Manipulation of the bioactivity of glucose oxidase via raft‐controlled surface modification

In this article, poly[poly(ethyleneglycol) acrylate] (polyPEG‐A) with mercaptothiazoline ester terminal group was synthesized directly by reversible addition fragmentation chain transfer (RAFT) polymerization using a mercaptothiazoline ester functional RAFT agent. The functional polyPEG‐A was then conjugated to glucose oxidase (GOx) via surface‐tethered amino groups through covalent amide coupling. Sorensenformaltitration assay revealed that GOx retained ～14 free amino groups available for covalent modification. The conjugation reaction turned out to be efficient and mild. Colorimetric method was applied to evaluate the enzymatic activity of native GOx and its derivatives by introducing another enzyme, horseradish peroxidase. The modified GOx with polymeric chains exhibited reduced enzymatic activity toward the catalytical oxidation of glucose, but with significantly increased thermal stability and elongated lifetime. When GOx was modified with polyPEG‐A [molecular weight (MW), 45,000; polydispersity index, 1.12] the enzymatic activity was decreased to 37 U/mg, only 29% left. However, when incubated at 25 °C the modified GOx still retained 9.6% of its original bioactivity after 60 days, whereas the native GOx only lived for 29 days. The more polymer chains or the longer polymer chain attached, the more reduction of the enzymatic activity resulted, however, the longer the lifetime of the enzyme obtained. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Temperature-regulated activity of responsive polymer-protein conjugates prepared by grafting-from via RAFT polymerization

A facile route to well-defined "smart" polymer-protein conjugates with tunable bioactivity is reported. Protein modification with a reversible addition-fragmentation chain transfer (RAFT) agent and subsequent room temperature polymerization in aqueous media led to conjugates of poly(N-isopropylacrylamide) and a model protein. Representing the first example of polymer-protein conjugation with RAFT agent immobilization via the "R-group" approach, high molecular weight and reductively stable conjugates were accessible without extensive purification or adverse effects on the protein structure. An increase in molecular weight with conversion was observed for the chains grafted from the protein surface, confirming the controlled nature of the polymerization. The responsive behavior of the immobilized polymer facilitated conjugate isolation and also allowed environmental modulation of bioactivity.     

Design of Enzyme Micelles with Controllable Concavo‐Convex Micromorphologies for Highly Enhanced Stability and Catalytical Activity

Concavo‐convex micelles with controllable sizes and nanostructures are prepared via self‐assembling polymer–enzyme (e.g., shellac enzyme) conjugates with heterogeneous polymer chains, which exhibit higher enzyme stability (300%) and bioactivity (760%) comparing with the well‐defined ones. The applied amphiphilic and negatively charged copolymer, poly (methyl methacrylate)‐block‐poly (sodium p‐styrene sulfonate), is synthesized via reversible addition–fragmentation chain transfer polymerization to modify shellac enzyme and immobilize the enzyme bioactivity inducer by covalent conjugation and electrostatic attraction, respectively. The degradation test of catechol confirms the application potential of as‐prepared micelles as an efficient and economical decontaminant.     

Thermoprecipitation of Glutathione S‐Transferase by Glutathione–Poly(N‐isopropylacrylamide) Prepared by RAFT Polymerization

Herein, we report an effective and rapid method to purify glutathione S‐transferase (GST) using glutathione (GSH)‐modified poly(N‐isopropylacrylamide) (pNIPAAm) and mild, thermal conditions. A chain transfer agent modified with pyridyl disulfide was employed in the reversible addition–fragmentation chain transfer (RAFT) polymerization of NIPAAm. The resulting polymer had a narrow molecular weight distribution (polydispersity index = 1.21). Conjugation of GSH to the pyridyl disulfide–pNIPAAm reached 95% within 30 min as determined by UV–Vis monitoring of the release of pyridine‐2‐thione. GST was successfully thermoprecipitated upon heating the GSH–pNIPAAm above the lower critical solution temperature (LCST). The pull down assay was repeated with bovine serum albumin (BSA) and T4 lysozyme (T4L), which demonstrated the specificity of the polymer for GST. Due to its simplicity and high efficiency, this method holds great potential for large‐scale purification of GST‐tagged proteins.

