Synthesis of profluorescent isoindoline nitroxides via palladium-catalysed Heck alkenylation

The synthesis of a new structural class of isoindoline nitroxides (aminoxyls), accessible via the palladium-catalysed Heck reaction, is presented. Reaction of the aryl bromoamine, 5-bromo-1,1,3,3-tetramethylisoindoline (4) or dibromoamine, 5,6-dibromo-1,1,3,3-tetramethylisoindoline (5) or the analogous bromonitroxides 6 and 7 with methyl acrylate gives the acrylate substituted tetramethylisoindoline amines 8 and 10 and nitroxides 12 and 14. Similarly, the reaction of the aryl bromides and dibromides 4–7 with methyl 4-vinylbenzoate gives the carboxystyryl substituted tetramethylisoindoline amines 9 and 11 and the analogous nitroxides 13 and 15. The carboxystyryl tetramethylisoindoline nitroxides demonstrate strongly suppressed fluorescence, which is revealed upon removal of the free radical by reduction or radical coupling.

The synthesis and application of novel profluorescent nitroxides as probes for polymer degradation

This PhD project has expanded the knowledge in the area of profluorescent nitroxides with regard to the synthesis and characterisations of novel profluorescent nitroxide probes as well as physical characterisation of the probe molecules in various polymer/physical environments. The synthesis of the first example of an azaphenalene-based fused aromatic nitroxide TMAO, [1,1,3,3-tetramethyl-2,3-dihydro-2-azaphenalen-2-yloxyl, was described. This novel nitroxide possesses some of the structural rigidity of the isoindoline class of nitroxides, as well as some properties akin to TEMPO nitroxides. Additionally, the integral aromatic ring imparts fluorescence that is switched on by radical scavenging reactions of the nitroxide, which makes it a sensitive probe for polymer degradation. In addition to the parent TMAO, 5 other azaphenalene derivatives were successfully synthesised. This new class of nitroxide was expected to have interesting redox properties when the structure was investigated by high-level ab initio molecular orbitals theory. This was expected to have implications with biological relevance as the calculated redox potentials for the azaphenalene ring class would make them potent antioxidant compounds. The redox potentials of 25 cyclic nitroxides from four different structural classes (pyrroline, piperidine, isoindoline and azaphenalene) were determined by cyclic voltammetry in acetonitrile. It was shown that potentials related to the one electron processes of the nitroxide were influenced by the type of ring system, ring substituents or groups surrounding the moiety. Favourable comparisons were found between theoretical and experimental potentials for pyrroline, piperidine and isoindoline ring classes. Substitution of these ring classes, were correctly calculated to have a small yet predictable effect on the potentials. The redox potentials of the azaphenalene ring class were underestimated by the calculations in all cases by at least a factor of two. This is believed to be due to another process influencing the redox potentials of the azaphenalene ring class which is not taken into account by the theoretical model. It was also possible to demonstrate the use of both azaphenalene and isoindoline nitroxides as additives for monitoring radical mediated damage that occurs in polypropylene as well as in more commercially relevant polyester resins. Polymer sample doped with nitroxide were exposed to both thermo-and photo-oxidative conditions with all nitroxides showing a protective effect. It was found that isoindoline nitroxides were able to indicate radical formation in polypropylene aged at elevated temperatures via fluorescence build-up. The azaphenalene nitroxide TMAO showed no such build-up of fluorescence. This was believed to be due to the more labile bond between the nitroxide and macromolecule and the protection may occur through a classical Denisov cycle, as is expected for commercially available HAS units. Finally, A new profluorescent dinitroxide, BTMIOA (9,10-bis(1,1,3,3- tetramethylisoindolin-2-yloxyl-5-yl)anthracene), was synthesised and shown to be a powerful probe for detecting changes during the initial stages of thermo-oxidative degradation of polypropylene. This probe, which contains a 9,10-diphenylanthracene core linked to two nitroxides, possesses strongly suppressed fluorescence due to quenching by the two nitroxide groups. This molecule also showed the greatest protective effect on thermo-oxidativly aged polypropylene. Most importantly, BTMIOA was found to be a valuable tool for imaging and mapping free-radical generation in polypropylene using fluorescence microscopy.

