Regiocontrol of the Bulk Polymerization of Lysine Ethyl Ester by the Selection of Suitable Immobilized Enzyme Catalysts.

The development of a green and facile method for the controlled synthesis of functional polypeptides is desired for sustainable material applications. In this study, the regioselective synthesis of poly(l-lysine) (polyLys) via enzyme-catalyzed aminolysis was achieved by bulk polymerization of l-lysine ethyl ester (Lys-OEt) using immobilized Candida antarctica lipase Novozym 435 (IM-lipase) or trypsin (IM-trypsin). Structural characterization of the obtained polyLys revealed that IM-lipase resulted solely in ε-linked amide bond formation, whereas IM-trypsin predominantly provided α-linked polyLys. Optimization of the conditions for the bulk polymerization using immobilized enzymes resulted in high monomer conversion and a high degree of polymerization, with excellent regioselectivity. Molecular docking simulations revealed different binding conformations of Lys-OEt to the catalytic pockets of lipase and trypsin, which putatively resulted in different amino moieties being used for amide bond formation. The immobilized enzymes were recovered and recycled for bulk polymerization, and the initial activity was maintained in the case of IM-trypsin. The obtained α- and ε-linked polyLys products exhibited different degradability against proteolysis, demonstrating the possibility of versatile applications as sustainable materials. This enzymatic regioregular control enabled the synthesis of well-defined polypeptide-based materials with a diverging structural variety.

High strength, epoxy cross-linked high sulfur content polymers from one-step reactive compatibilization inverse vulcanization.

Inverse vulcanization provides a simple, solvent-free method for the preparation of high sulfur content polymers using elemental sulfur, a byproduct of refining processes, as feedstock. Despite the successful demonstration of sulfur polymers from inverse vulcanization in optical, electrochemical, and self-healing applications, the mechanical properties of these materials have remained limited. We herein report a one-step inverse vulcanization using allyl glycidyl ether, a heterobifunctional comonomer. The copolymerization, which proceeds via reactive compatibilization, gives an epoxy cross-linked sulfur polymer in a single step, as demonstrated through isothermal kinetic experiments and solid-state 13C NMR spectroscopy. The resulting high sulfur content (≥50 wt%) polymers exhibited tensile strength at break in the range of 10-60 MPa (70-50 wt% sulfur), which represents an unprecedentedly high strength for high sulfur content polymers from vulcanization. The resulting high sulfur content copolymer also exhibited extraordinary shape memory behavior along with shape reprogrammability attributed to facile polysulfide bond rearrangement.

Chemoenzymatic Polymerization of l-Serine Ethyl Ester in Aqueous Media without Side-Group Protection.

Poly(l-serine) (polySer) has tremendous potential as a polypeptide-based functional material due to the utility of the hydroxyl group on its side chain; however, tedious protection/deprotection of the hydroxyl groups is required for its synthesis. In this study, polySer was synthesized by the chemoenzymatic polymerization (CEP) of l-serine ethyl ester (Ser-OEt) or l-serine methyl ester (Ser-OMe) using papain as a catalyst in an aqueous medium. The CEP of Ser-OEt proceeded at basic pH ranging from 7.5 to 9.5 and resulted in the maximum precipitate yield of polySer at an optimized pH of 8.5. A series of peaks detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed that the formed precipitate consisted of polySer with a degree of polymerization ranging from 5 to 22. Moreover, infrared spectroscopy, circular dichroism spectroscopy, and synchrotron wide-angle X-ray diffraction measurements indicated that the obtained polySer formed a ß-sheet/strand structure. This is the first time the synthesis of polySer was realized by CEP in aqueous solution without protecting the hydroxyl group of the Ser monomer.

Simultaneous characterization of poly(acrylic acid) and polysaccharide polymers and copolymers.

Copolymer products that result from grafting acrylic acid and other hydrophilic monomers onto polysaccharides have recently gained significant interest in research and industry. Originating from renewable sources, these biodegradable, low toxicity, and polar copolymer products exhibit potential to replace polymers from fossil sources in several applications and industries. The methods usually employed to characterize these copolymers are, however, quite limited, especially for the measurement of bulk properties. With more sophisticated applications, for example, in pharmaceutics requiring a more detailed analysis of the chemical structure, we describe a new approach for this kind of complex polymers. Our approach utilizes chromatography in combination with several detection methods to separate and characterize reaction products of the copolymerization of acrylic acid and chemically hydrolyzed starch. These samples consisted of a mixture of homopolymer poly (acrylic acid), homopolymer hydrolyzed starch, and - in a lower amount - the formed copolymers. Several chromatographic methods exist that are capable of characterizing either poly (acrylic acid) or hydrolyzed starch. In contrast, our approach offers simultaneous characterization of both polymers. The combination of LC and UV/RI offered insight into the composition and copolymer content of the samples. Size exclusion chromatography experiments revealed the molar mass distribution of homopolymers and copolymers. FTIR investigations confirmed the formation of copolymers while ESI-MS gave more details on the end groups of hydrolyzed starches and poly (acrylic acids). Evidence of copolymer structures was obtained through NMR measurements. Finally, two-dimensional chromatography led to the separation of the copolymers from both homopolymers as well as the additional separation of sodium clusters. The methods described in this work are a powerful toolset to characterize copolymerization products of hydrolyzed starch and poly(acrylic acid). Together, our approach successfully correlates the physicochemical properties of such complex mixtures with their actual composition.

