Well-defined protein-polymer conjugates via in situ RAFT polymerization

Biotechnology, biomedicine, and nanotechnology applications would benefit from methods generating well-defined, monodisperse protein-polymer conjugates, avoiding time-consuming and difficult purification steps. Herein, we report the in situ synthesis of protein-polymer conjugates via reversible addition-fragmentation chain transfer polymerization (RAFT) as an efficient method to generate well-defined, homogeneous protein-polymer conjugates in one step, eliminating major postpolymerization purification steps. A water soluble RAFT agent was conjugated to a model protein, bovine serum albumin (BSA), via its free thiol group at Cys-34 residue. The conjugation of the RAFT agent to BSA was confirmed by UV-visible spectroscopy, matrix-assisted laser desorption ionization--time of flight (MALDI-TOF), and 1H NMR. BSA-macroRAFT agent was then used to control the polymerization of two different water soluble monomers, N-isopropylacrylamide (NIPAAm) and hydroxyethyl acrylate (HEA), in aqueous medium at 25 degrees C. The growth of the polymer chains from BSA-macroRAFT agent was characterized by size exclusion chromatography (SEC), 1H NMR, MALDI-TOF, and polyacrylamide gel electrophoresis (PAGE) analyses. The controlled character of the RAFT polymerizations was confirmed by the linear evolution of molecular weight with monomer conversion. The SEC analyses showed no detectable free, nonconjugated polymer formation during the in situ polymerization. The efficiency of BSA-macroRAFT agent to generate BSA-polymer conjugates was found to be ca. 1 by deconvolution of the SEC traces of the polymerization mixtures. The structural integrity and the conformation-related esterase activity of BSA were found to be unaffected by the polymerization conditions and the conjugation of the polymer chain. BSA-poly(NIPAAm) conjugates showed hybrid temperature-dependent phase separation and aggregation behavior. The lower critical solution temperature values of the conjugates were found to increase with the decrease in molecular weight of poly(NIPAAm) block conjugated to BSA.     

Well‐Defined Polymer–Paclitaxel Prodrugs by a Grafting‐from‐Drug Approach

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.     

Efficient Synthesis of Poly(acrylic acid) in Aqueous Solution via a RAFT Process

The direct polymerization of acrylic acid (AA) in aqueous solution for high molecular weight by means of living radical polymerization is still difficult. Here, AA was polymerized homogeneously in water by a reversible addition-fragmentation transfer polymerization (RAFT) in the presence of a water-soluble trithiocarbonate as a RAFT agent. Various ratios [AA]:[RAFT agent] were investigated to aim at different molecular weights. The polymerization exhibited living free-radical polymerization characteristics at different ratios [AA]: [RAFT agent]: controlled molecular weight, low polydispersity and well-suited linear growth of the number-average molecular weight, M _n with conversion. The chain transfer to solvent or polymer was suppressed during the polymerization process, thus high linear PAA with high molecular weight and low PDI can be obtained. Moreover, using the generated PAA as a macro RAFT agent, the chain extension polymerization of PAA with fresh AA displayed controlled behavior, demonstrated the ability of PAA to reinitiate sequential polymerization.     

Reversible Addition Fragmentation Chain Transfer Mediated Dispersion Polymerization of Styrene

Polystyrene microspheres have been synthesized by the reversible addition-fragmentation chain transfer (RAFT) mediated dispersion polymerization in an alcoholic media in the presence of poly(N-vinylpyrrolidone) as stabilizer and 2,2′-azobisisobutyronitrile as a conventional radical initiator. In order to obtain monodisperse polystyrene particles with controlled architecture, the post–addition of RAFT agent was employed to replace the weak point from the pre-addition of RAFT. The feature of preaddition and postaddition of RAFT agent was studied on the polymerization kinetics, particle size and its distribution and on the particle stability. The living polymerization behavior as well as the particle stability was observed only in the postaddition of RAFT. The effects of different concentration on the postaddition of RAFT agent were investigated in terms of molecular weight, molecular weight distribution, particle size and its distribution. The final polydispersity index (PDI) value, particle size and the stability of the dispersion system were found to be greatly influenced by the RAFT agent. This result showed that the postaddition of RAFT agent in the dispersion polymerization not only controls the molecular weight and PDI but also produces stable monodisperse polymer particles.     