     

Accelerated brush growth on nanoparticle surfaces by reversible addition‐fragmentation chain transfer polymerization

It is now well established that controlling the grafted chain lengths and densities on nanoparticle surfaces determines the effective interactions between particles, and their assembly. Here, we present unusual kinetic results for achieving grafted chain lengths longer than the free chains using reversible addition‐fragmentation chain transfer (RAFT) polymerization and discuss the limitations to obtaining polymer grafting density higher than ～0.06 chains/nm². We observe that surface initiated polymerization grows faster than the free chains in solution with high RAFT agent coverage (1.95 agents/nm²) on nanoparticles. The time‐dependence of graft density suggests that activation of the anchored chain transfer agent (CTA) is limited by the diffusion of macro‐radicals within growing grafts. Thus, radical transfer and exchange reactions become inefficient between grafts and free polymer, and convert the surface‐initiated RAFT mechanism to a free radical polymerization. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 1700–1705     

Cyclic peptide–polymer conjugates: Grafting‐to vs grafting‐from

A systematic comparison between the grafting‐to (convergent) and grafting‐from (divergent) synthetic routes leading to cyclic peptide–polymer conjugates is described. The reversible addition–fragmentation chain transfer (RAFT) process was used to control the polymerizations and the couplings between cyclic peptide and polymer or RAFT agent were performed using N‐hydroxysuccinimide (NHS) active ester ligation. The kinetics of polymerization and polymer conjugation to cyclic peptides were studied for both grafting‐to and grafting‐from synthetic routes, using N‐acryloyl morpholine as a model monomer. The cyclic peptide chain transfer agent was able to mediate polymerization as efficiently as a traditional RAFT agent, reaching high conversion in the same time scale while maintaining excellent control over the molecular weight distribution. The conjugation of polymers to cyclic peptides proceeded to high conversion, and the nature of the carbon at the α‐position to the NHS group was found to play a crucial role in the reaction kinetics. The study was extended to a wider range of monomers, including hydrophilic and temperature responsive acrylamides, hydrophilic and hydrophobic acrylates, and hydrophobic and pH responsive methacrylates. Both approaches lead to similar peptide–polymer conjugates in most cases, while some exceptions highlight the advantages of one or the other method, thereby demonstrating their complementarity. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 1003–1011     

Preparation of biodegradable and thermoresponsive enzyme–polymer conjugates with controllable bioactivity via RAFT polymerization

The preparation of biodegradable and thermoresponsive enzyme–polymer bioconjugates with controllable enzymatic activity via reversible addition−fragmentation chain transfer (RAFT) polymerization and amidation conjugation reaction is presented. A new 2-mercaptothiazoline ester functionalized RAFT agent with intra-disulfide linkage was synthesized and used as chain transfer agent (CTA) to generate a biocompatible homopolymer, poly(ethyleneglycol) acrylate (polyPEG-A) and a thermoresponsive copolymer of poly(ethyleneglycol) acrylate with di(ethyleneglycol)ethyl ether acrylate [poly(PEG-A-co-DEG-A)]. These biodegradable and thermoresponsive polymers were then conjugated to the surface of glucose oxidase (GOx) under mild condition to afford the biodegradable and thermoresponsive enzyme–polymer conjugates. Cleavage of the polymer chains from the GOx surface obviously recovered the enzymatic activity. The thermoresponsive test of GOx-poly(PEG-A-co-DEG-A) revealed that the bioconjugate exhibited regular enzymatic activity fluctuation upon the temperature change below or above the lower critical solution temperature (LCST). The as-prepared enzyme–polymer conjugates were also characterized using ¹H NMR, UV–vis spectroscopy, polyacrylamide gel electrophoresis (PAGE) and biocatalytic activity tests. These smart enzyme–polymer conjugates would envision promising applications in biotechnology and biomedicine.     