Synthesis and Raman spectroscopic study of Mg/Al,Fe hydrotalcites with variable cationic ratios

Hydrotalcites of formula Mg6 (Fe,Al)2(OH)16(CO3).4H2O formed by intercalation with the carbonate anion as a function of divalent/trivalent cationic ratio have been successfully synthesised. The XRD patterns show variation in the d-spacing attributed to the size of the cation. Raman and infrared bands in the OH stretching region are assigned to (a) brucite layer OH stretching vibrations (b) water stretching bands and (c) water strongly hydrogen bonded to the carbonate anion. Multiple (CO3)2- symmetric stretching bands suggest that different types of (CO3)2- exist in the hydrotalcite interlayer. Increasing the cation ratio (Mg/Al,Fe) resulted in an increase in the combined intensity of the 2 Raman bands at around 3600 cm-1, attributed to Mg-OH stretching modes, and a shift of the overall band profile to higher wavenumbers. These observations are believed to be a result of the increase in magnesium in the structure. Raman spectroscopy shows a reduction in the symmetry of the carbonate, leading to the conclusion that the anions are bonded to the brucite-like hydroxyl surface and to the water in the interlayer. Water bending modes are identified in the infrared spectra at positions greater than 1630 cm-1, indicating the water is strongly hydrogen bonded to both the interlayer anions and the brucite-like surface.

Synthesis of a heparan sulfate mimetic disaccharide with a conformationally locked residue from a common intermediate

A simple mimetic of a heparan sulfate disaccharide sequence that binds to the growth factors FGF-1 and FGF-2 was synthesized by coupling a 2-azido-2-deoxy-D-glucosyl trichloroacetimidate donor with a 1,6-anhydro-2-azido-2-deoxy--D-glucose acceptor. Both the donor and acceptor were obtained from a common intermediate readily obtained from D-glucal. Molecular docking calculations showed that the predicted locations of the disaccharide sulfo groups in the binding site of FGF-1 and FGF-2 are similar to the positions observed for co-crystallized heparin-derived oligosaccharides obtained from published crystal structures.

