Reversible Termination Research Articles

Template-Independent Enzymatic RNA Synthesis.

A route to prepare ribonucleoside triphosphates featuring a 3'-aminoxy (3'-O-NH 2 ) removable blocking group is reported here. We then show that versions of two DNA polymerases, human DNA polymerase theta (Polθ) and mimiviral PrimPol, accept these triphosphates as substrates to add single nucleotides to an RNA primer under engineered conditions. Cleaving the O-N bond in the 3'-O-NH 2 group within the extended primer regenerates the 3'-OH group, facilitating subsequent polymerase cycles that add a second, selected, nucleotide. These enzymes and triphosphates together enable template-independent enzymatic RNA synthesis (TIERS) exploiting a cyclic reversible termination framework. The study shows that this process is ready for instrument adaptation by using it to add three ribonucleotides in three cycles using an engineered Polθ. This work creates a new way to synthesize RNA with a de novo defined sequence, without requiring the protecting groups, hazardous solvents, and sensitive reagents that bedevil phosphoramidite-based RNA synthesis.

Proton transfer anionic polymerization with C-H bond as the dormant species.

Living anionic polymerization-the most common living polymerization and the one with the longest history-generally requires stringent, water-free conditions and one metal initiator per polymer chain. Here we present the proton transfer anionic polymerization of methacrylates using acidic C-H bonds as the dormant species that are activated by base catalysts. The polymerization mechanism involves reversible chain transfer or termination of the growing enolate species. A weakly acidic compound, such as an alkyl isobutyrate, serves as the initiator or chain-transfer agent in the presence of a bulky potassium base catalyst to produce a polymer chain and, thereby, diminishes the metal compound per chain ratio. An added alcohol serves as a reversible terminator to tame the propagation. End-functionalized, star, block and graft polymers are easily accessible from compounds with C-H bonds.

Generalized Model for Inhibitor-Modulated 2D Polymer Growth to Understand the Controlled Synthesis of Covalent Organic Frameworks.

Two-dimensional (2D) polymers, also known as 2D covalent organic frameworks (COFs), are increasingly finding use in applications such as membrane separations, catalysis, and energy conversion. Current research is focused on the development of new synthesis routes for COFs and obtaining a mechanistic understanding of the growth process to control it in a better manner. In this regard, synthesis methods such as reversible polycondensation termination use monofunctional inhibitor species to achieve a controlled growth rate for COFs. However, so far, the role of the inhibitors in modulating the kinetics of COF growth is inadequately understood. In this work, inspired by the Mayo-Lewis framework, we develop a generalized kinetic model to describe the synthesis of a 2D COF monolayer. Our model involves six parameters corresponding to the rate constants of attachment and detachment of monomer and inhibitor species, as well as enhancement factors that quantify the effect of the local coordination environment of the attaching/detaching species on the reaction kinetics. We measure the inhibitor concentration-dependent growth kinetics of the COF experimentally and fit our model to experimental yield data, with the same parameters working across multiple inhibitor concentrations. As the growth process is inherently stochastic, we use this knowledge to develop a comprehensive kinetic Monte Carlo (KMC) simulation of 2D COF synthesis, demonstrating that scaled rate constants are required in the inherently local KMC simulations rather than those obtained from the global kinetic model. The KMC simulations point to an inverse flake size-inhibitor concentration relationship, in agreement with experiments, indicating that flake sizes could be precisely regulated by changing the inhibitor concentrations. Overall, our work promises to improve the understanding of 2D COF synthesis and will help in controlling the growth process to obtain the desired flake size distribution and product morphology.

A Hydrometalation Initiation Mechanism via a Discrete Cobalt-Hydride for a Rapid and Controlled Radical Polymerization.

Cobalt-mediated radical polymerization (CMRP) is a versatile technique for controlling the polymerization of vinyl monomers via reversible termination using CoII complexes as persistent radical deactivators. Here, we report a facile approach for the in situ generation of Co-H as a discrete initiator and mediator for CMRP of acrylate and acrylamide monomers, overcoming the limitations of existing initiation strategies. In situ oxidation of a CoII complex followed by transmetalation with silane generates a Co-H species, which initiates polymerization via hydrometalation of the monomer. This method precludes an induction period with excellent control over targeted molecular weight and dispersity. Strikingly, our approach allows complete polymerization when the induction period ends for conventional CMRP. A broad scope of monomers is amenable to this protocol, including acrylates and acrylamides. Tunable catalyst electronics afford tailored dispersity while maintaining agreement in molecular weight in stark contrast to conventional methods. Elimination of this induction period imbues polymerization behavior entirely to the catalyst electronic effects on reversible deactivation/activation rates.

