Optimized polymer-enzyme electrostatic interactions significantly improve penicillin G amidase efficiency in charged PEGA polymers

Hydrolytic yields as high as 80% were obtained by using penicillin G amidase (PGA) on substrates anchored on optimized positively charged PEGA polymers. By increasing the amount of permanent charges inside the polymer, electrostatic interactions between the positively charged PEGA⁺ and the negatively charged PGA (pI=5.2-5.4) were strengthened, thus favouring the accessibility of the bulky enzyme (MW=88 kDa) inside the pores. The effect of different amounts of charges on polymer swelling and protein retention inside the polymer was investigated and correlated to the enzyme efficiency demonstrating that electrostatic interactions predominate over swelling properties in determining enzyme accessibility.     

Significant enhancement of crystallization kinetics of polylactide in its immiscible blends through an interfacial effect from comb-like grafted side chains

A significant enhancement in isothermal crystallization kinetics of biodegradable polylactide (PLA) in its immiscible blends can be accomplished through blending it with a comb-like copolymer. PLA was blended with poly(ethylene glycol) methyl ether acrylate (PEGA) and poly[poly(ethylene glycol) methyl ether acrylate] (PPEGA, a comb-like copolymer), respectively. The results measured from phase contrast optical microscopy (PCOM) and differential scanning calorimetry (DSC) indicate that PLA and PEGA components are miscible, whereas PLA and PPEGA components are immiscible. The study of crystallization kinetics for PLA/PEGA and PLA/PPEGA blends by means of polarized optical microscopy (POM) and DSC indicates that both PEGA and PPEGA significantly increase the PLA spherulitic growth rates, G, although PLA/PPEGA blends are immiscible and the glass transition temperatures of PLA only have slight decreases. PPEGA component enhances nucleation for PLA crystallization as compared with PEGA component owing to the heterogeneous nucleation effect of PPEGA at the low composition of 20 wt%, while PLA crystallization-induced phase separation for PLA/PEGA blend might cause further nucleation at the high composition of 50 wt%. DSC measurement further demonstrates that isothermal crystallization kinetics can be relatively more enhanced for PLA/PPEGA blends than for PLA/PEGA blends. The “abnormal” enhancement in G for PLA in its immiscible blends can be explained by local interfacial interactions through the densely grafted PEGA side chains in the comb-like PPEGA, even though the whole blend system (PLA/PPEGA blends) represents an immiscible one.     

Behaviour of cationic polyelectrolytes upon binding of electrolytes: effects of polycation structure, counterions and nature of the solvent

 The effects of polycation structure, counterions and the nature of the solvent on the interaction between low-molecular-weight salts with some cationic polyelectrolytes in water and methanol were investigated. The polyelectrolytes used in this study were cationic polymers with quaternary nitrogen atoms in the backbone with or without a nonpolar side chain (polymer type PCA₅H₁, PCA₅D₁ and PCA₅) or tertiary amine nitrogen atoms in the main chain (polymer type PEGA). LiCl, NaCl, KCl, NaBr, NaI and Na₂SO₄ were used as low-molecular-weight salts. The interaction between polycations and salts was followed by viscometric and conductometric measurements. The study of the interaction of monovalent counterions with cationic polyelectrolytes emphasized an increase in the interaction with the decrease in the radius of the hydrated counterion, both for strong polycations and for weak polycations, suggesting that counterion binding is nonspecific. In the case of SO²⁻ ₄ anions, the Λ_m−c ^1/2 curve passes through a minimum at c _p values between 1 × 10⁻³ and 3 × 10⁻³ unit mol/l; this phenomenon can be explained by the maximum counterion interaction owing to the capacity of the polyvalent counterion to bind two charged groups by intra- or interchain bridges. The investigation of the influence of the polycation structure on the counterion binding indicated an increase in charged group–counterion interactions with a decrease in the nonpolar chain length and an increase in the quaternary ammonium salt group content (charge density) in the chain. The polyelectrolyte with tertiary amine groups in the chain, PEGA, showed, on one hand, a cation adsorption order as K⁺>Na⁺>Li⁺ and, on the other hand, a stronger association between ions and PEGA chains in methanol than in water owing to the poorer solvating effect of methanol on the cations. Received: 20 February 2001 Accepted: 29 June 2001     

