL synthesized at 90°C presents lower molecular weight and crystallinity than the one prepared at 70°C. It was detected by FTIR that PPDL synthesized at 90°C presents a large amorphous phase with more terminal OH groups. A difference in the melting and crystallization behavior was detected by differential scanning calorimetry, DSC, where the melting of the PPDL synthesized at 90°C presents multiple melting and crystallization events at lower temperature than those exhibit by PPDL synthesized at 70°C which presents a well defined single melting and crystallization event. The differences in melting behavior are attributed to the presence of a larger amorphous phase in PPDL synthesized at 90°C due to increased number of terminal OH groups that disrupt the crystalline structure. Thermal stability is also higher in PPDL synthesized at 70°C since the onset of decomposition starts 50°C above that observed in PPDL obtained at 70°C.

Enzymatic polymerization of cyclic monomers in ionic liquids as a prospective synthesis method for polyesters used in drug delivery systems.

Biodegradable or bioresorbable polymers are commonly used in various pharmaceutical fields (e.g., as drug delivery systems, therapeutic systems or macromolecular drug conjugates). Polyesters are an important class of polymers widely utilized in pharmacy due to their biodegradability and biocompatibility features. In recent years, there has been increased interest in enzyme-catalyzed ring-opening polymerization (e-ROP) of cyclic esters as an alternative method of preparation of biodegradable or bioresorbable polymers. Ionic liquids (ILs) have been presented as green solvents in enzymatic ring-opening polymerization. The activity, stability, selectivity of enzymes in ILs and the ability to catalyze polyester synthesis under these conditions are discussed. Overall, the review demonstrates that e-ROP of lactones or lactides could be an effective method for the synthesis of useful biomedical polymers.

Effect of reaction temperature on the physicochemical properties of poly(pentadecanolide) obtained by enzyme-catalyzed ring-opening polymerization

Differences on physicochemical properties of poly(pentadecanolide) (PPDL) synthesized by enzymatic ring-opening polymerization at two different temperatures, 70 and 90 °C, using Novozyme 435 were assessed. PPDL synthesized at 90 °C presents lower molecular weight and crystallinity than the one prepared at 70 °C. It was detected by FTIR that PPDL synthesized at 90 °C presents a large amorphous phase with more terminal OH groups. A difference in the melting and crystallization behavior was detected by differential scanning calorimetry, where the melting of the PPDL synthesized at 90 °C presents multiple melting and crystallization events at lower temperature than those exhibit by PPDL synthesized at 70 °C which presents a well-defined single melting and crystallization event. The differences in melting and crystallization behavior are attributed to the presence of a larger amorphous phase in PPDL synthesized at 90 °C due to increased number of terminal OH groups that disrupt the crystalline structure. Thermal stability is also higher in PPDL synthesized at 70 °C since the onset of decomposition starts 50 °C above that observed in PPDL obtained at 70 °C.

Enzyme-catalyzed ring-opening polymerization of ε-caprolactone using alkyne functionalized initiators

Well-defined poly(caprolactone)s were generated using alkyne-based initiating systems catalyzed by Candida antarctica Lipase B (CALB). Propargyl alcohol and 4-dibenzocyclooctynol (DIBO) were shown to efficiently initiate the polymerizations under metal free conditions and yielded 4 to 24 KDa polymers with relatively narrow molecular mass distribution. The alkyne bonds in the strained and unstrained initiating systems survived the polymerization conditions and the purification process. The resulting bonds can be sequentially functionalized post-polymerization using metal free and copper catalyzed click chemistry conditions.

Toward one-pot lipase-catalyzed synthesis of poly(ɛ-caprolactone) particles in aqueous dispersion

The preparation of polyester particles using enzyme-catalyzed (lipase from Candida antarctica B, CALB) ring-opening polymerization of ɛ-caprolactone (ɛ-CL) in aqueous dispersion was demonstrated for the first time. Immobilization of CALB enabled a significant increase of the number–average degree of polymerization of ɛ-CL oligomers (up to 38) as compared to dissolved CALB (8 at the maximum). The nature and amount of lipase, as well as the nature of the support material were identified as key parameters controlling ring-opening polymerization of ɛ-CL in aqueous dispersion. In addition, the involvement of solubilized monomers in polymerization elementary reactions was demonstrated and the consequences on oligomers average length were detailed. An overall mechanism of lipase-catalyzed ɛ-CL polymerization in aqueous dispersion taking into account the colloidal nature of reaction medium was proposed on the basis of experimental results.