Photoinduced organometallic mediating radical polymerization of acrylates mediated by CoII complexes of non-symmetrical tetradentate Schiff-base ligands

Three new cobalt(II) complexes with non-symmetrically-substituted N2O2 tetradentate Schiff-base ligands, prepared from the 1:1:1 condensation of 3-etoxy-salicylaldehyde, salicylaldehyde and ethylenediamine (H2Etsal − 1), o-phenylenediamine (H2Phsal − 2) or 1,2-cis,trans-cyclohexyldiamine (H2Cysal − 3), were synthesized. The complexes 1–3 were characterized by UV–Vis, FTIR, and MALDI-TOF spectroscopy, elemental analysis, molar conductance, cyclic voltammetry, and computational methods. The CoII-Schiff-base complexes (1–3) were used as controlling agents for the polymerization of n-butyl acrylate (BA) photoinitiated by diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO), according to an organometallic-mediated radical polymerization (OMRP) mechanism. Although, all complexes have presented good control ability, the polymerization mediated by complex 1 showed the best control over molecular weight (Mn matching with Mn,th) and dispersity (Ð < 1.50). Kinetic results and computational investigations support the activation/deactivation equilibrium, exerted by complexes 1–3, occurring by one face of the complexes via reversible termination (RT) mechanism.minim.

Photocontrolled reversible-deactivation radical polymerization of butyl acrylate mediated by Salen-type CoII complexes

Salen-type Co II complexes were applied in the photo-OMRP of BA and MA to synthetize a copolymer (poly[MA]– co -poly[BA]) under 390 and 400–800 nm LEDs. • Co II complexes with Schiff-base ligands were prepared. • The Co II complexes were applied in the photo-OMRP of butyl acrylate. • The photosystems proposed showed a living character being able to mediate the copolymerization of poly[MA]- b -[BA]. • Experimental and theoretical data suggest a majority reversible termination pathway for these photo-OMRP reactions. In this study, we synthesized the complex [Co(BuSalen)] using the novel tetradentate BuSalen ligand and analyzed its ability as a controlling agent for the photo-induced organometallic-mediated radical polymerization (photo-OMRP) of butyl acrylate (BA) using diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) as photo-initiator. BA polymerization with [Co(BuSalen)] reached significant control levels using the [BA]/[Co II ]/TPO molar ratio of 600/1/1. The [Co(Salen)] and [Co(Bu 2 Salen)] complexes were also obtained following literature methods aiming a comparison in the mediating ability in photo-OMRP. [Co(BuSalen)] and [Co(Bu 2 Salen)] showed better controlling ability, as observed in the copolymerization study (Đ= 2.64, 1.50, and 1.26 for complexes [Co(Salen)], [Co(BuSalen)] and [Co(Bu 2 Salen)], respectively). Kinetics studies and computational investigations support a reversible termination (RT) mechanism and a tailorable Co II complex reactivity mainly altered by Schiff-base steric factors. Calculated thermodynamic parameters agree with the controlling degree delivered by these complexes, since the polymerization control increases with increasing the ΔG values for the formation of dormant species.

Organo-Cobalt Complexes in Reversible-Deactivation Radical Polymerization.

Cobalt complexes have played an essential role in different chemical reactions. One of them that has attracted substantial attention in polymer science is cobalt mediated radical polymerization (CMRP), which is famous for its remarkable efficiency in controlling the radical polymerization of vinyl acetate (VAc) and other less active monomers (LAMs). Two pathways, reversible termination (RT) and degenerative transfer (DT), were recognized to control the polymerization in CMRP and could be further used to rationalize the mechanism of other RDRP methods. These control mechanisms were then found to be correlated to the redox potential of cobalt complexes and thus could be judged more quantitatively. The control of polymer composition and tacticity could also be achieved by using CMRP. The hybridization of CMRP and atom transfer radical polymerization (ATRP) could directly synthesize the vinyl acetate/methyl methacrylate and vinyl acetate/styrene block copolymers in one pot. The copolymer of acrylates and 1-octene could be obtained by visible-light-induced CMRP. With the addition of bulky Lewis acid, CMRP of N,N-dimethylacrylamide (DMA) showed high isotacticities with the contents of meso dyads (m) and meso triads (mm) up to 94 % and 87 %, respectively, and generated the crystalline PDMA with Tm as high as 276 °C. This personal account reviewed the development of CMRP with the mechanistic understanding, the control of composition and stereoselectivity of the polymeric products, and its perspective.