Synthesis of Structurally Well‐Defined Telechelic Polymers by Organostibine‐Mediated Living Radical Polymerization: In Situ Generation of Functionalized Chain‐Transfer Agents and Selective ω‐End‐Group Transformations

Several organostibine chain‐transfer agents possessing polar functional groups have been prepared by the reactions of azo initiators and tetramethyldistibine ( 1 ). Carbon‐centered radicals thermally generated from the azo initiators were trapped by 1 to yield the corresponding organostibine chain‐transfer agents. The high yields observed in the synthesis of the chain‐transfer agents strongly suggest that distibines have excellent radicophilic reactivity. As the reactions proceeded under neutral conditions, functional groups that are incompatible with ionic conditions were incorporated into the chain‐transfer agents. The chain‐transfer agents were used in living radical polymerization to synthesize the corresponding α‐functionalized polymers. As the functional groups in the chain‐transfer agents did not interfere with the polymerization reaction, well‐controlled polymers possessing number‐average molecular weights (M_ns) predetermined by the monomer/transfer agent ratios were synthesized with low polydispersity indices (PDIs). The organostibanyl ω‐polymer ends were transformed into a number of different functional groups by radical‐coupling, radical‐addition, and oxidation reactions. Therefore, it was possible to synthesize well‐controlled telechelic polymers with the same and also with different functional groups at their α‐ and ω‐polymer ends. Distibine 1 was also found to increase PDI control in the living radical polymerization of styrene and methyl methacrylate (MMA) using a purified organostibine chain‐transfer agent. Well‐controlled poly(methyl methacrylate)s with M_n values ranging from 10 000 to 120 000 with low PDIs (1.05–1.15) were synthesized by the addition of a catalytic amount of 1 . The results have been attributed to the high reactivity of distibine 1 towards polymer‐end radicals, which are spontaneously deactivated to yield organostibine dormant species.     

Synthesis of dual‐functional poly(6‐azidohexylmethacrylate) brushes by a RAFT agent carrying carboxylic acid end groups

Novel types of dual‐functional surface‐attached polymer brushes were developed by interface‐mediated reversible addition‐fragmentation chain transfer (RAFT) polymerization of 6‐azidohexylmethacrylate using the surface‐immobilized RAFT agent and the free initiator. The interface‐mediated RAFT polymerization produced silicon substrate coated with dual‐functional (azido groups from monomer and carboxylic acid groups from RAFT agent) poly(6‐azidohexylmethacrylate) [poly (AHMA)] with a grafting density as high as 0.59 chains/nm². Dual‐functional polymer brushes can represent an attractive chemical platform to deliberately introduce other molecular units at specific sites. The azido groups of the poly(AHMA) brushes can be modified with alkyl groups via click reaction, known for their DNA hybridization, while the carboxylic acid end groups can be reacted with amine groups via amide reaction, known for their antifouling properties. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2015 , 53, 1696–1706     

Selective Uptake of Cylindrical Poly(2‐Oxazoline) Brush‐AntiDEC205 Antibody‐OVA Antigen Conjugates into DEC‐Positive Dendritic Cells and Subsequent T‐Cell Activation

To achieve specific cell targeting by various receptors for oligosaccharides or antibodies, a carrier must not be taken up by any of the very many different cells and needs functional groups prone to clean conjugation chemistry to derive well‐defined structures with a high biological specificity. A polymeric nanocarrier is presented that consists of a cylindrical brush polymer with poly‐2‐oxazoline side chains carrying an azide functional group on each of the many side chain ends. After click conjugation of dye and an anti‐DEC205 antibody to the periphery of the cylindrical brush polymer, antibody‐mediated specific binding and uptake into DEC205⁺‐positive mouse bone marrow‐derived dendritic cells (BMDC) was observed, whereas binding and uptake by DEC205^? negative BMDC and non‐DC was essentially absent. Additional conjugation of an antigen peptide yielded a multifunctional polymer structure with a much stronger antigen‐specific T‐cell stimulatory capacity of pretreated BMDC than application of antigen or polymer–antigen conjugate.     