Living Free Radical Polymerization of Cyclodextrin Host‐Guest Complexes of Styrene via the Reversible Addition Fragmentation Chain Transfer (RAFT) Process in Aqueous Solution

Summary: Host‐guest complexes of styrene and randomly methylated β‐cyclodextrin (m‐β‐CD) were polymerized in aqueous medium via the reversible addition fragmentation chain transfer (RAFT) process. 3‐Benzylsulfanylthiocarbonylsulfanylpropionic acid (TTC) was used as trithiocarbonate‐type RAFT agent. The results indicate a controlled character of the polymerization of the styrene complexes as the number‐average molecular weight, , increases linearly with monomer to polymer conversion; however, the molecular weights of the obtained polystyrenes deviate to higher values than those theoretically predicted. Nevertheless, the molecular weights can be controlled by variation of the initial RAFT agent concentration. The polystyrenes produced in this system exhibited narrower polydispersities (1.23 < < 2.36) than those produced without RAFT agent (5.24 < < 9.21) under similar conditions. The present contribution represents the first example of RAFT polymerization of a m‐β‐CD‐complexed hydrophobic vinylmonomer (styrene) from homogenous aqueous solution.

Schematic presentation of complexation and RAFT polymerization of m‐β‐CD‐complexed styrene with TTC as RAFT agent and evolution of the full molecular weight distributions in the CD‐mediated styrene free radical RAFT polymerization.     

Reversible addition–fragmentation chain transfer/miniemulsion polymerization of butyl methacrylate in the presence of β‐cyclodextrin

In the presence of β‐cyclodextrin (β‐CD), reversible addition–fragmentation chain transfer (RAFT) polymerization has been successfully applied to control the molecular weight and polydispersity [weight‐average molecular weight/number‐average molecular weight (M_w/M_n)] in the miniemulsion polymerization of butyl methacrylate, with 2‐cyanoprop‐2‐yl dithiobenzoate as a chain‐transfer agent (or RAFT agent) and 2,2′‐azoisobutyronitrile (AIBN) as an initiator. β‐CD acted as both a stabilizer and a solubilizer, assisting the transportation of the water‐insoluble, low‐molecular‐weight RAFT agent into the polymerization loca (i.e., droplets or latex particles) and thereby ensuring that the RAFT agent was homogeneous in the polymerization loca. The polymers produced in the system of β‐CD exhibited narrower polydispersity (1.2 < M_w/M_n < 1.3) than those without β‐CD. Moreover, the number‐average molecular weight in the former case could be controlled by a definite amount of the RAFT agent. Significantly, β‐CD was proved to have a favorable effect on the stability of polymer latex, and no coagulum was observed. The effects of the concentrations of the RAFT agent and AIBN on the conversion, the molecular weight and its distribution, and the particle size of latices were investigated in detail. Furthermore, the influences of the variations of the surfactant (sodium dodecyl sulfate) and costabilizer (hexadecane) on the RAFT/miniemulsion polymerization were also studied. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 2931–2940, 2005     

Influence of a reversible addition–fragmentation chain transfer agent in the dispersion polymerization of styrene

Dispersion polymerization was applied to the controlled/living free‐radical polymerization of styrene with a reversible addition–fragmentation chain transfer (RAFT) polymerization agent in the presence of poly(N‐vinylpyrrolidone) and 2,2′‐azobisisobutyronitrile in an ethanol medium. The effects of the polymerization temperature and the postaddition of RAFT on the polymerization kinetics, molecular weight, polydispersity index (PDI), particle size, and particle size distribution were investigated. The polymerization was strongly dependent on both the temperature and postaddition of RAFT, and typical living behavior was observed when a low PDI was obtained with a linearly increased molecular weight. The rate of polymerization, molecular weight, and PDI, as well as the final particle size, decreased with an increased amount of the RAFT agent in comparison with those of traditional dispersion polymerization. Thus, the results suggest that the RAFT agent plays an important role in the dispersion polymerization of styrene, not only reducing the PDI from 3.34 to 1.28 but also producing monodisperse polystyrene microspheres. This appears to be the first instance in which a living character has been demonstrated in a RAFT‐mediated dispersion polymerization of styrene while the colloidal stability is maintained in comparison with conventional dispersion polymerization. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 348–360, 2007     

