Polymersome nanoreactors for enzymatic ring-opening polymerization

Polystyrene-polyisocyanopeptide (PS-PIAT) polymersomes containing CALB in two different locations, one in the aqueous inner compartment and one in the bilayer, were investigated for enzymatic ring-opening polymerization of lactones in water. It is shown that the monomers 8-octanolactone and dodecalactone yield oligomers with this polymersome system. It is also observed that the polymerization activity is dependent on the position of the enzyme in the polymersome. SEM investigations show that the polymersome structures were destabilized during the polymerization. Further investigations show that the vesicular morphology of the polymersomes was destabilized only in the case of polymer product formation.     

Modeling enzymatic kinetic pathways for ring-opening lactone polymerization

A unified kinetic pathway for the enzyme-catalyzed polymerization and degradation of poly(ε-caprolactone) was developed. This model tracks the complete distribution of individual chain lengths, both enzyme-bound and in solution, and successfully predicts monomer conversion and the molecular mass distribution as a function of reaction time. As compared to reported experimental data for polymerization reactions, modeled kinetics generate similar trends, with ring-opening rates and water concentration as key factors to controlling molecular mass distributions. Water is critically important by dictating the number of linear chains in solution, shifting the molecular mass distribution at which propagation and degradation equilibrate. For the enzymatic degradation of poly(ε-caprolactone), the final reaction product is also consistent with the equilibrium dictated by the propagation and degradation rates. When the modeling framework described here is used, further experiments can be designed to isolate key reaction steps and provide methods for improving the efficiency of enzyme polymerization.     

Novel biomineralization for hydrogels: electrophoresis approach accelerates hydroxyapatite formation in hydrogels

As a first step toward hydroxyapatite (HAp) formation in agarose hydrogels, we have tailored the internal chemistry using an electrophoresis approach. HAp was formed using aqueous solutions of calcium chloride and disodium hydrophosphate, which were set in a conventional agarose electrophoresis apparatus. Calcium and phosphate ions provided cations and anions, respectively, and were shown to migrate into the agarose hydrogel toward the corresponding electrode side. HAp was formed after colliding with each ion. The time needed to reach complete HAp formation was 30 min, and 130 ng of HAp was formed in 1 mg of agarose hydrogel when the equilibrium swelling state was reached. The electrophoresis approach accelerated the HAp formation, and the linear velocity of 1 mm/min was shown to be roughly 15 times larger than that of simple diffusion (0.06 mm/min).     

Living ring-opening polymerization of a carbohydrate-derived lactone for the synthesis of protein-resistant biomaterials

An efficient living ring-opening polymerization (ROP) of a permethoxylated epsilon-caprolactone [(OMe)CL] catalyzed by yttrium(III) isopropoxide was developed for the synthesis of degradable protein-resistant polymers [P(OMe)CL]. The lactone monomer was efficiently prepared from a reduced sugar, D-dulcitol. Kinetic studies of the ROP revealed a linear dependence of ln[M]0/[M] on polymerization time as well as a linear correlation between the number-averaged molecular weight (M(n)) and monomer conversion; both support it is a living polymerization. A series of block copolymers of our permethoxylated lactone with epsilon-caprolactone [P(OMe)CL-b-PCL] were synthesized and fully characterized. In thermal analyses only single T(g)s were observed in all the block copolymers, suggesting that P(OMe)CL and PCL blocks are fully miscible. Finally, surface plasmon resonance (SPR) sensograms demonstrated that both P(OMe)CL and the P(OMe)CL-b-PCL block copolymers exhibit excellent resistance to fibrinogen and lysozyme.     

Lipase/esterase-catalyzed ring-opening polymerization: A green polyester synthesis technique

In the last decade, there has been increased interest in lipase/esterase-catalyzed ring-opening polymerization as an alternative to metal-based catalytic processes. This review focuses on three components in the reaction system, namely biocatalysts, reaction medium and monomers. Novel lipases or esterases are described with particular emphasis on, those derived from thermophiles, immobilized enzymes and recombinant whole-cell biocatalysts. Green solvents in enzymatic ring-opening polymerization, including water, ionic liquids, supercritical carbon dioxide and hydrofluorocarbon solvents, are also discussed. Enzymatic ring-opening polymerization is reviewed with regard to the variety of polymers obtainable, such as polyesters, polycarbonates, polyphosphates and polythioesters. Among these, enzymatic synthesis of polyesters has been most widely investigated, and is discussed for lactones with small to large ring sizes. Finally, the mechanism of enzymatic ring-opening polymerization is described, which is generally accepted as a monomer-activated mechanism. Overall, the review demonstrates that lipase/esterase-catalyzed synthesis of polymers via ring-opening polymerization provides an effective platform for conducting “green polymer chemistry”.     