丙烯酸聚乙二醇酯-甲基丙烯酸共聚物对CaCO_3微粒形貌的调控

研究了两种聚合物-丙烯酸聚乙二醇酯（PEGA）和丙烯酸聚乙二醇酯-甲基丙烯 酸共聚物（PEGA-PMAA）对CaCO_3粒子生长的调控。发现PEGA调控下得到CaCO_3片 层状粒子的粒径比在纯水中的小，而PEGA-PMAA调控下的粒子粒径较大，并且长成 了奇特的花状形貌。这说明PEGA在CaCO_3粒子生长过程中起了阻碍作用，而双亲水 的PEGA-PMAA则促进了CaCO_3粒子的生长。     

Conjugation of siRNA with Comb‐Type PEG Enhances Serum Stability and Gene Silencing Efficiency

A thiol‐modified siRNA targeting the enhanced green fluorescence protein (eGFP) gene was conjugated with RAFT‐synthesized, pyridyl disulfide‐functional poly(PEG methyl ether acrylate)s (p(PEGA)s). siRNA‐p(PEGA) conjugates demonstrated significantly enhanced in vitro serum stability and nuclease resistance compared to the unmodified and thiol‐modified siRNA. The complexes of siRNA‐p(PEGA) conjugates with a fusogenic peptide, KALA ((+)/(–) = 2) inhibited the protein expression approximately 28‐fold more than the KALA complex of the unmodified siRNA. The protein inhibition caused by siRNA‐p(PEGA)‐KALA complexes (56 ± 5%–58 ± 3% of the fluorescence expressed in non‐treated cells) was comparable to the effect of the unmodified siRNA‐lipofectamine complex (77 ± 7%).

     

Polymeric cation-exchange monolithic columns containing phosphoric acid functional groups for capillary liquid chromatography of peptides and proteins

Two different monoliths, both containing phosphoric acid functional groups and polyethylene glycol (PEG) functionalities were synthesized for cation-exchange chromatography of peptides and proteins. Phosphoric acid 2-hydroxyethyl methacrylate (PAHEMA) and bis[2-(methacryloyloxy)ethyl] phosphate (BMEP) were reacted with polyethylene glycol diacrylate (PEGDA) and polyethylene glycol acrylate (PEGA), respectively, in 75-μm i.d. UV-transparent fused-silica capillaries by photo-initiated polymerization. The hydrophobicities of the monoliths were evaluated using propyl paraben under reversed-phase conditions and synthetic peptides under ion-exchange conditions. The resulting monoliths exhibited lower hydrophobicities than strong cation-exchange monoliths previously reported using PEGDA as cross-linker. Dynamic binding capacities of 31.2 and 269 mg/mL were measured for the PAHEMA–PEGDA and BMEP–PEGA monoliths, respectively. Synthetic peptides were eluted from both monoliths in 15 min without addition of acetonitrile to the mobile phase. Peak capacities of 50 and 31 were measured for peptides and proteins, respectively, using a PAHEMA–PEGDA monolith. The BMEP–PEGA monolith showed negligible hydrophobicity. A peak capacity of 31 was measured for the BMEP–PEGA monolith when a 20-min salt gradient rate was used to separate proteins. The effects of functional group concentration, mobile phase pH, salt gradient rate, and hydrophobicity on the retention of analytes were investigated. Good run-to-run [relative standard deviation (RSD) < 1.99%] and column-to-column (RSD < 5.64) reproducibilities were achieved. The performance of the monoliths in ion-exchange separation of peptides and proteins was superior to other polymeric monolithic columns reported previously when organic solvents were not added to the mobile phase.     

Antifouling properties of poly(methyl methacrylate) films grafted with poly(ethylene glycol) monoacrylate immersed in seawater

Biofouling of all structures immersed in seawater constitutes an important problem, and many strategies are currently being developed to tackle it. In this context, our previous work shows that poly(ethylene glycol) monoacrylate (PEGA) macromonomer grafted on preoxidized poly(methyl methacrylate) (PMMAox) films exhibits an excellent repellency against the bovine serum albumin used as a model protein. This study aims to evaluate the following: (1) the prevention of a marine extract material adsorption by the modified surfaces and (2) the antifouling property of the PEGA-g-PMMAox substrates when immersed in natural seawater during two seasons (season 1: end of April-beginning of May 2007, and season 2: end of October-beginning of November 2007). The antifouling performances of the PEGA-g-PMMAox films are investigated for different PEG chain lengths and macromonomer concentrations into the PEGA-based coatings. These two parameters are followed as a function of the immersion time, which evolves up to 14 days. The influence of the PEGA layer on marine compounds (proteins and phospholipids) adsorption is evidenced by time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS). It was found that the antifouling efficiency of the PEGA-grafted surfaces increases with both PEGA concentration and PEG chain length.     

