Protein-polymer conjugates exhibit superior properties to unmodified proteins, generating a high demand for these materials in the fields of medicine, biotechnology, and nanotechnology. Multimeric conjugates are predicted to surpass the activity of monomeric conjugates. Herein, we report a straightforward method to synthesize multimeric polymer-conjugates. Four armed poly(N-isopropylacrylamide) (pNIPAAm) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization in the presence of a tetra-functionalized trithiocarbonate chain transfer agent (CTA). The polymer molecular weight, architecture and polydispersity index (PDI) were verified by gel permeation chromatography (GPC), dynamic light scattering gel permeation chromatography (DLS-GPC), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This approach afforded well-defined polymers (PDI's < 1.06) and the ability to target various molecular weights. Maleimide functional groups were introduced at the chain ends by heating the polymers in the presence of a furan-protected azo-initiator. This allowed for site-specific conjugation of V131C T4 lysozyme to the polymers to generate multimeric protein-polymer conjugates. MALDI-TOF mass spectrometry, electrospray ionization gas-phase electrophoretic-mobility macromolecule analysis (ESI-GEMMA), gel electrophoresis, and liquid chromatography tandem mass spectrometry (LC-MS/MS) of the trypsin digests demonstrated that multimeric protein-polymer conjugates had formed. This simple strategy provides ready access to star protein-polymer conjugates for application in the fields of drug discovery, drug delivery, and nanotechnology. 相似文献
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. 相似文献
Protein-polymer conjugates are widely used in biotechnology and medicine, and new methods to prepare the bioconjugates would be advantageous for these applications. In this report, we demonstrate that bioactive "smart" polymer conjugates can be synthesized by polymerizing from defined initiation sites on proteins, thus preparing the polymer conjugates in situ. In particular, free cysteines, Cys-34 of bovine serum albumin (BSA) and Cys-131 of T4 lysozyme V131C, were modified with initiators for atom transfer radical polymerization (ATRP) either through a reversible disulfide linkage or irreversible bond by reaction with pyridyl disulfide- and maleimide-functionalized initiators, respectively. Initiator conjugation was verified by electrospray-ionization mass spectroscopy (ESI-MS), and the location of the modification was confirmed by muLC-MSMS (tandem mass spectrometry) analysis of the trypsin-digested protein macroinitiators. Polymerization of N-isopropylacrylamide (NIPAAm) from the protein macroinitiators resulted in thermosensitive BSA-polyNIPAAm and lysozyme-polyNIPAAm in greater than 65% yield. The resultant conjugates were characterized by gel electrophoresis and size exclusion chromatography (SEC) and easily purified by preparative SEC. The identity of polymer isolated from the BSA conjugate was confirmed by (1)H NMR, and the polydispersity index was determined by gel permeation chromatography (GPC) to be as low as 1.34. Lytic activities of the lysozyme conjugates were determined by two standard assays and compared to that of the unmodified enzyme prior to polymerization; no statistical differences in bioactivity were observed. 相似文献
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. 相似文献
Herein we report the synthesis of vinyl sulfone end functionalized PEGylated polymers by reversible addition-fragmentation chain transfer (RAFT) polymerization for conjugation to proteins. Poly(ethylene glycol) methyl ether acrylate (PEGA) was polymerized in the presence of 1-phenylethyl dithiobenzoate with 2,2'-azobis(2-methylpropionitrile) as the initiator to generate well-defined polyPEGAs with number-average molecular weights (M(n)) by gel permeation chromatography (GPC) of 6.7 kDa, 11.8 kDa and 16.1 kDa. Post-polymerization, the majority of polymer chains contained the dithioester functional group at the omega chain end, and the polydispersity indexes (PDI) of the polymers ranged from 1.08 to 1.24. The dithioester was subsequently reduced via aminolysis, and the resulting thiol was trapped with a divinyl sulfone in situ to produce semi-telechelic, vinyl sulfone polyPEGAs with efficiencies ranging between 85% and 99%. It was determined that the retention of vinyl sulfone was directly related to reaction time, with the maximum dithioester being transformed into a vinyl sulfone within 30 minutes. Longer reaction times resulted in slow decomposition of the vinyl sulfone end group. The resulting semi-telechelic vinyl sulfone polymers were then conjugated to a protein containing a free cysteine, bovine serum albumin (BSA). Gel electrophoresis demonstrated that the reaction was highly efficient and that conjugates of increasing size were readily prepared. After polymer attachment, the activity of the BSA was 92% of the unmodified biomolecule. 相似文献
Blue emitting dyes bearing a luciferin analogous chromophore were attached to a polystyrene backbone. For this purpose, 4-hydroxy-1,3-thiazoles were functionalized with a styrene unit and polymerized using the reversible addition–fragmentation chain transfer (RAFT) polymerization technique. Two different chain transfer agents were investigated and one monomer was studied in terms of its kinetic behavior. The polymerization kinetics are presented and discussed in detail, showing a controlled polymerization behavior, resulting in well-defined copolymers with polydispersity indices below 1.2. The obtained polymers were characterized by size exclusion chromatography (SEC), 1H NMR, MALDI-TOF MS and UV–vis absorption and fluorescence spectroscopy. In addition, the UV–vis absorption and emission behavior was investigated in thin films. 相似文献