RAFT Polymerization: Adding to the Picture

SUMMARY: Factors affecting the choice of RAFT agent [RSC(Z) = S] for a given polymerization are discussed. For polymerization of methyl methacrylate (MMA), tertiary cyanoalkyl trithiocarbonates provide very good control over molecular weight and distribution and polymerizations show little retardation. The secondary trithiocarbonate RAFT agents with R = CHPh(CN) also gives good control but an inhibition period attributed to slow reinitiation is manifest. Radical induced reduction with hypophosphite salts provides a clean and convenient process for removal of thiocarbonylthio end groups of RAFT-synthesized polymers. Two methods providing simultaneous control over stereochemistry and molecular weight distribution of chains formed by radical polymerization are reported. Polymerization of MMA in the presence of scandium triflate provides a more isotactic PMMA. A similar RAFT polymerization with trithiocarbonate RAFT agents also provides control and avoids issues of RAFT agent instability seen with dithiobenzoate RAFT agents in the presence of Lewis acids. RAFT polymerization of tetramethylammonium methacrylate at 45 °C provides a more syndiotactic PMMA of controlled molecular weight and distribution (after methylation; mm:mr:rr 2:21:77 compared to 3:35:62 when formed by bulk polymerization of MMA).     

两亲性RAFT试剂存在下丙烯酸六氟丁醋和苯乙烯的无皂乳液聚合

合成了具有两亲性结构的可逆加成断裂链转移(RAFT)试剂,在RAFT试剂的作用下,通过无皂乳液聚合方法合成了丙烯酸六氟丁酯与苯乙烯的共聚物.研究了RAFT试剂浓度和聚合温度对聚合动力学、聚合反应可控性及乳胶粒粒径的影响.通过红外光谱(FTIR)、核磁共振谱(1H NMR)、示差扫描量热仪(DSC)、凝胶渗透色谱仪(GPC)及表面张力仪表征了共聚物的结构、玻璃化转变温度(Tg)、分子量和分子量分布及乳胶膜表面性能.结果表明,得到的苯乙烯和丙烯酸六氟丁酯共聚物无皂乳液的乳胶粒粒径在100 nm左右且呈单分散分布.当RAFT试剂浓度高于0.016 mol/L时聚合体系有较好的可控性.共聚物乳液的乳胶膜对水和二碘甲烷的接触角都很高.     

Reversible addition-fragmentation chain transfer polymerization in microemulsion

This tutorial review first details the uncontrolled microemulsion polymerization mechanism, and the RAFT polymerization mechanism to provide the necessary background for examining the RAFT microemulsion polymerization mechanism. The effect of the chain transfer agent per micelle ratio and the chain transfer agent aqueous solubility on the RAFT microemulsion polymerization kinetics, polymer molecular weight and polydispersity, and polymer nanoparticle size are discussed with a focus on oil-in-water microemulsions. Modeling of RAFT microemulsion polymerization kinetics and the resulting final polymer molecular weight are presented to assist with the analysis of observed experimental trends. Lastly, the current significance of RAFT microemulsion polymerization and the future directions are discussed.     

Reversible Addition-Fragmentation Chain Transfer Polymerization of Methyl Methacrylate in Microemulsion: The Influence of Reaction Conditions on Polymerization

The reversible addition-fragmentation chain transfer (RAFT) polymerization of methyl methacrylate (MMA) using cetyltrimethylammonium bromide (CTAB) as surfactant and a difunctional RAFT agent S,S′-bis (α, α′-dimethylacetic acid) trithiocarbonate (BDAT) as chain transfer were conducted in microemulsion. The influence of polymerization temperature and concentration of RAFT agent on the polymerization were investigated, respectively. The results showed that the molecular weight of products increased linearly with conversion, the polydispersity indexes remained low value, and the polymerization processes were totally under control with increasing concentration of RAFT agent, the polymerization behavior exhibited living polymerization characters. In addition, the influence of RAFT concentration on the particle size was investigated by TEM. The results indicated that the particles were highly monodispersed and the particle size increased with increasing concentration of RAFT agent.     