Enzyme accessibility and solid supports: which molecular weight enzymes can be used on solid supports? An investigation using confocal Raman microscopy

The accessibility of various solid supports (TentaGel, PEGA 1900, and beaded controlled pore glasses (CPGs)) to a range of enzymes was investigated. The different beaded materials were loaded with the peptide 4-cyanobenzamide-Gly-Pro-Leu-Gly-Leu-Phe-Ala-Arg-OH and incubated with the enzymes MMP-12 (22 kDa), thermolysin (35 kDa), MMP-13 (42.5 kDa), clostridium collagenase (68 kDa), and NEP (90 kDa). The absence/presence of the cyano stretching frequency was measured by means of confocal Raman microscopy. It was found that none of the investigated enzymes could enter the polymer matrices of TentaGel. PEGA 1900 was compatible only with the two smallest enzymes, while beaded CPG was successful even with NEP (90 kDa), proving its superiority over other materials in terms of bio-compatibility.     

Microwave-assisted solid-phase synthesis of 2,5-diketopiperazines: solvent and resin dependence

Solid-phase synthesis of diketopiperazines (DKPs) was preformed using various combinations of resins (polystyrene, TentaGel, ArgoGel, and PEGA) and solvents (toluene, tert-butyl alcohol, water, and toluene/2-butanol (1:4, v/v). The DKPs were synthesized from solid-phase bound dipeptides via intramolecular aminolysis. Both thermal and microwave-assisted solid-phase synthesis of DKPs gave high yields of products independently of resin and organic solvent used; however, only the PEGA resin resulted in high yields of DKPs in water independent of heating method. The short reaction times, high yields, and the possibility to run reactions in water when an appropriate resin is used makes the microwave-assisted solid-phase synthesis the method of choice. The method should be suitable for solid-phase synthesis of diketopiperazine-based libraries.     

Kinetics of enzyme attack on substrates covalently attached to solid surfaces: influence of spacer chain length, immobilized substrate surface concentration and surface charge

The use of alpha-chymotrypsin to cleave covalently bound N-acetyl- l-tryptophan (Ac-Trp-OH) from the surfaces of aminopropylated controlled pore glass (CPG) and the polymer PEGA 1,900 was investigated. Oligoglycine spacer chains were used to present the covalently attached Ac-Trp-OH substrate to the aqueous enzyme. In the absence of the oligoglycine spacer chain, the rate of release was relatively slow, especially from the PEGA 1,900. These slow rates reflect the position of the amino group to which Ac-Trp-OH is covalently attached. On the glass there was a clear optimum with a chain of four glycine residues. For PEGA 1,900 there is no real apparent change beyond two glycine residues. The decline in rate beyond these optima are a possible result of changes in oligoglycine structure. Comparing different surface loadings of bound substrate the rate of release of Ac-Trp-OH from CPG with a pore diameter of 1,200 A was optimal when using 83% of the maximum that can be coupled, then fell again at higher loading. The rate of Ac-Trp-OH release from CPG was the same for surface coverages of 0.4 and 1.0. The introduction of permanent surface charges on CPG 1,200 exhibits a distinct influence on enzymatic cleavage with an increase in the rate of biocatalysis at the surface. Optimal presentation of covalently immobilized substrate on different supports by use of appropriate linkers leads to favorable biocatalysis from the support.     

Conformational studies of resin-bound vancomycin and the complex of vancomycin and Ac2-L-Lys-D-Ala-D-Ala

The molecular target of vancomycin, a commonly used glycopeptide antibiotic, is the D-Ala-D-Ala dipeptide subunit on the bacterial cell wall. The molecular basis of interaction between vancomycin and D-Ala-D-Ala in solution is well-known. However, there is no structural data on vancomycin, and its interaction with D-Ala-D-Ala when the drug is tethered to a solid support. In this Article, vancomycin was directly coupled onto TentaGel or PEGA resin through its C terminus. High-resolution magic angle spinning NMR studies indicated that conformation of PEGA bead-bound vancomycin is identical to that of the free drug. Broadening and shifts of the same proton resonances were observed in solution-phase vancomycin or PEGA-bound vancomycin when complexed with Ac(2)-L-Lys-D-Ala-D-Ala. This study demonstrates that bead-bound molecules can behave the same as solution-phase molecules in terms of molecular interaction with its target molecule, thus validating the on-bead screening approach of the "one-bead-one-compound" combinatorial library method.     