A radical approach to enzyme catalysis

Free radicals are generally perceived as highly reactive species which are harmful to biological systems. There are, however, a number of enzymes that use carbon-based radicals to catalyse a variety of important and unusual reactions. The most prominent example is ribonucleotide reductase, an enzyme which is crucial for the synthesis of DNA. In general, radicals are used to remove hydrogen from unreactive positions in the substrate, and in this way the substrate is activated to undergo chemical transformations that would otherwise be difficult to achieve. Several different mechanisms have evolved which allow enzymes to generate and maintain radicals in increasingly aerobic enviroments. An unexpected finding is the existance of stable protein-based radicals, residing on a variety of amino-acid side chains, which serve to link the radical-generating and catalytic sites and to store the radical between turnovers.     

Lipase-catalyzed ring-opening polymerization of lactones to polyesters and its mechanistic aspects.

Lipase catalysis induced a ring-opening polymerization of lactones with different ring-sizes. Small-size (four-membered) and medium-size lactones (six- and seven-membered) as well as macrolides (12-, 13-, 16-, and 17-membered) were subjected to lipase-catalyzed polymerization. The polymerization behaviors depended primarily on the lipase origin and the monomer structure. The macrolides showing much lower anionic polymerizability were enzymatically polymerized faster than epsilon-caprolactone. The granular immobilized lipase derived from Candida antartica showed extremely efficient catalysis in the polymerization of epsilon-caprolactone. Single-step terminal functionalization of the polyester was achieved by initiator and terminator methods. The enzymatic polymerizability of lactones was quantitatively evaluated by Michaelis-Menten kinetics.     

From interfacial ring-opening polymerization to melt processing of cellulose nanowhisker-filled polylactide-based nanocomposites

In the present work, cellulose nanowhiskers (CNWs), extracted from ramie fibers, were incorporated in polylactide (PLA)-based composites. Prior to the blending, PLA chains were chemically grafted on the surface of CNW to enhance the compatibilization between CNW and the hydrophobic polyester matrix. Ring-opening polymerization of l-lactide was initiated from the hydroxyl groups available at the CNW surface to yield CNW-g-PLA nanohybrids. PLA-based nanocomposites were prepared by melt blending to ensure a green concept of the study thereby limiting the use of organic solvents. The influence of PLA-grafted cellulose nanoparticles on the mechanical and thermal properties of the ensuing nanocomposites was deeply investigated. The thermal behavior and mechanical properties of the nanocomposites were determined using differential scanning calorimetry (DSC) and dynamical mechanical and thermal analysis (DMTA), respectively. It was clearly evidenced that the chemical grafting of CNW enhances their compatibility with the polymeric matrix and thus improves the final properties of the nanocomposites. Large modification of the crystalline properties such as the crystallization half-time was evidenced according to the nature of the PLA matrix and the content of nanofillers.     

Functionalized polycarbonate derived from tartaric acid: enzymatic ring-opening polymerization of a seven-membered cyclic carbonate

Enantiomerically pure functional polycarbonate was synthesized from a novel seven-membered cyclic carbonate monomer derived from naturally occurring L-tartaric acid. The monomer was synthesized in three steps and screened for polymerization with four commercially available lipases from different sources at 80 degrees C, in bulk. The ring-opening polymerization (ROP) was affected by the source of the enzyme; the highest number-average molecular weight, M(n) = 15500 g/mol (PDI = 1.7; [alpha]D(20) = +77.8, T(m) = 58.8 degrees C) optically active polycarbonate was obtained with lipase Novozyme-435. The relationship between monomer conversion, reaction time, molecular weight, and molecular weight distribution were investigated for Novozyme-435 catalyzed ROP. Deprotection of the ketal groups was achieved with minimal polymer chain cleavage (M(n) = 10000 g/mol, PDI = 2.0) and resulted in optically pure polycarbonate ([alpha]D(20) = +56) bearing hydroxy functional groups. Deprotected poly(ITC) shows T(m) of 60.2 degrees C and DeltaH(f) = 69.56 J/g and similar to that of the poly(ITC), a glass transition temperature was not found. The availability of the pendant hydroxyl group is expected to enhance the biodegradability of the polymer and serves in a variety of potential biomedical applications such as polymeric drug delivery systems.     