A novel Fmoc-protected chain transfer agent (CTA) was synthesized and applied in the reversible addition-fragmentation chain transfer (RAFT) polymerization of N-isopropylacrylamide (NIPAAm), resulting in well-defined Fmoc-protected PNIPAAm and the amino-end capped PNIPAAm by the subsequent hydrolysis. Poly(N-isopropylacrylamide)-b-poly(l-glutamic acid) (PNIPAAm-b-PLGA) with controlled molecular weight and narrow molecular weight distribution was synthesized successfully via ring-opening polymerization (ROP) of alpha-amino acid N-carboxyanhydrides (NCAs) by using PNIPAAm-NH2 as the macroinitiator. Both pH- and thermo-responsive micellization behaviors of the block copolymer PNIPAAm55-b-PLGA35 in dilute aqueous solution were investigated by means of the pyrene fluorescence, circular dichroism, 1H NMR, transmission electron microscopy and dynamic and static light scattering. Spherical PLGA-core and rod-like PNIPAAm-core micelles are formed in response to pH and temperature. The reversible transition between the PLGA-core and PNIPAAm-core micelles was observed. This work provides a versatile approach for synthesizing well-defined stimuli-responsive polypeptide-based double hydrophilic diblock copolymers (DHBCs), and is of great potential for generating useful stimuli-responsive materials in biomedical applications. 相似文献
This communication reports the first example of polymerization initiated from specific domains on proteins. Streptavidin was coupled with a biotinylated initiator for atom transfer radical polymerization (ATRP) and exposed to an aqueous solution of CuBr/2,2'-bipyridine and monomer. N-Isopropylacrylamide (NIPAAm) and poly(ethylene glycol) methyl ether methacrylate (PEGMA) were readily initiated by the modified streptavidin and polymerized from the protein at room temperature. Formation of streptavidin-polymer conjugates was confirmed by size exclusion chromatography (SEC) and gel electrophoresis. Polymer identity and biotinylation was verified using 1H NMR spectroscopy, gel permeation chromatography (GPC), and surface plasmon resonance (SPR) after dissociation of the biotin-streptavidin complex. This general approach is likely to be extended to other proteins and monomers and promises to enable easy synthesis and purification of a variety of polymer-protein conjugates. 相似文献
In this paper, we demonstrated an efficient and robust route to the preparation of well-defined molecularly imprinted polymer based on reversible addition-fragmentation chain transfer (RAFT) polymerization and click chemistry. The alkyne terminated RAFT chain transfer agent was first synthesized, and then click reaction was used to graft RAFT agent onto the surface of silica particles which was modified by azide. Finally, imprinted thin film was prepared in the presence of 2,4-dichlorophenol as the template. The imprinted beads were demonstrated with a homogeneous polymer films (thickness of about 2.27 nm), and exhibited thermal stability under 255°C. The as-synthesized product showed obvious molecular imprinting effects towards the template, fast template rebinding kinetics and an appreciable selectivity over structurally related compounds. 相似文献
Well‐defined “smart” block copolymer–protein conjugates were prepared by two consecutive “grafting‐from” reactions via reversible addition–fragmentation chain transfer (RAFT) polymerization. The initiating portion (R‐group) of the RAFT agent was anchored to a model protein such that the thiocarbonylthio moiety was readily accessible for chain transfer with propagating chains in solution. Well‐defined polymer‐protein conjugates of poly(N‐isopropylacrylamide) (PNIPAM) and bovine serum albumin (BSA) were prepared at room temperature in aqueous media. The retained trithiocarbonate moiety on the free end group of the immobilized polymer allowed the homopolymer conjugate to be extended by polymerization of N,N‐dimethylacrylamide. Polyacrylamide gel electrophoresis, size exclusion chromatography, and NMR spectroscopy confirmed the synthesis of the various conjugates and revealed that the polymerizations were well controlled. As expected, the resulting block copolymer–protein conjugates demonstrated thermoresponsive behavior due to the temperature‐sensitivity of the PNIPAM block, as evidenced by turbidity measurements and dynamic light scattering analysis.
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. 相似文献
We report on the design of a polymeric prodrug of the anticancer agent paclitaxel (PTX) by a grafting‐from‐drug approach. A chain transfer agent for reversible addition fragmentation chain transfer (RAFT) polymerization was efficiently and regioselectively linked to the C2′ position of paclitaxel, which is crucial for its bioactivity. Subsequent RAFT polymerization of a hydrophilic monomer yielded well‐defined paclitaxel–polymer conjugates with high drug loading, water solubility, and stability. The versatility of this approach was further demonstrated by ω‐end post‐functionalization with a fluorescent tracer. In vitro experiments showed that these conjugates are readily taken up into endosomes where native PTX is efficiently cleaved off and then reaches its subcellular target. This was confirmed by the cytotoxicity profile of the conjugate, which matches those of commercial PTX formulations based on mere physical encapsulation. 相似文献