The Effect of Hydrophile Topology in RAFT‐Mediated Polymerization‐Induced Self‐Assembly

Polymerization‐induced self‐assembly (PISA) was employed to compare the self‐assembly of different amphiphilic block copolymers. They were obtained by emulsion polymerization of styrene in water using hydrophilic poly(N‐acryloylmorpholine) (PNAM)‐based macromolecular RAFT agents with different structures. An average of three poly (ethylene glycol acrylate) (PEGA) units were introduced either at the beginning, statistically, or at the end of a PNAM backbone, resulting in formation of nanometric vesicles and spheres from the two former macroRAFT architectures, and large vesicles from the latter. Compared to the spheres obtained with a pure PNAM macroRAFT agent, composite macroRAFT architectures promoted a dramatic morphological change. The change was induced by the presence of PEGA hydrophilic side‐chains close to the hydrophobic polystyrene segment.     

A Useful Disulfide Linker for Single‐Bead Analysis of Peptide Libraries

A disulfide‐linker for conventional peptide synthesis, attached to a PEGA‐resin, has been developed. Reductive hydrolysis cleaves the linker within minutes, liberating the synthesized peptide for rapid sequencing by tandem mass spectrometry. The method has been tested for ten peptides in a single‐bead fashion.     

RAFT synthesis of an amphiphilic block copolymer bearing chlorin rings in the hydrophobic segment and its application in photodynamic therapy

RAFT polymerization of styrene (St) in the presence of 5,10,15,20‐tetrakis(pentafluorophenyl)porphyrin (TFPP) was conducted using 4‐cyano‐4‐(thiobenzoyl)thiopentanoic acid as a chain‐transfer agent and azobisisobutyronitrile as an initiator at 60 °C. The resulting polymer exhibited a chlorin‐like UV‐vis spectrum, which indicated that the polymer possessed a reduced TFPP structure. Furthermore, an SEC trace recorded using UV‐vis detector (λ = 410 nm), which selectively detected the TFPP‐incorporated polymer, shifted toward higher molecular mass as the polymerization progressed. This evidence indicated that TFPP acted as a vinylene‐type monomer, such as maleimide, to form a copolymer, namely, poly(St‐co‐TFPP). The mole fraction of TFPP units was estimated to be 0.74 × 10^?3, which was close to that in the feed (1 × 10^?3). Chain extension of poly(St‐co‐TFPP) with polyethylene glycol monomethyl ether acrylate (PEGA) was performed to afford the amphiphilic block copolymer poly(St‐co‐TFPP)‐b‐poly(PEGA). The degrees of polymerization of St and PEGA were determined to be 64 and 75, respectively. Poly(St‐co‐TFPP)‐b‐poly(PEGA) formed micelles following dialysis. The median diameter of the micelles in solution was determined to be 16 nm by DLS. The photocytotoxicity of the micelle solution was evaluated in a human glioblastoma cell line (U251) and an N‐methyl‐N'‐nitro‐N‐nitrosoguanidine‐induced mutant of a rat murine RGM‐1 gastric carcinoma mucosal cell line (RGK‐1). © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55 , 3395–3403     

Nitrophenol resins for facile amide and sulfonamide library synthesis

Novel nitrophenol solid supports based on various resin materials (polystyrene, TentaGel, macroporous, PEGA, and silica gel) are reported for facile amide and sulfonamide library synthesis. The broad choice of resin materials available will allow the reaction to occur successfully in solvents ranging from nonpolar organic solvents to aqueous media.     

Production of micron-sized polystyrene particles containing PEG near the particles surface

Polystyrene (PS) particles in the size range of 1-7 µm, containing poly(ethylene glycol) or PEG on the particles surface, were prepared by multi-step seeded polymerizations. Micron-sized PS particles were first prepared by dispersion polymerization using 2,2'-azobisisobutyronitrile as initiator and polyvinyl pyrrolidone as stabilizer. Conventional swelling method was then used to increase the size of the PS particles with a large amount of styrene in presence of oil soluble initiator, benzoyl peroxide. In the final step, the PS particles have been used to carry out seeded polymerization with small amount of styrene in presence of poly(ethylene glycol)-azo or PEGA initiator with average molecular weights of the PEG chains of 200 and 3000 g mol^-1 , respectively. The average size, size distribution, and surface morphology indicate that seeded polymerization in the final step with small amount of styrene in presence of PEGA is the best way to produce monodisperse polystyrene particles containing PEG near the particles surface.     