Synthesis of Polymers Containing Potassium Acyltrifluoroborates (KATs) and Post‐polymerization Ligation and Conjugation

We report the synthesis of monomers for atom‐transfer radical polymerization (ATRP) and a reversible addition‐fragmentation chain transfer (RAFT) agent bearing trifluoroborate iminiums (TIMs), which are quantitatively converted into potassium acyltrifluoroborates (KATs) after polymerization. The resulting KAT‐containing polymers are suitable for rapid amide‐forming ligations for both post‐polymerization modification and polymer conjugation. The polymer conjugation occurs rapidly, even under dilute (micromolar) aqueous conditions at ambient temperatures, thereby enabling the synthesis of a variety of linear and star‐shaped block copolymers. In addition, we applied post‐polymerization modification to the covalent linking of a photocaged cyclic antibiotic (gramicidin S) to the side chains of the KAT‐containing copolymer. Cellular assays revealed that the polymer–antibiotic conjugate is biocompatible and provides efficient light‐controlled release of the antibiotic on demand.     

High density cationic polymer brushes from combined “click chemistry” and RAFT‐mediated polymerization

A simple method for preparing cationic poly[(ar‐vinylbenzyl)trimethylammonium chloride)] [poly(VBTAC)] brushes was used by combined technology of “click chemistry” and reversible addition‐fragmentation chain transfer (RAFT) polymerization. Initially, silicon surfaces were modified with RAFT chain transfer agent by using a click reaction involving an azide‐modified silicon wafer and alkyne‐terminated 4‐cyanopentanoic acid dithiobenzoate (CPAD). A series of poly(VBTAC) brushes on silicon surface with different molecular weights, thicknesses, and grafting densities were then synthesized by RAFT‐mediated polymerization from the surface immobilized CPAD. The immobilization of CPAD on the silicon wafer and the subsequent polymer formation were characterized by X‐ray photoelectron spectroscopy, water contact angle measurements, grazing angle‐Fourier transform infrared spectroscopy, atomic force microscopy, and ellipsometry analysis. The addition of free CPAD was required for the formation of well‐defined polymer brushes, which subsequently resulted in the presence of free polymer chains in solution. The free polymer chains were isolated and used to estimate the molecular weights and polydispersity index of chains attached to the surface. In addition, by varying the polymerization time, we were able to obtain poly(VBTAC) brushes with grafting density up to 0.78 chains/nm² with homogeneous distributions of apparent needle‐like structures. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

A facile “graft from” method to prepare molecular‐level dispersed graphene–polymer composites

Graphene–polymer composites of positive‐charged poly(dimethyl aminoethyl acrylate), negative‐charged poly(acrylic acid), and neutral polystyrene were prepared by “graft from” methodology using reversible addition fragmentation chain transfer (RAFT) polymerization via a pyrene functional RAFT agent (PFRA) modified graphene precursor. Fluorescence spectroscopy and attenuated total reflection infrared (ATR‐IR) evidenced that the PFRA was attached on the graphene basal planes by π–π stacking interactions, which is strong enough to anti‐dissociation in the polymerization mixture up to 80°C. Atomic force microscopy (AFM) revealed that the thickness of a graphene–polymer sheet was about 4.0 nm. Graphene composites of different polymers with the same polymerization degree exhibited similar conductivity; however, when the polymer chain was designed as random copolymer the conductivity was significantly decreased. It was also observed that the longer the grafted polymer chains the lower the conductivity. ATR‐IR spectroscopy and thermogravimetric analysis were also performed to characterize the as‐prepared composites. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

RAFT‐based synthesis and the gelation property of telechelic polymers that can immobilize biomacromolecules

Telechelic polymers, macromolecules having two reactive end groups, can serve as building blocks for constructing polymers or polymeric materials that have complex architectures. Among the telechelic polymers, polymers bearing hydroxyl groups at two terminals have been used as components for preparation of functional materials. In the present study, RAFT polymerization of both N‐acryloylmorphorin and N‐succinimidyl acrylate by using a newly synthesized dithiobenzoate‐type chain transfer agent bearing hydroxyl groups at both terminals (HECPHD) was reported. After the acryloylation of the hydroxyl terminals of the obtained polymer, gelation was observed. Furthermore, the polymer could react with a protein via the conjugation of the succinimidyl esters‐containing polymers to the amino groups present on the protein surface. The results show that activated esters‐bearing polymers with hydroxyl groups at both terminals can be used as building blocks for constructing polymeric materials for an immobilization of biomacromolecules. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55 , 1356–1365     