Metal-ion catalyzed polymerization in the eutectic phase in water-ice: a possible approach to template-directed RNA polymerization

The emergence of an RNA world requires among other processes the non-enzymatic, template-directed replication of genetic polymers such as RNA or related nucleic acids, possibly catalyzed by metal-ions. The absence of uridilate derivative polymerization on adenine containing templates has been the main issue preventing an efficient template-directed RNA polymerization. We report here the investigation of template-directed RNA polymerization in the eutectic phase in water–ice. In particular, it was found that activated uridilate monomers in the presence of metal-ion catalysts could efficiently elongate RNA hairpins whose 5′-overhangs served as the templating sequence. The same applies for every other pyrimidine and purine nucleobase. Moreover, the initial elongation rates were always higher in the presence of a template complementary to the nucleotide than in systems without proper base-pairing opportunities. These results suggest that a template-directed RNA polymerization catalyzed by metal-ions could be carried out under eutectic phase in water–ice conditions.     

Solvent protection of the hammerhead ribozyme in the ground state: evidence for a cation-assisted conformational change leading to catalysis

Tertiary folding of the hammerhead ribozyme has been analyzed by hydroxyl radical footprinting. Three hammerhead constructs with distinct noncore sequences, connectivities, and catalytic properties show identical protection patterns, in which conserved core residues (G5, A6, U7, G8, and A9) and the cleavage site (C17, G1.1, and U1.2) become reproducibly protected from nucleolytic attack by radicals. Metal ion titrations show that all protections appear together, suggesting a single folding event to a common tertiary structure, rather than an ensemble of different folds. The apparent binding constants for folding and catalysis by Mg(2+) are lower than those for Li(+) by 3 orders of magnitude, but in each case the protected sites are identical. For both Mg(2+) and Li(+), the ribozyme folds into the protected tertiary structure at significantly lower cation concentrations than those required for cleavage. The sites of protection include all of the sites of reduced solvent accessibility calculated from two different crystal structures, including both core and noncore nucleotides. In addition, experimentally observed protected sites include additional sequences adjacent to those predicted by the crystal structures, suggesting that the solution structure may be folded into a more compact shape. A 2'-deoxy substitution at G5 abolishes all protection, indicating that the 2'-OH is essential for folding. Together, these results support a model in which low concentrations of metal ions fold the ribozyme into a stable ground state tertiary structure that is similar to the crystallographic structures, and higher concentrations of metal ions support a transient conformational change into the transition state for catalysis. These data do not themselves address the issue as to whether a large- or small-scale conformational change is required for catalysis.     

Observation of a hybrid random ping-pong mechanism of catalysis for NodST: a mass spectrometry approach

An efficient enzyme kinetics assay using electrospray ionization mass spectrometry (ESI-MS) was initially applied to the catalytic mechanism investigation of a carbohydrate sulfotransferase, NodST. Herein, the recombinant NodST was overexpressed with a His(6)-tag and purified via Ni-NTA metal-affinity chromatography. In this bisubstrate enzymatic system, an internal standard similar in structure and ionization efficiency to the product was chosen in the ESI-MS assay, and a single point normalization factor was determined and used to quantify the product concentration. The catalytic mechanism of NodST was rapidly determined by fitting the MS kinetic data into a nonlinear regression analysis program. The initial rate kinetics analysis and product inhibition study described support a hybrid double-displacement, two-site ping-pong mechanism of NodST with formation of a sulfated NodST intermediate. This covalent intermediate was further isolated and detected via trypsin digestion and Fourier transform ion cyclotron resonance mass spectrometry. To our knowledge, these are the first mechanistic data reported for the bacterial sulfotransferase, NodST, which demonstrated the power of mass spectrometry in elucidating the reaction pathway and catalytic mechanism of promising enzymatic systems.     