Polymer-based monolithic column with incorporated chiral metal-organic framework for enantioseparation of methyl phenyl sulfoxide using nano-liquid chromatography.

A new approach to the preparation of enantioselective porous polymer monolithic columns with incorporated chiral metal-organic framework for nano-liquid chromatography has been developed. While no enantioseparation was achieved with monolithic poly(4-vinylpyridine-co-ethylene dimethacrylate) column, excellent separations of both enantiomers of (±)-methyl phenyl sulfoxide were achieved with its counterpart prepared after admixing metal-organic framework [Zn2 (benzene dicarboxylate)(l-lactic acid)(dmf)], which is synthesized from zinc nitrate, l-lactic acid, and benzene dicarboxylic acid in the polymerization mixture. These novel monolithic columns combined selectivity of the chiral framework with the excellent hydrodynamic properties of polymer monoliths, may provide a great impact on future studies in the field of chiral analysis by liquid chromatography.

Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides.

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous polymer monoliths. The developed approach is based on the preparation of a macroporous polymer containing carboxylic acid functional groups and the subsequent step-by-step solution-based controlled growth of a layer of a porous coordination polymer on the surface of the pores of the polymer monolith. The prepared metal-organic polymer hybrid has a high specific micropore surface area. The amount of iron(III) sites is enhanced through metal-organic coordination on the surface of the pores of the functional polymer support. The increase of metal sites is related to the number of iterations of the coating process. The developed preparation scheme is easily adapted to a capillary column format. The functional porous polymer is prepared as a self-contained single-block porous monolith within the capillary, yielding a flow-through separation device with excellent flow permeability and modest back-pressure. The metal-organic polymer hybrid column showed excellent performance for the enrichment of phosphopeptides from digested proteins and their subsequent detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presented experimental protocol is highly versatile, and can be easily implemented to different organic polymer supports and coatings with a plethora of porous coordination polymers and metal-organic frameworks for multiple purification and/or separation applications.

Application of polymeric macroporous supports for temperature-responsive chromatography of pharmaceuticals.

A macroporous particulate support prepared previously by reactive gelation under shear and functionalized with poly(N-isopropylacrylamide), PNIPAM, brushes of variable length is applied for temperature-responsive chromatography, whereby temperature modulates hydrophobic interactions. Several different analytes, including small pharmaceuticals, peptides, proteins and monoclonal antibodies are employed. Contrary to the most commonly observed behavior in conventional chromatography, increasing retention is observed at elevated temperatures. Peak broadening is quantified using the peak standard deviation, which depends on both the polymer chain conformation and analyte adsorptivity. The favorable effect of grafted polymer thickness on retention becomes progressively less pronounced for thicker grafted PNIPAM layers. The effect of eluent composition on solute-sorbent interactions was investigated by introducing NaCl, methanol, dioxane and by varying the pH. Salt or organic solvent addition affects apart from the analytes solution properties, the hydrophobicity of the stationary phase itself. Frontal analyses performed at different temperatures to determine dynamic binding capacities, indicate small mass transfer resistances imposed by this novel packing material.

Perfusive ion-exchange chromatographic materials with high capacity.

In this work, novel macro-porous chromatographic stationary phases, combining low mass transfer resistance and high binding capacity, were thoroughly characterized in terms of porosity, HETP, resolution and binding capacity. These new stationary phases exhibited better performance compared to commercially available materials, i.e. Poros 50HS and Fractogel EMD SO3 (M). With the technique of reactive gelation under shear, it is possible to produce particles with pores from 100 nm to several microns, in which part of the flow can go through. This way, the mass transport inside the particles is significantly increased with perfusive flow faction values between 0.02 and 0.01. Despite the low pore surface area resulting from the large pore size, high binding capacity is obtained by functionalizing the pore surface with charged polymeric brushes resulting in a binding capacity in the range from 25 to 140 mg/mL col. This, together with the high mass transfer, gives excellent resolution performance and dynamic binding capacity compared to other commercial materials even at high flow rates.