Biodegradable Multiblock Poly[N-(2-hydroxypropyl)methacrylamide] via Reversible Addition-Fragmentation Chain Transfer Polymerization and Click Chemistry

A new bifunctional chain transfer agent (CTA) containing alkyne end groups was designed, synthesized and used for direct synthesis of clickable telechelic polymers. Good control of reversible addition-fragmentation chain transfer (RAFT) polymerization of N-(2-hydroxypropyl)methacrylamide (HPMA) was achieved by using the new CTA, as indicated by a linear increase of number average molecular weight (Mn) with conversion and low polydispersity (PDI) (<1.1). In particular, enzymatically degradable multiblock HPMA polymers were readily prepared by subsequent reaction with αω, -diazido oligopeptide (GFLG) sequence via Cu(I) catalyzed alkyne-azide cycloaddition. Upon exposure of high molecular weight fractions of multiblock polyHPMA to papain or cathepsin B, the polymer was degraded into segments of molecular weight and narrow polydispersity similar to those of the initial telechelic polyHPMA.     

Aluminum Alkyls as Highly Active Catalytic Chain Transfer Agents

During the production of free radical initiated low‐density polyethylene (LDPE), it was discovered that the addition of low levels of alkyl aluminum compounds caused the molecular weight of the LDPE to drop precipitously. Further investigation demonstrated that aluminum‐alkyl compounds are among the most effective chain transfer agents ever utilized. It was also shown that polymer chains, which transfer to Al alkyl species, contain almost exclusively vinyl terminated end groups. A catalytic chain transfer mechanism is proposed in which chain transfer occurs from a growing polymer chain to an aluminum center followed by beta hydride elimination to produce a vinyl terminated polymer chain and a new aluminum hydride bond. This new aluminum hydride bond can then undergo further chain transfer reactions. This is the first time such a catalytic chain transfer mechanism has been reported. As little as 10–20 mol ppm aluminum alkyl species decreased the degree of polymerization by a factor of 2 resulting in chain transfer constant (C_s) values as high as 1000–2000. Density functional theory (DFT) study elucidated the catalytic cycle of triethylaluminum (TEA). It is discovered that, depending on the reaction conditions, TEA can serve as a conventional as well as catalytic chain transfer agent.     

Stimuli‐responsive diblock copolymer brushes via combination of “click chemistry” and living radical polymerization

pH‐ and temperature‐responsive poly(N‐isopropylacrylamide‐block?4‐vinylbenzoic acid) (poly(NIPAAm‐b‐VBA)) diblock copolymer brushes on silicon wafers have been successfully prepared by combining click reaction, single‐electron transfer‐living radical polymerization (SET‐LRP), and reversible addition‐fragmentation chain‐transfer (RAFT) polymerization. Azide‐terminated poly(NIPAAm) brushes were obtained by SET‐LRP followed by reaction with sodium azide. A click reaction was utilized to exchange the azide end group of a poly(NIPAAm) brushes to form a surface‐immobilized macro‐RAFT agent, which was successfully chain extended via RAFT polymerization to produce poly(NIPAAm‐b‐VBA) brushes. The addition of sacrificial initiator and/or chain‐transfer agent permitted the formation of well‐defined diblock copolymer brushes and free polymer chains in solution. The free polymer chains were isolated and used to estimate the molecular weights and polydispersity index of chains attached to the surface. Ellipsometry, contact angle measurements, grazing angle‐Fourier transform infrared spectroscopy, and X‐ray photoelectron spectroscopy were used to characterize the immobilization of initiator on the silicon wafer, poly(NIPAAm) brush formation via SET‐LRP, click reaction, and poly(NIPAAm‐b‐VBA) brush formation via RAFT polymerization. The poly(NIPAAm‐b‐VBA) brushes demonstrate stimuli‐responsive behavior with respect to pH and temperature. The swollen brush thickness of poly(NIPAAm‐b‐VBA) brush increases with increasing pH, and decreases with increasing temperature. These results can provide guidance for the design of smart materials based on copolymer brushes. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 2677–2685     