Reversible-deactivation radical polymerization of vinyl acetate mediated by tralen, an organomediator

An organic compound, tralen, has been developed as a mediator to control the radical polymerization of vinyl acetate, methyl acrylate, and N-vinyl pyrrolidone via the reversible termination mechanism.

Merging New Organoborane Chemistry with Living Radical Polymerization

In this issue of Cell Reports Physical Science, Pan and co-workers synthesize alkylcatecholboranes that can be oxidized by oxygen to drive the release of carbon-centered radicals for initiating reversible addition-fragmentation chain transfer (RAFT) polymerization. These organoboranes can slowly decompose to radicals that enable RAFT polymerization with exceptional control yielding ultra-high molecular weight (UHMW) polymers.

Enzymatic Synthesis of Designer DNA Using Cyclic Reversible Termination and a Universal Template.

Phosphoramidite chemistry remains the industry standard for DNA synthesis despite significant limitations on the length and yield of the oligonucleotide, time restrictions, and hazardous waste production. Herein, we demonstrate the synthesis of single-stranded oligos on a solid surface by DNA polymerases and reverse transcriptases. We report the extension of surface-bound oligonucleotides enabled by transient hybridization of as few as two bases to a neighboring strand. When multiple hybridization structures are possible, each templating a different base, a DNA polymerase or reverse transcriptase can extend the oligonucleotide with any of the complementary bases. Therefore, the sequence of the newly synthesized fragment can be controlled by adding only the desired base as a substrate to the reaction solution. We used this enzymatic approach to synthesize a 20 base oligonucleotide by incorporating reversible terminator dNTPs through a two-step cyclic reversible termination process with a corrected stepwise efficiency over 98%. In our approach, a nascent DNA strand that serves as both primer and template is extended through polymerase-controlled sequential addition of 3'-reversibly blocked nucleotides followed by subsequent cleavage of the 3'-capping group. This process enables oligonucleotide synthesis in an environment not permitted by traditional phosphoramidite methods, eliminates the need for hazardous chemicals, has the potential to provide faster and higher yield results, and synthesizes DNA on a solid support with a free 3' end.

Kinetic Features of Elementary Events in the Radical Polymerization of Methyl Methacrylate under Conditions of Nitroxide-Mediated Reversible Inhibition

The kinetic features of the elementary events of initiation, reinitiation, and reversible termination in the radical polymerization of methyl methacrylate mediated by piperidine and imidazoline nitroxides are studied. The rate constants of these reactions and the constant of equilibrium between the dormant and growing chains are determined. In all the systems studied, the reinitiation rate constant and the equilibrium constant decrease during polymerization. The activity of imidazoline nitroxides in the termination reaction turns out to be an order of magnitude lower than that of piperidine ones, and the constant of interaction of both types of nitroxides with methyl methacrylate growing radicals is two and a half orders of magnitude lower than that with their low molecular weight counterparts.

A versatile approach for the preparation of end‐functional polymers and block copolymers by stable radical exchange reactions

ABSTRACTA versatile strategy for the preparation of end‐functional polymers and block copolymers by radical exchange reactions is described. For this purpose, first polystyrene with 2,2,6,6‐tetramethylpiperidine‐1‐oxyl end group (PS‐TEMPO) is prepared by nitroxide‐mediated radical polymerization (NMRP). In the subsequent step, these polymers are heated to 130 °C in the presence of independently prepared TEMPO derivatives bearing hydroxyl, azide and carboxylic acid functionalities, and polymers such as poly(ethylene glycol) (TEMPO‐PEG) and poly(ε‐caprolactone) (TEMPO‐PCL). Due to the simultaneous radical generation and reversible termination of the polymer radical, TEMPO moiety on polystyrene is replaced to form the corresponding end‐functional polymers and block copolymers. The intermediates and final polymers are characterized by 1H NMR, UV, IR, and GPC measurements. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019, 57, 2387–2395