Kinetic modeling of controlled living microemulsion polymerizations that use reversible addition–fragmentation chain transfer

Reversible addition–fragmentation chain transfer (RAFT) polymerization is a useful technique for the formation of polymers with controlled architectures and molecular weights. However, when used in the polymerization of microemulsions, RAFT agents are only able to control the polymer molecular weight only at high RAFT concentrations. Here, a kinetic model describing RAFT microemulsion polymerizations is derived that predicts the reaction rates, molecular weight polydispersities, and particle size. The model predicts that at low RAFT concentrations, the RAFT agent will be consumed early in the reaction and that this will result in uncontrolled polymerization in particles nucleated late in the reaction. The higher molecular weight polydispersity that is observed in RAFT microemulsion polymerizations is the result of this uncontrolled polymerization. The model also predicts a shift in the conversion at which the maximum reaction rate occurs and a decrease in the particle size with increasing RAFT concentration. Both of these trends are also consistent with those observed experimentally. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 6055–6070, 2006     

A simple methodology for the synthesis of heterotelechelic protein–polymer–biomolecule conjugates

A synthetic protocol for the preparation of hetero‐biofunctional protein–polymer conjugates is described. A chain transfer agent, S,S‐bis (α,α′‐dimethyl‐α″‐acetic acid) trithiocarbonate was functionalized with α,ω‐pyridyl disulfide (PDS) groups, Subsequently, one of the PDS groups was covalently attached to bovine serum albumin (BSA) at the specific free thiol group on the cysteine residue through a disulfide linkage. The second PDS group remained intact, as it was found to be inaccessible to further BSA functionalization. The BSA‐macro‐reversible addition‐fragmentation chain transfer (RAFT) agent was then used to prepare BSA‐polymer conjugates via in situ polymerization of oligo (ethyleneglycol) acrylate and N‐(2‐hydroxypropyl) methacrylamide using an ambient temperature initiator, 4,4′‐azobis [2,9‐imidazolin‐2‐ethyl)propane] dihydrochloride in an aqueous medium. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) confirmed that the in situ polymerization occurred at the protein surface where the RAFT agent was attached and the molecular weights of the BSA–polymer conjugates were found to increase concomitantly with monomer conversion and polymerization time. After polymerization the remaining terminal PDS groups were then utilized to attach thiocholesterol and a flurophore, rhodamine B to the protein–polymer conjugates via disulfide coupling. UV–Vis and fluorescence analyses revealed that ～80% of the protein conjugates were found to retain integral PDS end groups for further attachment to free thiol‐tethered precursors. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 1399–1405, 2010     

端三吡啶基聚异丙基丙烯酰胺的合成及其金属配合物

合成出三吡啶官能化的二硫代酯并以其作为链转移剂进行了异丙基丙烯酰胺(NIPAAm)的可逆加成断裂链转移(RAFT)聚合,得到分子量可控和窄分子量分布的端三吡啶基聚异丙基丙烯酰胺(tpy-PNIPAAm),聚合反应为对单体浓度的一级动力学关系.Tpy-PNIPAAm在水中既保持了与聚异丙基丙烯酰胺类似的相转变行为,又拥有三吡啶(tpy)的强络合能力.将tpy-PNIPAAm与金属核素模型进行络合得到金属配合物,所得金属配合物在水中拥有与PNIPAAm类似的相转变行为,同时tpy-PNIPAAm的金属配合物由于其两亲性,在水中可以形成纳米粒子,由于纳米粒子间的静电排斥作用,这种纳米粒子即使在高于相转变温度时,仍能稳定于水中.     

Controlled bimodal molecular-weight-distribution polymers: facile synthesis by RAFT polymerization

The RAFT agents RAFT-1 and RAFT-2 were used for RAFT polymerization to synthesize well-defined bimodal molecular-weight-distribution (MWD) polymers. The system showed excellent controllability and "living" characteristics toward both the higher- and lower-molecular-weight fractions. It is important that bimodal higher-molecular-weight (HMW) polymers and block copolymers with both well-controlled molecular weight (MW) and MWD could be prepared easily due to the "living" features of RAFT polymerization. The strategy realized a mixture of higher/lower-molecular-weight polymers at the molecular level but also preserved the features of living radical polymerization (LRP) of the RAFT polymerization.     

Synthesis of well‐defined three‐armed polystyrene having thiourethane–isocyanurate as the core structure derived from trifunctional five‐membered cyclic dithiocarbonate

The synthesis of a three‐armed polymer with an isocyanurate–thiourethane core structure is described. Monofunctional reversible addition–fragmentation chain transfer (RAFT) agent 2 and trifunctional RAFT agent 5 were prepared from mercapto‐thiourethane and tris(mercapto‐thiourethane), which were obtained from the aminolysis of mono‐ and trifunctional five‐membered cyclic dithiocarbonates, respectively. The radical polymerization of styrene in the presence of 2,2′‐azobis(isobutyronitrile) and RAFT agent 2 in bulk at 60 °C proceeded in a controlled fashion to afford the corresponding polystyrene with desired molecular weights (number‐average molecular weight = 3000–10,100) and narrow molecular weight distributions (weight‐average molecular weight/number‐average molecular weight < 1.13). On the basis of the successful results with the monofunctional RAFT agents, three‐armed polystyrene with thiourethane–isocyanurate as the core structure could be obtained with trifunctional RAFT agent 5 in a similar manner. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 5498–5505, 2005     

