Chain Transfer Activity Research Articles

A novel method to quantify TSG‐6 protein levels in synovial fluid from osteoarthritis patients

AbstractTSG‐6, the protein product of Tumor Necrosis Factor‐stimulated gene‐6, is a potential biomarker of disease activity/progression in osteoarthritis (OA). By ELISA, TSG‐6 is elevated in synovial fluid (SF) from OA patients and its catalytic activity, for the covalent transfer of inter‐α‐inhibitor heavy chains onto hyaluronan (HA), is reported to correlate with progression to total knee replacement. Here, quantitative western blot (qWB) analysis, following sequential digestion with hyaluronidase and chondroitinase enzymes, was developed for the accurate determination of TSG‐6 levels in SFs. qWB analysis of 125 OA SFs yielded values of 1.0–45.2 μg/mL (median: 3.9 μg/mL), 100–1000‐fold higher than those previously reported; TSG‐6 concentration was positively correlated with age and pain/outcome scores in male patients. The values determined, by ELISA or a heavy chain transfer activity assay, following the same digestion conditions (in 23 SFs), were both ≥10‐fold lower. Apparent TSG‐6 catalytic activity was significantly affected by the concentration of HA present in SF or added exogenously. Thus, the interactions of TSG‐6 with components of OA SF prevent accurate determination of its concentration/activity when using existing assays. However, the quantitative western blot method developed here appears more suitable for further evaluation of the utility of TSG‐6 as a biomarker in OA.

Prooxidative chain transfer activity by thiol groups in biological systems

Cysteine is arguably the best-studied biological amino acid, whose thiol group frequently participates in catalysis or ligand binding by proteins. Still, cysteine's unusual biological distribution has remained mysterious, being strikingly underrepresented in transmembrane domains and on accessible protein surfaces, particularly in aerobic life forms (“cysteine anomaly”). Noting that lipophilic thiols have been used for decades as radical chain transfer agents in polymer chemistry, we speculated that the rapid formation of thiyl radicals in hydrophobic phases might provide a rationale for the cysteine anomaly. Hence, we have investigated the effects of dodecylthiol and related compounds in isolated biomembranes, cultivated human cells and whole animals (C. elegans). We have found that lipophilic thiols at micromolar concentrations were efficient accelerators, but not inducers of lipid peroxidation, catalyzed fatty acid isomerization to trans-fatty acids, and evoked a massive cellular stress response related to protein and DNA damage. These effects were specific for lipophilic thiols and were absent with thioethers, alcohols or hydrophilic compounds. Catalytic chain transfer activity by thiyl radicals appears to have deeply influenced the structural biology of life as reflected in the cysteine anomaly. Chain transfer agents represent a novel class of biological cytotoxins that selectively accelerate oxidative damage in vivo.

Metal Ion-dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix

The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.

Synthesis of Block Copolymers Based on N-alkyl Substituted Acrylamide via Combination of Reversible Addition-Fragmentation Transfer Polymerization and Click Chemistry

Chain transfer agents of azido terminated trithiocarbonate and alkyne terminated trithiocarbonate were synthesized by esterification reaction with trithiocarbonate (2-dodecylsulfanylthiocarbonylsulfanyl-2-methylpropionic acid, DMP). The kinetics of reversible addition-fragmentation chain transfer (RAFT) polymerization of N-isopropylacrylamide (NIPAAm) with the above three kinds of thiocarbonate as RAFT agents showed that chain transfer activity of azido-DMP and alkyne-DMP was similar, which was lower than that of DMP. Homopolymer (poly(N,N-dimethylacrylamide) (PDMA-C ≡ CH)) and block copolymer (P(DMA-b-NIPAAm)-N3) were successfully prepared by RAFT polymerization with desired structure. The subsequent preparation of triblock P(DMA-b-NIPAAm-b-DMA) was carried out by the click reaction. The molecular weight of P(DMA-b-NIPAAm-b-DMA) was the summation of molecular weight of reactant polymers and the molecular weight distribution was small (PDI<1.15), proving the successful click reaction.

