A 'click approach' to the generation of novel profluorescent nitroxides

This project focused on the first application of the copper catalyzed azide alkyne cycloaddition reaction for the generation of novel profluorescent systems. Through this approach four novel profluorescent nitroxides were prepared both rapidly and in good yield from coumarin and nitroxide CuAAC coupling partners. Specifically, 7-hydroxy, 7-diethylamino, 6-bromo and unsubstituted coumarin analogues bearing an azide group in the 3-position were prepared and conjugatively joined to an alkyne isoindoline nitroxide previously reported by our group. To explore the impact of the nitroxide moiety on the fluorescence of these systems, methoxyamine analogues of the corresponding nitroxide analogues were prepared. Spectrophotometric analysis of these methoxyamine analogues revealed that the aromatic systems possessed high quantum yields. However, the quantum yield efficiency was found to be dependent on the presence of electron donating substituents in the 7-position of the coumarin motif, which enhanced the charge-transfer character of the system. Furthermore, spectrophotometric analysis of nitroxide analogues demonstrated that the triazole effectively mediated fluorophore-nitroxide communication, as evidenced by the low quantum yield values of the nitroxide analogues. These results suggest that this technique can be used to conjugatively join any azide bearing fluorescent system with the key alkyne isoindoline coupling partner allowing for the rapid generation of diverse profluorescent systems.

Photoclickable surfaces for profluorescent covalent polymer coatings

The nitrile imine-mediated tetrazole-ene cycloaddition reaction (NITEC) is introduced as a powerful and versatile conjugation tool to covalently ligate macromolecules onto variable (bio)surfaces. The NITEC approach is initiated by UV irradiation and proceeds rapidly at ambient temperature yielding a highly fluorescent linkage. Initially, the formation of block copolymers by the NITEC methodology is studied to evidence its efficacy as a macromolecular conjugation tool. The grafting of polymers onto inorganic (silicon) and bioorganic (cellulose) surfaces is subsequently carried out employing the optimized reaction conditions obtained from the macromolecular ligation experiments and evidenced by surface characterization techniques, including X-ray photoelectron spectroscopy and FT-IR microscopy. In addition, the patterned immobilization of variable polymer chains onto profluorescent cellulose is achieved through a simple masking process during the irradiation. Photoinduced nitrile imine-alkene 1,3-dipolar cycloaddition (NITEC) is employed to covalently bind well-defined polymers onto silicon oxide or cellulose. A diaryl tetrazole-functionalized molecule is grafted via silanization or amidification, respectively. Under UV light, a reactive nitrile imine rapidly forms and reacts with maleimide-functionalized polymers yielding a fluorescent linkage. Via a masking method, polymeric fluorescent patterns are achieved.

Application of profluorescent nitroxides for measurements of oxidative capacity of combustion generated particles

Oxidative stress caused by generation of free radicals and related reactive oxygen species (ROS) at the sites of deposition has been proposed as a mechanism for many of the adverse health outcomes associated with exposure to particulate matter (PM). Recently, a new profluorescent nitroxide molecular probe (BPEAnit) developed at QUT was applied in an entirely novel, rapid and non-cell based assay for assessing the oxidative potential of particles (i.e. potential of particles to induce oxidative stress). The technique was applied on particles produced by several combustion sources, namely cigarette smoke, diesel exhaust and wood smoke. One of the main findings from the initial studies undertaken at QUT was that the oxidative potential per PM mass significantly varies for different combustion sources as well as the type of fuel used and combustion conditions. However, possibly the most important finding from our studies was that there was a strong correlation between the organic fraction of particles and the oxidative potential measured by the PFN assay, which clearly highlights the importance of organic species in particle-induced toxicity.

Investigation of the chemical and physical basis of oxidative stress generated by particulate matter using the profluorescent probe technique

Following the growing need for adoption of alternative fuels, this project aimed at getting more information on the oxidative potential of biodiesel particulate matter. Within this scope, the physical and chemical characteristics of biodiesel PM were analysed which lead to identification of reactive organic fractions. An in-house developed proflurescent nitroxide probe was used. This project further developed in-depth understanding of the chemical mechanisms following the detection of the oxidative potential of PM. This knowledge made a significant contribution to our understanding of processes behind negative health effects of pollution and enabled us to further develop new techniques to monitor it.