Living radical miniemulsion polymerization by RAFT in the presence of beta-cyclodextrin

Living radical polymerization of styrene in a miniemulsion by reversible addition–fragmentation chain transfer (RAFT) was successfully realized in the presence of beta-cyclodextrin (CD), using sodium dodecyl sulfate and hexadecane as surfactant and costabilizer, respectively. The drawback of instability (red layer formation) encountered in the living radical polymerization in emulsion or miniemulsion was overcome. The linear relationship between the monomer conversion and the molecular weight, as well as lower molecular weight distribution (MWD), shows that the polymerization process was under control. The addition of CD was found to have little influence on the polymerization rate. However, MWD of the polymer synthesized is obviously decreased. The mechanism of stability and controllability improvement in the presence of CD proposed that the complex formation between CD and RAFT agent or RAFT agent-ended oligomer increased their diffusion ability from monomer droplet to polymerization locus and improved the homogeneity of the RAFT agent level among the polymerization loci.     

Well-defined higher-molecular-weight polyacrylonitrile via RAFT technique in the presence of disulfide compounds as a source of chain transfer agent

Well-defined polyacrylonitrile with a higher number-average molecular weight () up to 200,000 and a lower polydispersity index (PDI, 1.7-2.0) was firstly obtained via reversible addition-fragmentation chain transfer (RAFT) process. This was achieved by selecting a stable, easy way to prepare disulfide compound intermediates including bis(thiobenzoyl) disulfide (BTBDS) and bis(thiophenylacetoyl) disulfide (BTPADS) to react with azobis(isobutyronitrile) to directly synthesize RAFT agents in situ. The polymerization of acrylonitrile (AN) displays the characteristics of controlled/living radical polymerization as evidenced by pseudo first-order kinetics of polymerization, linear evolution of molecular weight with increasing monomer conversion, and narrow PDIs. The polymerization rate and the efficiency for producing RAFT agent of BTPADS system are obviously higher than those of BTBDS system, whereas the control of the latter over the polymerization is superior to that of the former. ¹H NMR analysis has confirmed the dithioester chain-end functionality of the resultant polymer. The RAFT copolymerizations of AN and the comonomers including methyl acrylate, itaconic acid, methyl methacrylate, n-butyl acrylate, 2-hydroxyethyl acrylate, and acrylamide were also successfully carried out using the same polymerization system.     

两亲性RAFT试剂存在下丙烯酸六氟丁醋和苯乙烯的无皂乳液聚合

合成了具有两亲性结构的可逆加成断裂链转移(RAFT)试剂,在RAFT试剂的作用下,通过无皂乳液聚合方法合成了丙烯酸六氟丁酯与苯乙烯的共聚物.研究了RAFT试剂浓度和聚合温度对聚合动力学、聚合反应可控性及乳胶粒粒径的影响.通过红外光谱(FTIR)、核磁共振谱(1H NMR)、示差扫描量热仪(DSC)、凝胶渗透色谱仪(GP...     

SET‐RAFT of MMA mediated by ascorbic acid‐activated copper oxide

In this work, cupric oxide (CuO) or cuprous oxide (Cu₂O) was used as the catalyst for the single electron transfer‐reversible addition‐fragmentation chain transfer (SET‐RAFT) polymerization of methyl methacrylate in the presence of ascorbic acid at 25 °C. 2‐Cyanoprop‐2‐yl‐1‐dithionaphthalate (CPDN) was used as the RAFT agent. The polymerization occurred smoothly after an induction period arising from the slow activation of CuO (or Cu₂O) and the “initialization” process in RAFT polymerization. The polymerizations conveyed features of “living”/controlled radical polymerizations: linear evolution of number‐average molecular weight with monomer conversion, narrow molecular weight distribution, and high retention of chain end fidelity. From the polymerization profile, it was deduced that the polymerization proceeded via a conjunct mechanism of single electron transfer‐living radical polymerization (SET‐LRP) and RAFT polymerization, wherein CPDN acting as the initiator for SET‐LRP and chain transfer agent for RAFT polymerization. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012