H-형태 양친매성 펜타블록 공중합체의 화학효소적 합성과 자기회합거동 평가

H-shaped amphiphilic pentablock copolymers (PSt)(2)-b-PCL-b-PEO-b-PCL-b-(PSt)2 was synthesized via chemoenzymatic method by combining enzyme-catalyzed ring-opening polymerization (eROP) of epsilon-caprolactone (epsilon-CL) and atom transfer radical polymerization (ATRP) of styrene. By this process, we obtained copolymers with controlled molecular weight and low polydispersity. The structure and composition of the obtained copolymers were characterized by nuclear magnetic resonance (NMR), gel permeation chromatography (GPC) and infrared spectroscopy analysis (IR). The crystallization behavior of the copolymers was analyzed by differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The crystallization behavior of the H-shaped block copolymers-demonstrated a PCL dominate crystallization. The self-assembly behavior of the copolymers was investigated in aqueous media. The hydrodynamic diameters of the copolymer micelles in aqueous solution were measured by dynamic light scattering (DLS). The morphology of the copolymer micelles was observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The hydrodynamic diameters of spherical micelles declined gradually with the increase of the hydrophobic chain lengths of the copolymers. The critical micelle concentration (CMC) values were determined from fluorescence emission, and it was found that the CMCs decreased with an increase of PSt hydrophobic block lengths.

High-pressure phase equilibrium data for the l-lactic acid + (propane + ethanol) and the l-lactic acid + (carbon dioxide + ethanol) systems

Biodegradable polymers have received increased attention due to their potential applications in the medicine and food industries; in particular, poly(l-lactic acid) (PLA) is of primary importance because of its biocompatibility and resorbable features. Recently, the synthesis of this biopolymer through the enzyme-catalyzed ring-opening polymerization of l-lactic acid in a compressed fluid has been considered promising. The aim of this work was to report the phase equilibrium data (cloud points) of the l-lactic acid+(propane+ethanol) and the l-lactic acid+(carbon dioxide+ethanol) systems. The phase equilibrium experiments were conducted in a variable-volume view cell employing the static synthetic method. These experiments were conducted in the temperature range of 323.15–353.15K and at pressures up to 25MPa; the mass ratio of ethanol to either CO2 or propane was maintained at 1:9. The l-lactic acid+(propane+ethanol) system exhibited vapor–liquid, liquid–liquid and vapor–liquid–liquid transitions, whereas the l-lactic acid+(carbon dioxide+ethanol) system only exhibited liquid–liquid type transitions.

Increasing Molecular Mass in Enzymatic Lactone Polymerizations.

Using a model developed for the enzyme-catalyzed polymerization and degradation of poly(caprolactone), we illustrate a method and the kinetic mechanisms necessary to improve molecular mass by manipulating equilibrium reactions in the kinetic pathway. For these polymerization/degradation reactions, a water/linear chain equilibrium controls the number of chains in solution. Here, we control the equilibrium by adding water-trapping molecular sieves in the batch polymerization reactions of ε-caprolactone. While ring-opening rates were mostly unaffected, the molecular mass shifted to higher molecular masses after complete conversion was reached, and a good agreement between the experimental and modeling results was found. These results provide a framework to improve the molecular mass for enzyme-catalyzed ring-opening polymerization of lactone.

Continuous Flow Enzyme-Catalyzed Polymerization in a Microreactor

Enzymes immobilized on solid supports are increasingly used for greener, more sustainable chemical transformation processes. Here, we used microreactors to study enzyme-catalyzed ring-opening polymerization of ε-caprolactone to polycaprolactone. A novel microreactor design enabled us to perform these heterogeneous reactions in continuous mode, in organic media, and at elevated temperatures. Using microreactors, we achieved faster polymerization and higher molecular mass compared to using batch reactors. While this study focused on polymerization reactions, it is evident that similar microreactor based platforms can readily be extended to other enzyme-based systems, for example, high-throughput screening of new enzymes and to precision measurements of new processes where continuous flow mode is preferred. This is the first reported demonstration of a solid supported enzyme-catalyzed polymerization reaction in continuous mode.

Atomistic Model for the Polyamide Formation from β-Lactam Catalyzed by Candida antarctica Lipase B

Candida antarctica lipase B (CALB) is an established biocatalyst for a variety of transesterification, amidation, and polymerization reactions. In contrast to polyesters, polyamides are not yet generally accessible via enzymatic polymerization. In this regard, an enzyme-catalyzed ring-opening polymerization of β-lactam (2-azetidinone) using CALB is the first example of an enzymatic polyamide formation yielding unbranched poly(β-alanine), nylon 3. The performance of this polymerization, however, is poor, considering the maximum chain length of 18 monomer units with an average length of 8, and the molecular basis of the reaction so far is not understood. We have employed molecular modeling techniques using docking tools, molecular dynamics, and QM/MM procedures to gain insight into the mechanistic details of the various reaction steps involved. As a result, we propose a catalytic cycle for the oligomerization of β-lactam that rationalizes the activation of the monomer, the chain elongation by additional β-lactam molecules, and the termination of the polymer chain. In addition, the processes leading to a premature chain termination are studied. Particularly, the QM/MM calculation enables an atomistic description of all eight steps involved in the catalytic cycle, which features an in situ-generated β-alanine as the elongating monomer and which is compatible with the experimental findings.