Catalytic Chain Transfer in Continuous Emulsion Polymerization

AbstractIt is demonstrated that CCT‐mediated emulsion polymerization can successfully be carried out in a PSPC. Proper molecular weight control is achieved with only minor quantities of a cobalt(II) complex, illustrating the robustness and scalability of CCT. Despite the successful implementation of CCT in the PSPC, differences in the course of the polymerization and the final latex properties (in terms of the PSD/MWD) for batchwise and PSPC operation are observed. Backmixing occurs to some extend in the PSPC, which increases the steady state CCTA concentration in the column. Consequently, a higher chain transfer activity is observed than expected based on the corresponding batch experiments, which affects the course of the polymerization and the final latex properties.magnified image

Polymer Architecture Control in Emulsion Polymerization via Catalytic Chain Transfer Polymerization

Hyperbranched polymers are synthesized via a cobalt(II) mediated emulsion copolymerization of methyl methacrylate and diethylene glycol dimethacrylate. The concentration of divinyl monomer and chain transfer activity have to be balanced in order to prevent gelation of the polymer particles. The physiochemical properties of the cobalt(II) complex, specifically the intrinsic chain transfer activity and the partitioning behavior, play a crucial role in governing the polymer architecture (i.e., branched versus cross-linked). Furthermore, an empirical correlation, based on the chain transfer frequency and the divinyl monomer mol fraction, is presented and validated with the results obtained in emulsion polymerization as well as with available literature data.

Radical Polymerization of Vinyl Acetate with Bis(tetramethylheptadionato)cobalt(II): Coexistence of Three Different Mechanisms

The complex [Co(II)(tmhd)(2)] (4; tmhd = 2,2,6,6-tetramethylhepta-3,5-dionato) has been investigated as a mediator for controlled radical polymerization of vinyl acetate (VAc) and compared with the analogue [Co(II)(acac)(2)] (1; acac = acetylacetonato). A relatively well controlled process occurs, after an induction time, with 2,2'-azobis(4-methoxyl-2,4-dimethylvaleronitrile) (V-70) as radical initiator at 30 degrees C. However, whereas the polymerization essentially stops after about six initiator half-lives in the presence of 1, it continues with a first-order rate law in the presence of 4. The successful simulation of the kinetic data shows that 4 operates simultaneously by associative (degenerative transfer, DT) and dissociative (organometallic radical polymerization, OMRP) mechanisms. The occurrence of OMRP was confirmed by an independent polymerization experiment starting from an isolated and purified [Co(tmhd)(2)](PVAc) macroinitiator. The polymer molecular weight evolves linearly with conversion in accordance with the expected values for one chain per Co atom when DT is the predominant mechanism and also during the pure OMRP process; however, observation of stagnating molecular weights at long reaction times with concomitant breakdown of the first-order rate law for monomer consumption indicates a competitive chain-transfer process catalyzed by an increasing amount of Co(II). In the presence of external donors L (water, pyridine, triethylamine) the DT pathway is blocked and the OMRP pathway is accelerated, and polymerization with complex 4 is then about five times slower than with complex 1. The reversal of relative effective OMRP rate constants k(eff) (4>1 in the absence of external donors, 4<1 in their presence) is rationalized through competitive steric effects on Co(III)-C and Co(II)-L bond strengths. These propositions are supported by (1)H NMR studies and by DFT calculations.

Chain Transfer Activity of ω-Unsaturated Methacrylic Oligomers in Polymerizations of Methacrylic Monomers

Chain transfer constants have been determined for an unsaturated methyl methacrylate trimer, CH2C(CO2Me)CH2[C(CO2CH3)(CH3)CH2]2H (MMA3), in polymerizations of methacrylate esters (ethyl, EMA; n-butyl, BMA; tert-butyl, tBMA; 2-ethylhexyl, EHMA) and for the analogous trimers of butyl methacrylate (BMA3) and methacrylic acid (MAA3), a hydroxyethyl methacrylate dimer (HEMA2), a hydroxyethyl methacrylate−methyl methacrylate−hydroxyethyl methacrylate trimer (HEMA−MMA−HEMA), and a HEMA macromonomer in methyl methacrylate (MMA) polymerization. These data have been assessed with reference to our previously reported data on chain transfer of MMA macromonomers in MMA polymerizations. The transfer constants (Ctr) for MMA3 in polymerizations of methyl, ethyl, n-butyl, tert-butyl, and 2-ethylhexyl methacrylate at 60 °C are similar (∼0.18). The Ctr of MAA3 (0.28 at 60 °C in 2-butanone solvent) in MMA polymerization is ca. 50% higher than that of MMA3 under similar conditions. Other trimers (BMA3, HEMA−MMA−HEMA) and a lo...