Isolation and use of chromosome 1 probes for linkage studies on Charcot-Marie-Tooth disease

Nine probes were isolated from a human chromosome 1 enriched library and mapped to regions of chromosome 1 using somatic cell hybrid lines. One clone, LR67, which mapped 1q12→q23 detected a BglI RFLP. This probe, as well as 4 other known chromosome 1 markers, α-spectrin, Factor XIIIB, DR10 and DR78, were used for linkage studies in 15 Charcot-Marie-Tooth disease (CMT1) families. Close linking of CMT1 to any of the 5 markers was not indicated. Total lod scores excluded linkage of CMT1 to LR67 and to DR10 at 5 cM or less, to DR78 and 10 cM or less, α-spectrin at 15 cM or less and Factor XIIIB at 20 cM or less. Possible linkage, however, was shown between LR67 and CMT1 at a distance of 30 cM. Also linkage at a distance of 5 cM was detected between this probe and α-spectrin.

DNA probes in Charcot-Marie-Tooth neuropathy

Results of Duffy (Fy) linkage confirm genetic heterogeneity in Charcot-Marie-Tooth disease type 1 (CMT1). Of 11 families informative for Fy, four showed probable linkage with CMT1, seven showed probable non-linkage and two showed definite non-linkage. These results suggest that Fy linked CMT1 may be less common than previously thought. These results combined with those of another DNA probe for the antithrombin III gene confirm that there are at least two gene loci for CMT1, termed 1A and 1B.

Organizational probes : exploring playful interactions in work environment

Playfulness, with non-intrusive elements, can be considered a useful resource for enhancing social awareness and community building within work organizations. Taking inspirations from the cultural probes approach, we developed organizational probes as a set of investigation tools that could provide useful information about employees’ everyday playful experiences within their work organizations. In an academic work environment, we applied our organizational probes over a period of three weeks. Based on the collected data we developed two design concepts for playful technologies in work environments.

Catalytic probes with nanostructured surface for gas/discharge diagnostics : a study of a probe signal behaviour

Catalytic probes are used for plasma diagnostics in order to quantify the density of neutral atoms. The probe response primarily depends on the probe material and its surface morphology. Here we report on the design, operation and modelling of the response of niobium pentoxide sensors with a flat and nanowire (NW) surfaces. These sensors were used to detect neutral oxygen atoms in the afterglow region of an inductively coupled rf discharge in oxygen. A very different response of the flat-surface and NW probes to the varying densities of oxygen atoms was explained by modelling heat conduction and taking into account the associated temperature gradients. It was found that the nanostructure probe can measure in a broader range than the flat oxide probe due to an increase in the surface to volume ratio, and the presence of nanostructures which act as a thermal barrier against sensor overheating. These results can be used for the development of the new generation of catalytic probes for gas/discharge diagnostics in a range of industrial and environmental applications.

Modular design of profluorescent polymer sensors

A simple modular strategy for the synthesis of profluorescent nitroxide containing polymers is described. The incorporation of an epoxide as a pendant functionality on a polymer backbone synthesized using ATRP and subsequent nucleophilic ring-opening with sodium azide gave hydroxyl and azide functionality within a 3-bond radius. Orthogonal coupling chemistry then allowed the independent attachment of fluorophore and nitroxide groups in close proximity, giving rise to a profluorescent polymer. Validation of the viability of these materials as fluorescent sensors is demonstrated through efficient fluorescence switch-on observed when the materials are exposed to a model reductant or carbon-centred radical source.

Synthesis and properties of fullerene C60 and C70 spin probes containing isoindoline nitroxides

Three fullerene isoindoline nitroxides N-methyl-3,4-fulleropyrrolidine-2-spiro-5′- (1′,1′,3′,3′-tetramethylisoindolin-2′-yloxyl), (C60-(TMIO)m, and C70-(TMIO)n) were synthesized by the covalent bonding of 5-formyl-1,1,3,3-tetramethyl isoindolin-2-yloxyl to the fullerenes C60 and C70. Significantly, the X-ray photoelectron spectra indicated the characteristic N 1s signals of NO. at 402 eV. The atomic force microscope morphologies showed that the average particle sizes of C60-(TMIO)m and C70-(TMIO)n were 38 and 15 nm. The electrochemical experiments indicated that fullerene bound isoindoline nitroxides retained similar electrochemical properties and redox reaction mechanisms as the parent nitroxides. The electron paramagnetic resonance spectra of the fullerene isoindoline nitroxides all exhibited the hyperfine splittings and characteristic spectra of tetramethyl isoindoline nitroxides, with typical nitroxide g-values and nitrogen isotropic hyperfine coupling constants. Therefore, these fullerene isoindoline nitroxides may be considered as potential candidates for novel biological spin probes using electron paramagnetic resonance spectroscopy.