The genetic basis of human height : the role of estrogen

Height is a complex physical trait that displays strong heritability. Adult height is related to length of the long bones, which is determined by growth at the epiphyseal growth plate. Longitudinal bone growth occurs via the process of endochondral ossification, where bone forms over the differentiating cartilage template at the growth plate. Estrogen plays a major role in regulating longitudinal bone growth and is responsible for inducing the pubertal growth spurt and fusion of the epiphyseal growth plate. However, the mechanism by which estrogen promotes epiphyseal fusion is poorly understood. It has been hypothesised that estrogen functions to regulate growth plate fusion by stimulating chondrocyte apoptosis, angiogenesis and bone cell invasion in the growth plate. Another theory has suggested that estrogen exposure exhausts the proliferative capacity of growth plate chondrocytes, which accelerates the process of chondrocyte senescence, leading to growth plate fusion. The overall objective of this study was to gain a greater understanding of the molecular mechanisms behind estrogen-mediated growth and height attainment by examining gene regulation in chondrocytes and the role of some of these genes in normal height inheritance. With the heritability of height so well established, the initial hypothesis was that genetic variation in candidate genes associated with longitudinal bone growth would be involved in normal adult height variation. The height-related genes FGFR3, CBFA1, ER and CBFA1 were screened for novel polymorphisms using denaturing HPLC and RFLP analysis. In total, 24 polymorphisms were identified. Two SNPs in ER (rs3757323 C>T and rs1801132 G>C) were strongly associated with adult male height and displayed an 8 cm and 9 cm height difference between homozygous genotypes, respectively. The TC haplotype of these SNPs was associated with a 6 cm decrease in height and remarkably, no homozygous carriers of the TC haplotype were identified in tall subjects. No significant associations with height were found for polymorphisms in the FGFR3, CBFA1 or VDR genes. In the epiphyseal growth plate, chondrocyte proliferation, matrix synthesis and chondrocyte hypertrophy are all major contributors to long bone growth. As estrogen plays such a significant role in both growth and final height attainment, another hypothesis of this study was that estrogen exerted its effects in the growth plate by influencing chondrocyte proliferation and mediating the expression of chondrocyte marker genes. The examination of genes regulated by estrogen in chondrocyte-like cells aimed to identify potential regulators of growth plate fusion, which may further elucidate mechanisms involved in the cessation of linear growth. While estrogen did not dramatically alter the proliferation of the SW1353 cell line, gene expression experiments identified several estrogen regulated genes. Sixteen chondrocyte marker genes were examined in response to estrogen concentrations ranging from 10-12 M to 10-8 M over varying time points. Of the genes analysed, IHH, FGFR3, collagen II and collagen X were not readily detectable and PTHrP, GHR, ER, BMP6, SOX9 and TGF1 mRNAs showed no significant response to estrogen treatments. However, the expression of MMP13, CBFA1, BCL-2 and BAX genes were significantly decreased. Interestingly, the majority of estrogen regulated genes in SW1353 cells are expressed in the hypertrophic zone of the growth plate. Estrogen is also known to regulate systemic GH secretion and local GH action. At the molecular level, estrogen functions to inhibit GH action by negatively regulating GH signalling. GH treated SW1353 cells displayed increases in MMP9 mRNA expression (4.4-fold) and MMP13 mRNA expression (64-fold) in SW1353 cells. Increases were also detected in their respective proteins. Treatment with AG490, an established JAK2 inhibitor, blocked the GH mediated stimulation of both MMP9 and MMP13 mRNA expression. The application of estrogen and GH to SW1353 cells attenuated GH-stimulated MMP13 levels, but did not affect MMP9 levels. Investigation of GH signalling revealed that SW1353 cells have high levels of activated JAK2 and exposure to GH, estrogen, AG490 and other signalling inhibitors did not affect JAK2 phosphorylation. Interestingly, AG490 treatment dramatically decreased ERK2 signalling, although GH did stimulate ERK2 phosphorylation above control levels. AG490 also decreased CBFA1 expression, a transcription factor known to activate MMP9 and MMP13. Finally, GH and estrogen treatment increased expression of SOCS3 mRNA, suggesting that SOCS3 may regulate JAK/STAT signalling in SW1353 cells. The modulation of GH-mediated MMP expression by estrogen in SW1353 cells represents a potentially novel mechanism by which estrogen may regulate longitudinal bone growth. However, further investigation is required in order to elucidate the precise mechanisms behind estrogen and GH regulation of MMP13 expression in SW1353 cells. This study has provided additional evidence that estrogen and the ER gene are major factors in the regulation of growth and the determination of adult height. Newly identified polymorphisms in the ER gene not only contribute to our understanding of the genetic basis of human height, but may also be useful in association studies examining other complex traits. This study also identified several estrogen regulated genes and indicated that estrogen modifies the expression of genes which are primarily expressed in the hypertrophic region of the epiphyseal growth plate. Furthermore, synergistic studies incorporating GH and estrogen have revealed the ability of estrogen to attenuate the effects of GH on MMP13 expression, revealing potential pathways by which estrogen may modulate growth plate fusion, longitudinal bone growth and even arthritis.

Synthesis, characterization of palygorskite supported zero-valent iron and its application for methylene blue adsorption

In this work, natural palygorskite impregnated with zero-valent iron (ZVI) was prepared and characterised. The combination of ZVI particles on surface of fibrous palygorskite can help to overcome the disadvantage of ultra-fine powders which may have strong tendency to agglomerate into larger particles, resulting in an adverse effect on both effective surface area and catalyst performance. There is a significant increase of methylene blue (MB) decolourized efficiency on acid treated palygorskite with ZVI grafted, within 5 mins, the concentration of MB in the solution was decreased from 94 mg/L to around 20 mg/L and the equilibration was reached at about 30 to 60 mins with only around 10 mg/L MB remained in solution. Changes in the surface and structure of prepared materials were characterized using X-ray diffraction (XRD), infrared (IR) spectroscopy, surface analysing and scanning electron microscopy (SEM) with element analysis and mapping. Comparing with zero-valent iron and palygorskite, the presence of zero-valent iron reactive species on the palygorskite surface strongly increases the decolourization capacity for methylene blue, and it is significant for providing novel modified clay catalyst materials for the removal of organic contaminants from waste water.