Degenerative chain‐transfer process: Controlling all chain‐growth polymerizations and enabling novel monomer sequences

ABSTRACTThe use of dormant species has opened a new era in precision polymerization and has changed the concept of living polymerization. The dormant species can be exchanged into the active species via reversible termination or via reversible chain transfer. Professor Mitsuo Sawamoto has greatly contributed to the establishment of the concepts of living cationic and radical polymerizations based on the reversible activation of dormant species. This highlight, dedicated to Professor Sawamoto on his retirement from Kyoto University, provides an overview of reversible or degenerative chain‐transfer (DT) processes, which are effective in controlling all chain‐growth polymerizations, including radical, cationic, anionic, coordination, ring‐opening metathesis, and ring‐opening polymerizations. In addition, structures with novel sequences accessible only by a combination of different propagating species with a common DT agent are reviewed. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019, 57, 243–254

Enzyme-Free Replication with Two or Four Bases.

All known forms of life encode their genetic information in a sequence of bases of a genetic polymer and produce copies through replication. How this process started before polymerase enzymes had evolved is unclear. Enzyme-free copying of short stretches of DNA or RNA has been demonstrated using activated nucleotides, but not replication. We have developed a method for enzyme-free replication. It involves extension with reversible termination, enzyme-free ligation, and strand capture. We monitored nucleotide incorporation for a full helical turn of DNA, during both a first and a second round of copying, by using mass spectrometry. With all four bases (A/C/G/T), an "error catastrophe" occurred, with the correct sequence being "overwhelmed" by incorrect ones. When only C and G were used, approximately half of the daughter strands had the mass of the correct sequence after 20 copying steps. We conclude that enzyme-free replication is more likely to be successful with just the two strongly pairing bases than with all four bases of the genetic alphabet.

Continuous Flow Photoinduced Reversible Deactivation Radical Polymerization

AbstractPhotoinduced reversible deactivation radical polymerization (RDRP) is a versatile and robust method to yield well‐defined polymers under mild conditions with additional advantages of spatial and temporal control. The process and scalability of photoinduced RDRP in the batch mode, however, are limited by the inherent light gradient effect according to the Beer–Lambert law. The continuous flow technique, as an alternative methodology, provides a solution to improve the irradiation efficiency and scale up the polymerization. This Minireview highlights recent progress in continuous flow photoinduced RDRP since 2016. Reversible termination RDRP, degenerative transfer RDRP and others are reviewed in consecutive order. An outlook for continuous flow photoinduced RDRP is also discussed.

Catalyzed Chain Transfer in Vinyl Acetate Polymerization Mediated by 9-Oxyphenalenone Cobalt(II) Complexes.

The vinyl acetate (VAc) radical polymerization initiated by V-70 at 30 °C in the presence of [CoII(OPN)2] (OPN = deprotonated 9-oxyphenalenone), 1, leads to PVAc of lower molecular weight (MW) than expected for organometallic-mediated radical polymerization (OMRP), whether reversible termination or degenerate transfer conditions are used. This represents the first clear evidence of catalyzed chain transfer (CCT) in VAc polymerization. The bis-pyridine adduct [CoII(OPN)2(py)2], 2, shows a marginally lower polymerization rate and an increased CCT activity relative to 1, whereas the activity decreases with marginal effect on the polymerization rate upon addition of excess py. However, raising the temperature to 80 °C (with AIBN as initiator) led to a low MW polymer even in the presence of a large py excess. The CCT was confirmed by 1H NMR characterization of the chain ends and by a MALDI-TOF MS analysis of the recovered polymer. The collective trends are consistent with greater CCT activity for the 5-coordinate complex [CoII(OPN)2(py)] relative to 1 and 2. The presence of py association/dissociation equilibria relating these three complexes was confirmed by a 1H NMR investigation.

RAS testing in metastatic colorectal cancer: advances in Europe.