Macromolecular brushes synthesized by “grafting from” approach based on “click chemistry” and RAFT polymerization

Well‐defined macromolecular brushes with poly(N‐isopropyl acrylamide) (PNIPAM) side chains on random copolymer backbones were synthesized by “grafting from” approach based on click chemistry and reversible addition‐fragmentation chain transfer (RAFT) polymerization. To prepare macromolecular brushes, two linear random copolymers of 2‐(trimethylsilyloxy)ethyl methacrylate (HEMA‐TMS) and methyl methacrylate (MMA) (poly(MMA‐co‐HEMA‐TMS)) were synthesized by atom transfer radical polymerization and were subsequently derivated to azide‐containing polymers. Novel alkyne‐terminated RAFT chain transfer agent (CTA) was grafted to polymer backbones by copper‐catalyzed 1,3‐dipolar cycloaddition (azide‐alkyne click chemistry), and macro‐RAFT CTAs were obtained. PNIPAM side chains were prepared by RAFT polymerization. The macromolecular brushes have well‐defined structures, controlled molecular weights, and molecular weight distributions (M_w/M_n ≦ 1.23). The RAFT polymerization of NIPAM exhibited pseudo‐first‐order kinetics and a linear molecular weight dependence on monomer conversion, and no detectable termination was observed in the polymerization. The macromolecular brushes can self‐assemble into micelles in aqueous solution. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 443–453, 2010     

Thermosensitive graphene nanocomposites formed using pyrene‐terminal polymers made by RAFT polymerization

Thermosensitive graphene‐polymer composites have been prepared by attaching poly(N‐isopropylacrylamide) (PNIPAAm) onto the basal plane of graphene sheets via π‐π stacking. Pyrene‐terminated PNIPAAm was synthesized using reversible addition fragmentation chain transfer (RAFT) polymerization via a pyrene‐functional RAFT agent. Aqueous solutions of the graphene‐polymer composites were stable and thermosensitive. The lower critical solution temperature (LCST) of pyrene‐terminated PNIPAAm was measured to be 33 °C. When the pyrene‐functional polymer was attached to graphene the resultant composites were also thermosensitive in aqueous solutions exhibiting a reversible suspension behavior at 24 °C. Atomic force microscopy (AFM) analysis revealed that the thickness of a graphene‐PNIPAAm (M_n: 10,000 and PDI: 1.1) sheet was ～5.0 nm. The surface coverage of polymer chains on the graphene basal plane was calculated to be 7.2 × 10^?11 mol cm^?2. The graphene‐PNIPAAm composite material was successfully characterized using X‐ray photoelectron spectroscopy (XPS), attenuated total reflection infrared (ATR‐IR) spectroscopy, and thermogravimetric analysis (TGA). © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 425–433, 2010     

Energy transfer along a sequence controlled hybrid polymer

The present work provides an ideal model for intra‐chain energy transfer study in conjugated polymer through shielding the polymer backbone by using bulky polyhedral oligomeric silsesquioxanes (POSS). POSS provides a circumference shielding of the polymer backbone to prevent closed packing of the polymer chains, allowing the intra‐chain energy transfer dominating in large concentration range. Bi‐functional POSS (B‐POSS) is specially designed to separate donor (fluorene) and acceptor (benzothiadiazole) within the polymer chain. The dynamics of energy transfer in poly(fluorene‐POSS‐alt‐POSS‐benzothiodiazole) (PTBtTbOFl₃) is studied by steady state as well as time resolved fluorescence spectroscopy at different donor/acceptor ratios. Results reveal that POSS can effectively shield inter‐chains energy transfer of the polymers, suggesting it is an effective model for energy transfer study with less inter‐chains effects. PTBtTbOFl₃ works as a chemosensors is also reported in the detection of explosive derivatives. These results provide insights for optimizing nanostructured materials for use in optoelectronic devices. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 1225–1233