The stimulation of healing within a rat calvarial defect by mPCL–TCP/collagen scaffolds loaded with rhBMP-2

Bone morphogenetic proteins (BMPs) have been widely investigated for their clinical use in bone repair and it is known that a suitable carrier matrix to deliver them is essential for optimal bone regeneration within a specific defect site. Fused deposited modeling (FDM) allows for the fabrication of medical grade poly 3-caprolactone/tricalcium phosphate (mPCL–TCP) scaffolds with high reproducibility and tailor designed dimensions. Here we loaded FDM fabricated mPCL–TCP/collagen scaffolds with 5 mg recombinant human (rh)BMP-2 and evaluated bone healing within a rat calvarial critical-sized defect. Using a comprehensive approach, this study assessed the newly regenerated bone employing microcomputed tomography (mCT), histology/histomorphometry, and mechanical assessments. By 15 weeks, mPCL–TCP/collagen/rhBMP-2 defects exhibited complete healing of the calvarium whereas the non- BMP-2-loaded scaffolds showed significant less bone ingrowth, as confirmed by mCT. Histomorphometry revealed significantly increased bone healing amongst the rhBMP-2 groups compared to non-treated scaffolds at 4 and 15 weeks, although the % BV/TV did not indicate complete mineralisation of the entire defect site. Hence, our study confirms that it is important to combine microCt and histomorphometry to be able to study bone regeneration comprehensively in 3D. A significant up-regulation of the osteogenic proteins, type I collagen and osteocalcin, was evident at both time points in rhBMP-2 groups. Although mineral apposition rates at 15 weeks were statistically equivalent amongst treatment groups, microcompression and push-out strengths indicated superior bone quality at 15 weeks for defects treated with mPCL–TCP/collagen/rhBMP-2. Consistently over all modalities, the progression of healing was from empty defect < mPCL–TCP/collagen < mPCL–TCP/collagen/rhBMP-2, providing substantiating data to support the hypothesis that the release of rhBMP-2 from FDM-created mPCL–TCP/collagen scaffolds is a clinically relevant approach to repair and regenerate critically-sized craniofacial bone defects. Crown Copyright 2008 Published by Elsevier Ltd. All rights reserved.

Synthesis and characterization of boehmite nanofibers

Boehmite nanofibers of high quality were synthesized through a wet-gel conversion process without the use of a surfactant. The long nanofibers of boehmite with clear-cut edges were obtained by steaming the wet-gel precipitate at 170 ºC for 2 days under a pH 5. Hydrothermal treatment of the boehmite gels enabled self-assembly through directed crystal growth. Detailed characterization using X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), Infrared Emission Spectroscopy (IES) and Raman Spectroscopy is presented.

Synthesis of 4-arm star poly(L-Lactide) oligomers using an in situ-generated calcium-based initiator

Using an in situ-generated calcium-based initiating species derived from pentaerythritol, the bulk synthesis of well-defined 4-arm star poly(L-lactide) oligomers has been studied in detail. The substitution of the traditional initiator, stannous octoate with calcium hydride allowed the synthesis of oligomers that had both low PDIs and a comparable number of polymeric arms (3.7 – 3.9) to oligomers of similar molecular weight. Investigations into the degree of control observed during the course of the polymerization found that the insolubility of pentaerythritol in molten L-lactide resulted in an uncontrolled polymerization only when the feed mole ratio of L-lactide to pentaerythritol was 13. At feed ratios of 40 and greater, a pseudo-living polymerization was observed. As part of this study, in situ FT-Raman spectroscopy was demonstrated to be a suitable method to monitor the kinetics of the ring-opening polymerization (ROP) of lactide. The advantages of using this technique rather than FT-IR-ATR and 1H NMR for monitoring L-lactide consumption during polymerization are discussed.