The environmental influence in enzymatic polymerization of aliphatic polyesters in bulk and aqueous mini-emulsion

The catalytic effect of enzymes in different environments has been compared. Biodegradable polyesters and corresponding nanoparticles have been synthesized by an “eco-friendly” technique; enzyme-catalyzed ring-opening polymerization of lactones in bulk and in an aqueous mini-emulsion. Lipases from Burkholderia cepacia (lipase PS), B. cepacia immobilized on ceramic, Pseudomonas fluorescens and Candida Antarctica have been used as catalysts in the polymerization of l-Lactide (LLA), pentadecanolide (PDL) and hexadecanolide (HDL). The reaction conditions during the bulk polymerization of LLA were varied by adding different amounts of ethylene glycol at 100 °C or 125 °C. A number average molecular weight (Mn) of 78,100 was obtained when lipase PS was used at 125 °C. Lipase PS had a high catalytic activity in an aqueous environment with 100% conversion in 4 h, and the nanoparticles obtained from mini-emulsion polymerization were between 113 and 534 nm in size. The amount of hydrophobe affected the size of the PDL nanoparticles produced, less than the amount of surfactant in both systems.

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.

Enzyme-Catalyzed Ring-Opening Polymerization of Seven-Membered Ring Lactones Leading to Terminal-Functionalized and Triblock Polyesters

Terminal-functionalized polyesters and triblock polyesters were synthesized by lipase-CA-catalyzed ring-opening polymerization of seven-membered ring lactones, i.e., 1,5-dioxepan-2-one (DXO) and ε-caprolactone (CL), in the bulk in the presence of an appropriate alcohol that acts as an initiator. To introduce a double bond at the chain end, 4-pentene-2-ol was used to initiate the polymerization of the lactones. The unsaturation introduced at the chain end in this way is a useful approach for synthesizing comb polymers. Two different dihydroxyl compounds, viz. poly(caprolactone diol) and poly(ethylene glycol), were used as macro-initiators. Triblock copolymers were synthesized in this way, where the macro-initiator formed the middle block. Polymers having different degrees of polymerization were synthesized by varying the molar feed ratio of monomer to initiator. DXO and CL showed significant differences in reactivity toward lipase-CA-catalyzed polymerization initiated by different alcohols as initiators. The polymers were characterized by FTIR, NMR, SEC, optical microscopy, and DSC techniques.

Immobilized lipase on porous silica beads: preparation and application for enzymatic ring-opening polymerization of cyclic phosphate

Porcine pancreas lipase (PPL) immobilized on porous silica beads and employed successfully for ring-opening polymerization of ethylene isobutyl phosphate (EIBP) was investigated. The factors affecting the activity of the immobilized lipase were studied. A good coupling yield was achieved — 41.88 mg of native lipase/g of porous silica beads. We also studied the thermal properties of this immobilized lipase. The optimum temperature of this immobilized lipase was 70°C. After incubation at 90°C for 1 h, the activity of immobilized lipase remained above 70%. An enzyme-catalyzed ring-opening polymerization was achieved in bulk with Mn values ranging from 1642 to 5783 g/mol. It was found that recovered immobilized lipase was more active for the polymerization of EIBP than in the first use and Mn was significantly increased. The higher molecular weight can be gained with the propriety amount of immobilized lipase and a higher temperature.

Enzyme-catalyzed Ring-opening Polymerization of β-Butyrolactone Using PHB Depolymerase

Abstract Poly(3-hydroxybutyrate) [P(3HB)] was prepared by the ring-opening polymerization of (R,S)-β-butyrolactone (BL) using two types of PHB depolymerase with or without substrate-binding domains (SBD) as the catalyst. The SBD lacking PHB depolymerase exhibited better catalytic activities for the ring-opening polymerization of BL.

Synthesis of metal-free poly(1,4-dioxan-2-one) by enzyme-catalyzed ring-opening polymerization

To avoid the harmful effects of metallic residues in poly(1,4-dioxan-2-one) (PPDO) for medical applications, the enzymatic polymerization of 1,4-dioxan-2-one (PDO) was carried out at 60 °C for 15 h with 5 wt % immobilized lipase CA. The lipase CA, derived from Candida antarctica, exhibited especially high catalytic activity. The highest weight-average molecular weight (M w = 41,000) was obtained. The PDO polymerization by the lipase CA occurred because of effective enzyme catalysis. The water component appeared to act not only as a substrate of the initiation process but also as a chain cleavage agent. A slight amount of water enhanced the polymerization, but excess water depressed the polymerization. PPDO prepared by enzyme-catalyzed polymerization is a metal-free polyester useful for medical applications.