Catalytic Chain Transfer in a Miniemulsion Copolymerization of Methyl Methacrylate and n-Butyl Acrylate

This paper reports on the application of catalytic chain transfer (CCT) in miniemulsion copolymn. It has been found that Co(Et)4BF has a similar chain transfer activity in bulk as CoBF. CCT copolymns. of MMA and BA can be performed in miniemulsion; large mol. wt. redns. are achieved, and at high conversions significant macromer incorporation occurs. [on SciFinder (R)]

Understanding Substrate Specificity of Polyketide Synthase Modules by Generating Hybrid Multimodular Synthases

Modular polyketide biosynthesis can be harnessed to generate rationally designed complex natural products through bioengineering. A detailed understanding of the features that govern transfer and processing of polyketide biosynthetic intermediates is crucial to successfully engineer new polyketide pathways. Previous studies have shown that substrate stereochemistry and protein-protein interactions between polyketide synthase modules are both important factors in this process. Here we investigated the substrate tolerance of different polyketide modules and assessed the relative importance of inter-module chain transfer versus chain elongation activity of some of these modules. By constructing a variety of hybrid modular polyketide synthase systems and assaying their ability to generate polyketide products, it was determined that the substrate tolerance of each individual ketosynthase domain is an important parameter for the successful recombination of polyketide synthase modules. Surprisingly, however, failure by a module to process a candidate substrate was not due to its inability to bind to it. Rather, it appeared to result from a blockage in carbon-carbon bond formation, suggesting that proper orientation of the initially formed acyl thioester in the ketosynthase active site was important for the enzyme-catalyzed decarboxylative condensation reaction.

Polymerization and copolymerization of dimethyl(1-ethoxycarbonyl)vinyl phosphate

Dimethyl(1-ethoxycarbonyl)vinyl phosphate (DMEVP), prepared in nearly quantitative yield from the reaction of ethyl bromopyruvate with trimethyl phosphite, polymerizes easily by free radical initiated polymerization and copolymerizes readily with styrene (S). Reactivity ratios determined at 60 and 135 °C were r S=0.21±0.02, r DMEVP=0.35±0.05 and r S=0.31±0.03, r DMEVP=0.31±0.04, respectively. Glass transition temperatures and thermal stabilities of the copolymers decreased but char yields obtained upon thermolysis increased as the DMEVP contents of the copolymers increased. 1H, 13C and 31P NMR spectra of the copolymers are reported. DMEVP exhibited mild chain transfer activity ( C tr∼0.01) in styrene polymerizations conducted at 135 °C and in emulsion polymerizations at 50 °C.

New applications of catalytic chain transfer polymerization to waterborne binders for automotive paint systems

Catalytic chain transfer polymerization (CCTP) is a conventional free radical polymerization technique that allows the preparation of macromonomers in a one step process. Acid functional macromonomers can be copolymerized with acrylate backbone monomers to form graft copolymers that, after neutralization with a base, are water dispersible. Low molecular weight oligomers from CCTP act as chain transfer agents themselves for methacrylate monomers via addition–fragmentation mechanism and lead to (semi) AB-block copolymers. Group transfer polymerization (GTP) is another polymerization technique to make AB-block copolymers but economically less attractive for functional comonomers since they interfere with the initiation mechanism. Graft and AB-block copolymer dispersions offer advantages in waterborne coatings compared to linear polymers of the same overall composition and molecular weight. Examples discussed in this paper are pigment dispersants and dispersion resins in which the backbone or A-segment of the copolymer has specific groups to anchor to the pigment surface and the side chains or B-segment give both charge and steric stabilization. AB-block copolymers with one block water soluble or dispersible also function as copolymerizable surfactants in an emulsion polymerization process and allow the synthesis of surfactant-free emulsions with low amounts of hydrophilic groups. The catalytic chain transfer agents (CCTAs) used in CCTP do have a high chain transfer activity for methacrylate monomers at very low concentrations so that low molecular weight oligomers (e.g. dimers) can be made. This chain transfer activity is lost in an emulsion polymerization process if the CCTA is (partly) water soluble. The paper will further demonstrate the use of dimers in the control of molecular weight in emulsion copolymerization.