The utility of a 3-O-allyl group as a protective group for ring-opening polymerization of alpha-D-glucopyranose 1,2,4-orthopivalate derivatives

To clarify the utility as a protective group of 3-O-allyl group on ring-opening polymerization of alpha-D-glucopyranose 1,2,4-orthopivalate derivatives, four orthopivalate derivatives, 3-O-allyl-6-O-pivaloyl- (1), 3-O-allyl-6-O-benzyl- (2), 3,6-di-O-allyl- (3), and 3-O-allyl-6-O-methyl-alpha-D-glucopyranose 1,2,4-orthopivalates (4), were selected as starting monomers and were polymerized under -30 degrees C in CH2Cl2 using BF3.Et2O as a catalyst. All the orthopivalate derivatives 1-4 were found to give stereoregular polysaccharides, (1-->4)-beta-D-glucopyranans. Thus, it was concluded that the allyl group as a protective group at 3-O position of glucose othropivalate is acceptable to yield stereoregular (1-->4)-beta-D-glucopyranans, cellulose derivatives.     

Amino-acid containing metallomonomers copolymerized into porous organic polymers: applicability to allylic alkylation catalysis

This paper reports the synthesis of a polymerizable dppe-derivative with eight pendent styrenyl units linked to the dppe core through four tyrosine residues. This diphosphine ligand was utilized in the synthesis of a series of P₂PdX₂ complexes (X₂=(R)-BINOL, (S)-BINOL, Cl₂ and π-1,3-Ph₂-allyl⁺). The compounds were used as comonomers for the synthesis of porous organic polymers (poly EDMA). Molecular imprinting effects on X₂-ligand removal were investigated. Overall, the presence of a cavity of significant size was found to be beneficial to the rate of the allylic alkylation reaction, however, chiral BINOL shaped cavities did not influence the enantio-selectivity of the reaction.     

Synthesis of D-xylopyranan by the ring-opening polymerization of 3-O-benzyl-alpha-D-xylopyranose 1,2,4-orthopivalate. Attempts to synthesize a stereoregular polymer.

3-O-Benzyl-alpha-D-xylopyranose 1,2,4-orthopivalate (1) was newly synthesized and polymerized under cationic polymerization reaction conditions in order to synthesize stereoregular (1-->4)-beta-D-xylopyranan. Although the polymerization of orthopivalate 1 was carried out under various reaction conditions, a non-stereoregular polymer, but mainly consisting of (1-->4)-beta-xylopyranose units, was obtained. Comparing these results with those of glucose 1,2,4-orthopivalates, it was revealed that not only the substituents in the C-2 and C-3 positions, but also the CH(2)OR group in glucose 1,2,4-orthopivalate, largely contribute to (1-->4)-beta-glucosidic bond formation by the ring-opening polymerization.     

The design of organic catalysis for epoxidation by hydrogen peroxide

The potential of various organic species to catalyze epoxidation of ethene by hydrogen peroxide is explored with B3LYP/6-31G* DFT calculations. Electronic Supplementary Materials Supplementary material is available for this article at http://dx.doi.org/10.1007/s00894-005-0044-4.Dedicated to Professor Dr. Paul von Ragué Schleyer on the occasion of his 75th birthday     

Continuous screening system for inhibited enzyme catalysis: A membrane reactor approach

The screening of catalysts, substrates or conditions in the early stages of bioprocess development requires an enormous number of experiments and is a tedious, expensive and time-consuming task. Currently available screening systems can only be operated in batch or fed-batch mode, which can lead to severe misinterpretations of screening results. For example, catalysts that are inhibited by substrates or accumulating products will be excluded from further investigations in the early stages of process development despite the fact that they might be superior to other candidates in a different operational mode. Important and advantageous properties such as turnover stability can also be overshadowed by product inhibition. The aim of this study was to develop a novel screening system that enables continuous feeding of substrates and continuous removal of products. A prototype based on the membrane reactor concept was designed and operated for a model reaction, the hydrolysis of cellulose.     

Preparation of highly active alpha-chymotrypsin for catalysis in organic media

A simple one step process for the preparation of free alpha-chymotrypsin, using an organic solvent to precipitate the enzyme from a buffered solution, followed by washing with organic solvents, is described. This preparation gave 132 times greater esterification activity than lyophilized powder.     

Quantitative and predictive correlations for peroxidase catalysis in organic media

Summary Quantitative and predictive relationships have been developed for horseradish peroxidase catalysis in both aqueous and organic media. These relationships take into account the physicochemical characteristics of both substrate (e.g., hydrophobicity and electronic characteristics) and solvent (e.g., hydrophobicity and polarity). The results suggest that solvent effects on hydrophobic interactions within proteins are secondary to effects on electrostatic forces.