Daniel J. Keddie

A novel profluorescent nitroxide as a sensitive probe for the cellular redox environment.

Changes to the redox status of biological systems have been implicated in the pathogenesis of a wide variety of disorders. Sensitive quantification of these changes has been developed using a novel fluorescent probe containing a redox-sensitive nitroxide moiety. As well as being able to selectively detect the superoxide radical in vitro, this method can measure overall changes to the cellular redox environment using flow cytometry on the basis of nitroxide reduction. The reversible nature of the probes detection mechanism offers the unique advantage of being able to monitor redox changes in both oxidizing and reducing directions in real time.

The scope for synthesis of macro-RAFT agents by sequential insertion of single monomer units

The scope for synthesis of new macro-RAFT agents (Z–C(S)S–(M)–R) by sequential insertion of monomers (M) ‘one at a time’ into an initial RAFT agent (Z–C(S)S–R) has been explored. The process is illustrated with the preparation of a styrene-N-isopropylacrylamide (NIPAM) co-dimer macro-RAFT agent [(CH3)3C(CN)–CH2CH(Ph)–CH2CH(CONHiPr)–SC(S)–S-alkyl] by successive single unit monomer insertions into a cyanoisopropyl trithiocarbonate. Critical factors for success are a high transfer constant for the RAFT agent and a high rate of addition of the radical (R·) to monomer relative to further propagation. With these conditions satisfied, the rate of reaction is largely determined by the rate of R· adding to monomer. Initiator-derived by-products (Z–C(S)S–(M)–I) become an issue when R· is different from the initiator-derived radical (I·).

Nitric Oxide and Nitroxides Can Act as Efficient Scavengers of Protein-Derived Free Radicals

Nitric oxide ((*)NO) may act as either a pro-oxidant or an antioxidant in biological systems. Although (*)NO and nitroxide radicals react slowly with most molecules, they react at near diffusion-controlled rates with other radicals and may therefore be efficient protective agents. This study assessed the ability of (*)NO and nitroxides to intercept specific protein-derived radicals and compared the efficacy of these species. Three protein radical systems were investigated as follows: BSA-derived radicals generated via radical transfer from H(2)O(2)-activated horseradish peroxidase, radicals formed on myoglobin via reaction with H(2)O(2), and carbon-centered radicals formed from amino acid hydroperoxides on exposure to Fe(2+)-EDTA. In each case, radicals were generated in the absence or presence of (*)NO or nitroxides of different size and charge. Concentration-dependent loss of the protein radicals was detected by electron paramagnetic resonance with both (*)NO and nitroxides and time-dependent consumption of (*)NO using an (*)NO electrode. The protein oxidation product dityrosine was significantly reduced by (*)NO and nitroxides, and 3,4-dihydroxyphenylalanine levels were reduced by nitroxides but not (*)NO. Overall, these studies demonstrate that (*)NO and nitroxides are efficient near-stoichiometric scavengers of protein radicals and, hence, are potential protective agents against protein oxidation reactions and resulting damage. These reactions show little dependence on nitroxide structure or charge.

One pot synthesis of higher order quasi-block copolymer libraries via sequential RAFT polymerization in an automated synthesizer

Recently developed sequential reversible addition–fragmentation chain transfer (RAFT) polymerization protocols allow the rapid, fully unattended preparation of quasi-block copolymer libraries that cover a wide range of copolymer compositions in an automated synthesizer. This contribution explores the scope and limitations of this sequential approach for the synthesis of higher order quasi-multiblock copolymers (including copolymer sequences of BAB, CBABC, ABC and ABCD). These syntheses illustrate the utility of this high-throughput approach for the one pot synthesis of functional polymers of increased complexity. Additionally, the use of this experimental technique for method development is highlighted.

Rapid and systematic access to quasi-diblock copolymer libraries covering a comprehensive composition range by sequential RAFT polymerization in an automated synthesizer

A versatile, cost-effective approach to the rapid, fully unattended preparation of systematic quasi-diblock copolymer libraries via sequential RAFT polymerization in an automated synthesizer is reported. The procedure is demonstrated with the synthesis of a 23 member library of low dispersity poly(butyl methacrylate)-quasiblock-poly(methyl methacrylate) covering a wide (fivefold) range of molar mass for the second block in a one-pot, sequential, RAFT polymerization.

Quasi-block copolymer libraries on demand via sequential RAFT polymerization in an automated parallel synthesizer

A convenient synthetic method for the systematic preparation of quasi-diblock copolymer libraries utilizing a sequential RAFT polymerization strategy is described. This method utilizes a parallel synthesizer and allows the unattended and fully automated synthesis of this type of library in a short period of time. The materials obtained in this investigation have shown properties very similar to those expected in “pure” diblock copolymers as determined by differential scanning calorimetry. The described method can be a useful and less expensive alternative for the rapid preparation and screening of block copolymer libraries.