Enzymatic Attachment of Glycosaminoglycan Chain to Peptide Using the Sugar Chain Transfer Reaction with Endo-β-xylosidase

Endo-beta-xylosidase from the mid-gut gland of the molluscus Patinopecten is an endo-type glycosidase that hydrolyzes the xylosyl serine linkage between a core protein and a glycosaminoglycan (GAG) chain, releasing the intact GAG chain from proteoglycan. In this study, we investigated GAG chain transfer activity of this enzyme, in order to develop a method for attaching GAG chains to peptide. Peptidochondroitin sulfate (molecular mass of sugar chain, 30 kDa) from bovine tracheal cartilage as a donor and butyloxycarbonyl-leucyl-seryl-threonyl-arginine-(4-methylcoumaryl-7-amide) as an acceptor were incubated with endo-beta-xylosidase. As a result, a reaction product with the same fluorescence as the acceptor peptide was observed. High pressure liquid chromatography analysis, cellulose acetate membrane electrophoresis, and enzymatic digestion showed that this reaction product had the chondroitin sulfate (ChS) from the donor. Furthermore, the acceptor peptide was released from this reaction product after hydrolysis by endo-beta-xylosidase. Therefore, it was confirmed that the ChS chain released from the donor was transferred to the acceptor peptide by the GAG chain transfer reaction of endo-beta-xylosidase. The optimal pH for hydrolysis by this enzyme was found to be about 4.0, whereas that for this reaction was about 3.0. Not only the ChS but also the dermatan sulfate and the heparan sulfate were transferred to the acceptor peptide by this reaction. By using this reaction, the GAG chain could be attached to the peptide in one step. The GAG chain transfer reaction of endo-beta-xylosidase should be a significant glycotechnological tool for the artificial synthesis of proteoglycan.

Minimizing tocopherol-mediated radical phase transfer in low-density lipoprotein oxidation with an amphiphilic unsymmetrical azo initiator.

The antioxidant alpha-tocopherol (alpha-TOH) has been found to act as a pro-oxidant under many in vitro conditions. The observed tocopoherol-mediated peroxidation (TMP) is dependent on two primary factors. (1) Chain transfer: alpha-TO. radical reacts with lipid to form lipid peroxyl radicals. (2) Phase transfer: alpha-TOH can transport radical character into the lipoprotein. Given the limitations of existing initiators, there is a need for new compounds that avoid the requirement for alpha-TOH to act as a phase-transfer agent. We report here a study showing that the new unsymmetrical azo compound, C-8, initiates LDL lipid peroxidation without requirement for alpha-TOH. This initiator provides a steady source of free amphiphilic peroxyl radicals that efficiently initiates oxidation of alpha-TOH-depleted LDL at a rate comparable to that reported for the very reactive hydroxyl radical (.OH). With other initiators tested, unsymmetrical C-12 and C-16 and symmetrical C-0 and MeOAMVN, alpha-TOH-depleted LDL displayed significant resistance to oxidation. Results indicate that the amphiphilic nature of the unsymmetrical initiators increases their partitioning into lipoprotein depending on the hydrocarbon chain length, and the symmetrical azo initiators C-0 and MeOAMVN primarily remain in the aqueous phase. Evidence suggests that even when the phase-transfer activity of alpha-TOH is limited, with the use of an initiator such as C-8, the mechanism of peroxidation remains controlled by TMP chain-transfer activity.

Catalytic chain transfer copolymerization of methyl methacrylate and methyl acrylate

Bis(aqua)bis((difluoroboryl)dimethylglyoximate)cobalt(II) (COBF) has proven to be a very effective catalytic chain transfer agent in the copolymerization of MA and MMA. The chain transfer activity depends on the fraction of MMA in the monomer feed and the total radical concentration. The polymerization can be described by a model that combines features of catalytic chain transfer for MMA homopolymerization and cobalt mediated controlled radical polymerization of MA. According to the model part of the COBF is covalently bonded to MA-ended polymeric radicals and cannot take part in the chain transfer step. The model can also account for the observed inhibition time that occurs at high chain transfer agent concentration and low fraction of MMA in the monomer feed.

Synthesis of fluorinated telomers. Part 6. Telomerisation of chlorotrifluoroethylene with methanol

Bulk radical telomerisation of chlorotrifluoroethylene (CTFE) with methanol was investigated for the synthesis of fluorinated alcohols. Improvements in the conversion of CTFE and of the selectivity of the reaction were sought by using different radical initiators (peroxides, percarbonates, peresters) and an excess of methanol. These reactions were compared in terms of CTFE conversion, yield of monoadduct and formation of higher molecular weight telomers. Under these conditions, radical initiated telomerisations systematically led to low molecular weight CTFE telomers. 2,5-Bis(tert-butylperoxy)-2,5-dimethylhexane (DHBP) proved to be the most effective radical initiator. All these experiments revealed the low chain transfer activity of methanol in the presence of CTFE. A thorough structural study of typical CTFE–methanol telomers was performed by 19F, 1H and 13C NMR spectroscopies. These analytical methods allowed us to prove the highly regioselective formation of HOCH2CF2CFClH (produced with a 95% selectivity), showing the regioselectivity of the addition of hydroxymethyl radicals onto the difluoromethylene side of CTFE.