Reversible addition–fragmentation chain transfer synthesis of amidine‐based,CO2‐responsive homo and AB diblock (Co)polymers comprised of histamine and their gas‐triggered self‐assembly in water

Well‐defined homopolymers of pentafluorophenyl acrylate (PFPA) and AB diblock copolymers of N,N‐dimethylacrylamide (DMA) and poly(ethylene glycol) methyl ether acrylate (PEGA) with PFPA were prepared by reversible addition–fragmentation chain transfer (RAFT) radical polymerization. Three PFPA homopolymers of different molecular weights were reacted with the commercially available amidine and guanidine species histamine (HIS) dihydrochloride and L ‐arginine methyl ester (ARG) dihydrochloride in the presence of S‐methyl methanethiosulfonate to yield, quantitatively, the corresponding amidine and guanidine‐based acrylamido homopolymers. Both the HIS and ARG homopolymers are known to reversibly bind CO₂ with, in the case of the former, CO₂ fixation being accompanied with a switch from a hydrophobic to hydrophilic state. The RAFT synthesis of PFPA‐DMA and PEGA‐PFPA diblock copolymers yielded well‐defined materials with a range of molar compositions. These precursor materials were converted to the corresponding HIS and ARG block copolymers whose structure was confirmed using ¹H NMR spectroscopy. Employing a combination of dynamic light scattering and transmission electron microscopy, we demonstrate that the DMA‐HIS and PEGA‐HIS diblock copolymers are able to undergo reversible and cyclable self‐directed assembly in aqueous media using CO₂ and N₂ as the triggers between fully hydrophilic and amphiphilic (assembled) states. For example, in the case of the 54:46 DMA‐HIS diblock, aggregates with hydrodynamic diameters of about 40.0 nm are readily formed from the molecularly dissolved state. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

Screening of polymeric supports and enzymes for the development of an endo enzyme cleavable linker

Several polymeric supports possessing an ester moiety were prepared and a range of enzymes was investigated to hydrolyse the ester linkage and release a signalling group into solution for applications in immunoassays. Pseudomonas lipases were found to most readily cleave the solution-phase analogue and this observation translated well to the corresponding polymeric supports, where the most effective were PEGA resins and the LPOS support PEG-6000.     

Direct site-selective covalent protein immobilization catalyzed by a phosphopantetheinyl transferase

Immobilization of proteins onto solid supports is important in the preparation of functional protein microarrays and in the development of bead-based bioassays, biosensors, and industrial biocatalysts. In order to generate the stable, functional, and homogeneous materials required for these applications, attention has focused on methods that enable the efficient and site-specific covalent immobilization of recombinant proteins onto a wide range of platforms. To this end, the phosphopantetheinyl transferase Sfp was employed to catalyze the direct immobilization of recombinant proteins bearing the small, genetically encoded ybbR tag onto surfaces functionalized with CoA. Using mass spectrometry, it was shown that the Sfp catalyzes immobilization of a model acyl carrier protein (ACP) onto CoA-derivatized PEGA resin beads through specific covalent bond formation. Luciferase (Luc) and glutathione-S-transferase (GST) ybbR-fusion proteins were similarly immobilized onto PEGA resin retaining high levels of enzyme activity. This strategy was also successfully applied for the immobilization of the ACP, as well as ybbR-Luc, -GST, and -thioredoxin fusion proteins, on hydrogel microarray slides. Overall, the Sfp-catalyzed surface ligation is mild, quantitative, and rapid, occurring in a single step without prior chemical modification of the target protein. Immobilization of the target proteins directly from a cell lysate mixture was also demonstrated.     

Solid-phase synthesis for the identification of high-affinity bivalent lectin ligands

The development of carbohydrate-based therapeutics has been frustrated by the low affinities that characterize protein-carbohydrate complexation. Because of the oligomeric nature of most lectins, the use of multivalency may offer a successful strategy for the creation of high-affinity ligands. The solid-phase evaluation of libraries of peptide-linked multivalent ligands facilitates rapid examination of a large fraction of linker structure space. If such solid-phase assays are to replicate solution binding behavior, the potential for intermolecular bivalent binding on bead surfaces must be eliminated. Here we report the solid-phase synthesis and analysis of peptide-linked, spatially segregated mono- and bivalent ligands for the legume lectin concanavalin A. Bead shaving protocols were used for the creation of beads displaying spatially segregated binding sequences on the surface of Tentagel resins. The same ligands were also synthesized on PEGA resin to determine the effect of ligand presentation on solid-phase binding. While we set out to determine the lower limit of assay sensitivity, the unexpected observation that intermolecular bivalent ligand binding is enhanced for bivalent ligands relative to monovalent ligands allowed direct observation of the level of surface blocking required to prevent intermolecular bivalent ligand binding. For a protein with binding sites separated by 65 A, approximately 99.9% of Tentagel(1) surface sites and 99.99% of the total sites on a PEGA bead must be blocked to prevent intermolecular bivalent binding. We also report agglutination and calorimetric solution-phase binding studies of mono- and bivalent peptide-linked ligands.