The reactivity of N-vinylcarbazole in RAFT polymerization: trithiocarbonates deliver optimal control for the synthesis of homopolymers and block copolymers

The use of various RAFT agents (ZC(S)SR) including dithiobenzoates (Z = Ph), trithiocarbonates (Z = SR′), xanthates (Z = OR′), and conventional and switchable N-aryldithiocarbamates (Z = NR′Ar) in RAFT polymerization of N-vinylcarbazole (NVC) has been explored with a view to establishing which is most effective. Consistent with earlier work, we find that xanthates and N-aryldithiocarbamates give adequate control (dispersities (Đ) < 1.3) while dithiobenzoates give marked retardation. However, contrary to popular belief, we find that the polymerization of NVC is best controlled with trithiocarbonate RAFT agents, which provide both good molecular weight control, very narrow dispersities (Đ < 1.1), and high end-group fidelity. The results demonstrate that NVC has intermediate reactivity, i.e. between that of the traditional more activated (MAMs; styrene, acrylates) and less activated monomers (LAMs; vinyl acetate, N-vinylpyrrolidone). A further key to good control is the selection of RAFT agent R substituent to be both a good leaving group and a good initiating radical. The cyanomethyl group meets these criteria whereas phenylethyl is a poor initiating radical for NVC polymerization. A further demonstration of the intermediate reactivity of NVC and the derived propagating radical was the successful preparation of both poly(n-butyl acrylate)-block-poly(N-vinylcarbazole) and poly(N-vinylcarbazole)-block-poly(n-butyl acrylate) with a trithiocarbonate RAFT agent (the sequence of block synthesis is not important). Two-dimensional, liquid chromatography near critical conditions-gel permeation chromatography (LCCC-GPC) has been applied to demonstrate block purity. The corresponding styrene-based blocks can also be successfully synthesized, however, the reinitiation of NVC polymerization by the polystyryl radical proved to be a constraint on the purity of polystyrene-block-poly(N-vinylcarbazole).

Automated parallel freeze-evacuate-thaw degassing method for oxygen-sensitive reactions: RAFT polymerization.

An automated and parallel freeze-evacuate-thaw degassing method in a commercially available synthesizer is disclosed and tested for its applicability to reversible addition-fragmentation chain transfer (RAFT) polymerization. The effectiveness of this method to eliminate oxygen in polymerization reactions is demonstrated by directly comparing it against experiments performed using conventional laboratory techniques. Apart from the demonstrated accuracy, the proposed method has also shown significant precision when performing RAFT polymerizations. The reported experimental technique can be easily adapted to other chemical systems where the removal of oxygen is mandatory. This new high-throughput method has the potential to significantly increase the productivity and/or research outcomes in laboratories where oxygen-sensitive reactions are carried out.

Novel steric stabilizers for lyotropic liquid crystalline nanoparticles: PEGylated-phytanyl copolymers.

Lyotropic liquid crystalline nanostructured particles (e.g., cubosomes and hexosomes) are being investigated as delivery systems for therapeutics in biomedical and pharmaceutical applications. Long term stability of these particulate dispersions is generally provided by steric stabilizers, typically commercially available amphiphilic copolymers such as Pluronic F127. Few examples exist of tailored molecular materials designed for lyotropic liquid crystalline nanostructured particle stabilization. A library of PEGylated-phytanyl copolymers (PEG-PHYT) with varying PEG molecular weights (200-14K Da) was synthesized to assess their performance as steric stabilizers for cubosomes and to establish structure-property relationships. The PEGylated-lipid copolymers were first found to self-assemble in excess water in the absence of cubosomes and also displayed thermotropic liquid crystal phase behavior under cross-polarized light microscopy. An accelerated stability assay was used to assess the performance of the copolymers, compared to Pluronic F127, for stabilizing phytantriol-based cubosomes. Several of the PEGylated-lipid copolymers showed steric stabilizer effectiveness comparable to Pluronic F127. Using synchrotron small-angle X-ray scattering and cryo-transmission electron microscopy, the copolymers were shown to retain the native internal lyotropic liquid crystalline structure, double diamond cubic phase (Q2(D)), of phytantriol dispersions; an important attribute for controlling downstream performance.

Amphiphilic silicone architectures via anaerobic thiol-ene chemistry.

Despite broad application, few silicone-based surfactants of known structure or, therefore, surfactancy have been prepared because of an absence of selective routes and instability of silicones to acid and base. Herein the synthesis of a library of explicit silicone-poly(ethylene glycol) (PEG) materials is reported. Pure silicone fragments were generated by the B(C(6)F(5))(3)-catalyzed condensation of alkoxysilanes and vinyl-functionalized hydrosilanes. The resulting pure products were coupled to thiol-terminated PEG materials using photogenerated radicals under anaerobic conditions.