Dynamic, invivo, real-time detection of retinal oxidative status in a model of elevated intraocular pressure using a novel, reversibly responsive, profluorescent nitroxide probe

Changes to the redox status of biological systems have been implicated in the pathogenesis of a wide variety of disorders including cancer, Ischemia-reperfusion (I/R) injury and neurodegeneration. In times of metabolic stress e.g. ischaemia/reperfusion, reactive oxygen species (ROS) production overwhelms the intrinsic antioxidant capacity of the cell, damaging vital cellular components. The ability to quantify ROS changes in vivo, is therefore essential to understanding their biological role. Here we evaluate the suitability of a novel reversible profluorescent probe containing a redox-sensitive nitroxide moiety (methyl ester tetraethylrhodamine nitroxide, ME-TRN), as an in vivo, real-time reporter of retinal oxidative status. The reversible nature of the probe's response offers the unique advantage of being able to monitor redox changes in both oxidizing and reducing directions in real time. After intravitreal administration of the ME-TRN probe, we induced ROS production in rat retina using an established model of complete, acute retinal ischaemia followed by reperfusion. After restoration of blood flow, retinas were imaged using a Micron III rodent fundus fluorescence imaging system, to quantify the redox-response of the probe. Fluorescent intensity declined during the first 60 min of reperfusion. The ROS-induced change in probe fluorescence was ameliorated with the retinal antioxidant, lutein. Fluorescence intensity in non-Ischemia eyes did not change significantly. This new probe and imaging technology provide a reversible and real-time response to oxidative changes and may allow the in vivo testing of antioxidant therapies of potential benefit to a range of diseases linked to oxidative stress

Perylene-based profluorescent nitroxides for the rapid monitoring of polyester degradation upon weathering: An assessment

A profluorescent nitroxide possessing an isoindoline nitroxide moiety linked to a perylene fluorophore was developed to monitor radical mediated degradation of melamine-formaldehyde crosslinked polyester coil coatings in an industry standard accelerated weathering tester. Trapping of polyester-derived radicals (most likely C-radicals) that are generated during polymer degradation leads to fluorescent closed-shell alkoxy amines, which was used to obtain time-dependent degradation profiles to assess the relative stability of different polyesters towards weathering. The nitroxide probe couples excellent thermal stability and satisfactory photostability with high sensitivity and enables detection of free radical damage in polyesters under conditions that mimic exposure to the environment on a time scale of hours rather than months or years required by other testing methods. There are indications that the profluorescent nitroxide undergoes partial photo-degradation in the absence of polymer-derived radicals. Unexpectedly, it was also found that UV-induced fragmentation of the NO–C bond in closed-shell alkoxy amines leads to regeneration of the profluorescent nitroxide and the respective C-radical. The maximum fluorescence intensity that could be achieved with a given probe concentration is therefore not only determined by the amount of polyester radicals formed during accelerated weathering, but also by the light-driven side reactions of the profluorescent nitroxide and the corresponding alkoxy amine radical trapping products. Studies to determine the optimum probe concentration in the polymer matrix revealed that aggregation and re-absorption effects lowered the fluorescence intensity at higher concentrations of the profluorescent nitroxide, but too low probe concentrations, where these effects would be avoided, were not sufficient to trap the amount of polyester radicals formed upon weathering. The optimized experimental conditions were used to assess the impact of temperature and UV irradiance on polymer degradation during accelerated weathering.

ESR measurements of the partitioning of some new spin probes in n-octanol-water

The stable free radical 1,1,3,3-tetramethylisoindolin-2-yloxyl (TMIO) has proved to be very suitable for use as a spin probe for a number of applications. Because it is soluble mainly in non-polar liquids, there is a need for new derivatives that can be used in a variety of environments. This has been done by introducing substituents in the 5-position of the aromatic ring, namely carboxyl (CTMIO), trimethylamino (TMTMIOI) and sodium sulphonate (NaTMIOS). An accurate ESR method was developed for the measurement of partition coefficients in n-octanol–water. For comparison purposes the method was also applied to some Tempo derivatives. The effect of temperature on the rotational correlation times and the nitrogen-14 hyperfine coupling constant of some of the spin probes was investigated. There is evidence for dimerization of CTMIO to form a biradical

Chemical probes into the active centre of a heme thiolate monoxygenase

Linalool-8-monoxygenase, a typical bacterial P-450 heme thiolase, shows a high degree of substrate specificity towards linalool. The active site of the pure enzyme has been probed with a large number of substrate analogues with systematic alterations or conformational variations in the linalool molecule. The comparison of three parameters, the mo→mos conversion of the enzyme as a result of substrate binding monitored at 392 nm, theK D of the analogues giving information about energies of association and the relative turnover as substrate have given information about the space-filling characteristics of the substrates in the enzyme cleft, the number of contacts the molecules make with the respective domains of the enzyme and the distance of the site undergoing hydroxylation from the oxygen site, respectively. The data permit the conclusion that linalool makes contact with the enzyme by hydrogen bonding with the hydroxyl group as well through hydrophobic association with all the eight carbons carrying hydrogen in the molecules.