Personalized medicine shows promise for maximizing efficacy and minimizing toxicity of anti-cancer treatment. KRAS exon 2 mutations are predictive of resistance to epidermal growth factor receptor-directed monoclonal antibodies in patients with metastatic colorectal cancer. Recent studies have shown that broader RAS testing (KRAS and NRAS) is needed to select patients for treatment. While Sanger sequencing is still used, approaches based on various methodologies are available. Few CE-approved kits, however, detect the full spectrum of RAS mutations. More recently, “next-generation” sequencing has been developed for research use, including parallel semiconductor sequencing and reversible termination. These techniques have high technical sensitivities for detecting mutations, although the ideal threshold is currently unknown. Finally, liquid biopsy has the potential to become an additional tool to assess tumor-derived DNA. For accurate and timely RAS testing, appropriate sampling and prompt delivery of material is critical. Processes to ensure efficient turnaround from sample request to RAS evaluation must be implemented so that patients receive the most appropriate treatment. Given the variety of methodologies, external quality assurance programs are important to ensure a high standard of RAS testing. Here, we review technical and practical aspects of RAS testing for pathologists working with metastatic colorectal cancer tumor samples. The extension of markers from KRAS to RAS testing is the new paradigm for biomarker testing in colorectal cancer.

The Mechanism and Thermodynamic Studies of CMRP: Different Control Mechanisms Demonstrated by CoII(TMP), CoII(salen*), and CoII(acac)2 Mediated Polymerization, and the Correlation of Reduction Potential, Equilibrium Constant, and Control Mechanism

The mechanism study of cobalt mediated radical polymerization (CMRP) is performed with CoII(TMP), CoII(salen*), and CoII(acac)2 at 60 °C to demonstrate that the control of vinyl acetate radical polymerization is achieved via a degenerative transfer pathway when CoII(TMP) and CoII(salen*) are used but via a reversible termination process with equilibrium constant (Keq) equal to 1.4 × 106 m−1 when CoII(acac)2 is the mediator. Given by the measured Keq in CMRP with varied cobalt complexes and the reduction potential (E1/2) of the corresponding cobalt complexes, a linear correlation between log(Keq), and E1/2 is found and can be used to estimate the Keq of other CMRP systems. With the enriched database of Keq values, the control mechanisms of CMRP are quantitatively defined by CoII/CoIII equilibrium constant. The connection of reduction potential, equilibrium constant, and control mechanism would contribute to the in‐depth understanding of CMRP process. image

Cobalt Bipyridine Bisphenolate Complex in Controlled/Living Radical Polymerization of Vinyl Monomers

Cobalt(II) bipyridine bisphenolate (CoII(BpyBph)) was applied to mediate the controlled/living radical polymerization of vinyl acetate (VAc), methyl acrylate (MA), and other vinyl monomers such as N-vinylpyrrolidone (NVP) and acrylonitrile (AN). The living characters of linear increased molecular weight with monomer conversion, relatively narrow molecular weight distribution, and the synthesis of block copolymer demonstrated by the formation of PVAc-b-PNVP were all observed. The polymerization of VAc was proposed to be mediated by the degenerative transfer (DT) process, but the polymerization of MA should be mainly controlled by the reversible termination (RT) process since the equilibrium constant (Keq = [CoIII–R]/[CoII][R•]) between cobalt(II) and organocobalt(III) was measured as 8.6 × 107 M–1. The correlation between Keq and the redox potential (E1/2) of cobalt complexes was preliminarily observed as a higher E1/2 leading to a larger Keq.

UV Light as External Switch and Boost of Molar-Mass Control in Iodine-Mediated Polymerization

UV-initiated iodine-mediated polymerization of n-butyl methacrylate employing an iodo chain-transfer agent and a photoinitiator was studied with respect to its kinetics and mechanism. UV irradiation was found to be a significant boost for molar-mass control compared to thermally initiated systems, drastically lowering dispersities from above 1.5 down to 1.1. The reason for this, as proven by extensive kinetic simulations, is the incessant C–I-bond cleavage and formation of free iodine on the nanomolar scale, which leads to a reversible termination mechanism coexisting to degenerative chain-transfer. In experiments with intermittent irradiation, the system was perfectly dormant with no polymerization occurring during the dark time, illustrating the potential as photoswitchable controlled polymerization system. Photopolymerization at elevated temperatures is presented as a tool for increasing polymerization rate while fully retaining molar-mass control. The presented system is highly robust and proceeds without the use of smelly or toxic additives, offering a cheap and green way to produce externally triggered well-defined polymer and extending the scope of iodine-mediated polymerizations.