Chain Transfer in the Sulfur-Centered Free Radical Ring-Opening Polymerization of 3-Methylene-6-methyl-1,5-dithiacyclooctane

3-Methylene-6-methyl-1,5-dithiacyclooctane (MDTO, 1) was polymerized in both the presence and absence of a number of chain transfer agents, viz. 1-butanethiol (BuSH), dibutyl disulfide (BuSSBu), and dibutyl sulfide (BuSBu). Mark−Houwink−Sakurada (MHS) constants for poly(MDTO) were found to be K = 23 × 10-5 dL g-1 and α = 0.67. Using these parameters, chain transfer constants were obtained at 60 °C and are reported as 0.13 (BuSH), 0.19 (BuSSBu), and 0.0025 (BuSBu). These values are compared to the corresponding chain transfer constants obtained for styrene (STY) and methyl methacrylate (MMA). Subsequently, the chain transfer activity was measured over a range of temperatures (40−80 °C), and Arrhenius parameters were determined. Significant differences in chain transfer activity between carbon- and sulfur-centered radicals were observed. In addition, the extent of transfer to monomer was determined across the range of temperatures 40−100 °C, yielding a value for CM of 55 × 10-4 at 60 °C. The likely extent o...

Study of activity enhancement by hydrogen in propylene polymerization using stopped-flow and conventional methods

The activity enhancement effect of hydrogen in propylene polymerization with a MgCl 2-supported Ziegler catalyst was studied by using the stopped-flow polymerization method, by which a reaction can be conducted within an extremely short period (ca. 0.1 s). Kinetic study using this method suggested that the hydrogen did not affect the propagation rate constant and did not lead to the formation of new active sites. The results of the stopped-flow polymerization with a pre-treated catalyst indicated that activity enhancement by hydrogen did not take place in the region, under which hydrogen acts as a chain transfer agent and the reduction of the titanium species is believed to occur to produce dead sites. Slurry polymerization with prolonged polymerization time indicated that hydrogen affected both chain transfer reaction and activity enhancement, which may be due to the reactivation of the dormant sites by hydrogen.

33] Assessment of prooxidant activity of vitamin E in human low-density lipoprotein and plasma

This chapter discusses the assessment of pro-oxidant activity of vitamin E in human low-density lipoprotein and plasma. The chapter discusses a series of simple tests that manipulate both the phase- and the chain-transfer activity of α-tocopherol (α-TOH) in low-density lipoprotein (LDL). While these tests can provide useful information on the mechanism of action of α -TOH in vitro , they do not give information on the action of vitamin E in vivo , nor propose that α -TOH acts as a prooxidant in vivo . The chapter presents various procedures for testing the pro-oxidant activity of α-TOH in lipoproteins and lipid emulsions. The methods described in the chapter allow investigations on the mechanism of in vitro lipoprotein lipid peroxidation and the way this process is affected by various (co-)antioxidants surrounding or associated with lipoproteins. The methods discussed also allow investigation of a possible role of tocopherol-mediated peroxidation (TMP) in cellular lipid peroxidation and antioxidation, particularly once analytical tools comparable to those for lipoproteins are available for the assessment of cellular lipid peroxidation.

Pressure and Temperature Dependence of Butyl Acrylate Propagation Rate Coefficients in Fluid CO2

Propagation kinetics of butyl acrylate (BA) free-radical homopolymerization in fluid CO2 has been studied by the PLP−SEC (pulsed laser polymerization−size-exclusion chromatography) method at pressures between 200 and 2000 bar and temperatures between −9 and 20 °C. The CO2 content was close to 45 wt % in all experiments. As a consequence of the poor chain-transfer activity in solution of CO2, the molecular-weight distributions of the polymer samples show well-resolved PLP structures with at least two, but in most cases three, distinct inflection points. At the p and T conditions of this work, the observed propagation rate coefficients (kp) of BA in fluid carbon dioxide at CO2 contents around 45 wt % are found to be about 40% below the corresponding bulk polymerization values. The activation volume and activation energy of kp are, however, identical for bulk and solution (in CO2) free-radical BA polymerizations within experimental accuracy. The origin of the solvent influence on the propagation reaction is ...