Macroporous polymer particles via reactive gelation under shear: effect of primary particle properties and operating parameters.

Reactive gelation under shear is a recently developed procedure toward rigid macroporous polymeric particles that does not require the use of any porogen. A comprehensive study of the effect of individual parameters on the resulting material characteristics is presented. Primary particle properties are found to be pivotal, namely, the primary particles size, cross-linking degree, and outer shell composition. Operating parameters also play a significant role; specifically, the effects of applied shear rate, salt feeding rate, swelling degree of primary particles, waiting time before postpolymerization, and postpolymerization temperature are investigated. By varying the operating conditions, the size, internal structure, as well as porosity of the fabricated microclusters may be controlled. The pores are invariably micrometer-sized, with pore size distributions exhibiting adjustable maxima. Thanks to the sequential character of the procedure, different parameters may be tuned individually at different stages along the preparation route, allowing thus for high versatility in the control of different properties of the final material.

Synthesis of macroporous polymer particles using reactive gelation under shear.

By combining elements from colloidal and polymer reaction engineering a new approach toward macroporous, mechanically robust polymer particles is presented, which does not require any porogenic additives. Specifically, aggregation and breakage in turbulent conditions of aggregates originating from fully destabilized primary latex particles is applied to produce compact, micrometer-sized clusters. Post-polymerization of monomer introduced initially to swell the primary particles is imparting mechanical rigidity and permanence to the internal structure. The resulting microclusters exhibit an internal porosity on the order of 70% and relatively broad pore size distribution, with exceptionally large pores, ranging from about 50 nm to 10 µm in diameter. These particulate microclusters, produced via reactive gelation under shear, are fractal objects with fractal dimension around 2.7, as opposed to the more open fractal structure of a monolith produced via stagnant reactive gelation, with fractal dimension of 1.9. Such macroporous particles are thought to be useful in applications requiring pores on the micrometer scale, e.g., in the chromatography of biomolecules or for packing beds perfusive to convective flow.

Shear-induced gelation of soft strawberry-like particles in the presence of polymeric P(BA-b-AA) surfactants.

The effect of polymeric surfactants, copolymers of n-butyl acrylate (BA) and acrylic acid (AA), on shear-induced gelation of a colloidal system in the absence of additional electrolytes is studied. One random (PBA-co-PAA) and two block copolymer (PBA-b-PAA, with different PAA lengths) surfactants have been synthesized by ATRP and used in this work. The colloidal system is composed of strawberry-like particles with a rubbery core, partially covered by a few grafted plastic patches. In the absence of any surfactant, as the colloidal system passes through a microchannel at a Peclet number of 220 and a particle volume fraction of 0.15, shear-induced gelation occurs and the particles coalesce partially, due to the rubbery core, leading to a fractal dimension of the clusters constituting the gel equal to 2.78. On the other hand, in the presence of any of the three polymeric surfactants, shear-induced gelation occurs only in the range of low surfactant surface density. Meanwhile, the fractal dimension of the clusters decreases with adsorption of the two block surfactants, reaching a plateau value of about 2.58, while for the random surfactant it remains constant and equal to 2.78, like in the absence of any surfactant. This indicates that adsorption of the block surfactants can reduce the particle coalescence, while adsorption of the random surfactant cannot. Moreover, for all three surfactants, as their surface density increases progressively, a transition from solid-like gel to a liquid-like state occurs and finally no shear-induced gelation or even aggregation occurs. Since the three surfactants comprise carboxylic groups, considering also the results in the literature (Zaccone et al., J. Phys. Chem. B, 2008, 112, 1976; 6793), we can reach a general conclusion that carboxylic groups on the particle surface not only stabilize the particles through electrostatic repulsion, but also generate very short-range, strongly repulsive (e.g. hydration, steric) forces, which when high enough protect the particles from intense shear-activated aggregation.

Quantification of a single aggregate inner porosity and pore accessibility using hard X-ray phase-contrast nanotomography.

The 3D structure of three individual aggregates composed of 165 nm polystyrene primary particles is revealed nondestructively by hard X-ray phase-contrast synchrotron nanotomography. Three-dimensional image analysis allows us for the first time to obtain the complex inner porosity of the entire aggregate. It is demonstrated that despite their rather compact structure, characterized by a fractal dimension equal to 2.7, the produced aggregates are still porous, with porosity increasing with its size. Generated pores have diameters from 100 nm to 3 µm and are almost completely interconnected.