Summary: Partially chlorosulphated derivatives were synthesised for the purpose of examining the reactions of the chlorosulphonyloxy group in the presence of free hydroxyl groups. The behaviour of the chlorosulphonyloxy group was investigated under acidic conditions. Since sterically favoured chlorosulphonyloxy groups undergo facile replacement by chlorine to form chlorodeoxy sugars, some compounds possessing chlorosulphonyloxy groups which,due to polar and steric effects are not replaced by chloride,were investigated with a view to possible activation of the unfavourable centres towards nucleophilic substitution, thereby making available previously inaccessible chlorodeoxy sugars.

Sugar chlorosulphates of furanoid and pyranoid derivatives bearing chlorosulphonyloxy groups at primary and secondary positions, were synthesized and examined mainly with a view to determine their extent of reactivity in terms of nucleophilic substitution reactions, especially with azide. Inversion of configuration occurred at reactive chiral centres, whereas intermediate azidosulphonyloxy derivatives (azidosulphates) were formed via S-Cℓ bond fission of the chlorosulphonyloxy group at less reactive primary or secondary centres, e.g. 1,2:3,4-diO̲isopropyl idene-α-D-galactopyranose 6-azidosulphate, 1,2-O̲isopropylidenea- D-xylofuranose 3-azidosulphate and 1,2:5,6-di-O̲-isopropyl idene-α-Dglucofuranose 3-azidosulphate. 1,2:3,4-Di-O̲-isopropylidene-α-Dgalactopyranose 6-azidosulphate ultimately afforded the 6-azidodeoxy derivative probably by an SN2 mechanism. Some SNi characteristics were,however, evident when substitution occurred at a reactive primary centre (e.g. methyl 2,3,4-tri-O̲-methyla- D-glucopyranoside 6-chlorosulphate), as the 6-azidodeoxy derivative obtained, appeared to be contaminated with a trace amount of the corresponding 6-chlorodeoxy sugar, which had presumably formed via an internal SNi mechanism, while no intermediate azidosulphonyloxy derivative was isolated. In another study, the reaction pathways for the synthesis of benzylated chlorodeoxy sugars having potential biological properties as exemplified by the multivalent drug, tribenoside, were also investigated

Internodes 5-8, 10, 13 and 15 of Saccharum sp. var. NCo376 were examined for evidence of symplasmic phloem unloading of sucrose from the phloem, via the bundle sheath to the storage parenchyma. The vascular bundle possesses wellisolated phloem comprised of large diameter sieve elements and small diameter companion cells. A layer of phloem parenchyma surrounds the phloem, except where the phloem abuts the crushed protophloem. Outside this is a sclerenchymatous sheath, directly endarch to a parenchymatous bundle sheath, which is surrounded by storage parenchyma. The bundle sheath is interrupted at the centrifugal pole of the vascular bundle by a phloem fibre cap. Scanning Electron Microscopy revealed plasmodesmal fields throughout the bundle sheath and pith tissue. Transmission Electron Microscopy studies provided evidence of plasmodesmal occlusion, but not in all tissues. Aniline blue reactions under UV light indicate the presence of occluded plasmodesmal fields at the phloem parenchyma / sclerenchymatous sheath interface, and in localised regions of cells which are smaller than the surrounding storage parenchyma cells. This suggests a symplasmic transport pathway at these locations, and, based on these positive aniline blue reactions, with regulation via callose-mediated transplasmodesmal transport. Osmotic stress experiments, which included the addition of Ca2+, did not reveal further callose occlusion in the parenchyma, suggesting that the plasmodesmata in these regions may be closed via a noncallosic mechanism. Dye-coupling studies, using Lucifer Yellow (LYCH), which was iontophoretically injected following turgor-pressure equalisation, showed only rare, limited symplastic transport, usually only between the injected cell and one adjacent cell. Most injections did not result in transport of LYCH, suggesting either a lack of plasmodesmal connectivity, occlusion, or gating of any plasmodesmata present. This limited symplasmic transport, combined with the presence of occluded plasmodesmata at the phloem parenchyma / sclerenchymatous sheath interface suggests the presence of a two-domain phloem-unloading pathway. While symplastic transport may occur from the phloem to the sclerenchymatous sheath, further sucrose transport to the storage parenchyma appears to proceed apoplasmically from the sclerenchymatous sheath / bundle sheath interface, and into storage parenchyma cells across the cell wall and cell membrane via specialised sucrose transporters.

M.S.

UDP-sugar metabolizing pyrophosphorylases provide the primary mechanism for de novo synthesis of UDP-sugars, which can then be used for myriads of glycosyltranferase reactions, producing cell wall carbohydrates, sucrose, glycoproteins and glycolipids, as well as many other glycosylated compounds. The pyrophosphorylases can be divided into three families: UDP-Glc pyrophosphorylase (UGPase), UDP-sugar pyrophosphorylase (USPase) and UDP-N-acety lglucosamine pyrophosphorylase (UAGPase), which can be discriminated both by differences in accepted substrate range and amino acid sequences. This thesis focuses both on experimental examination (and re-examination) of some enzymatic/ biochemical properties of selected members of the UGPases and USPases and UAGPase families and on the design and implementation of a strategy to study in vivo roles of these pyrophosphorylases using specific inhibitors. In the first part, substrate specificities of members of the Arabidopsis UGPase, USPase and UAGPase families were comprehensively surveyed and kinetically analyzed, with barley UGPase also further studied with regard to itspH dependency, regulation by oligomerization, etc. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Frc-1-P, whereas USPase reacted with arange of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P, β-L-Ara-1-P and α-D-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P and, to some extent, with D-Glc-1-P. A structure activity relationship was established to connect enzyme activity, the examined sugar-1-phosphates and the three pyrophosphorylases. The UGPase/USPase/UAGPase active sites were subsequently compared in an attempt to identify amino acids which may contribute to the experimentally determined differences in substrate specificities. The second part of the thesis deals with identification and characterization of inhibitors of the pyrophosphorylases and with studies on in vivo effects of those inhibitors in Arabidopsis-based systems. A novel luminescence-based high-throughput assay system was designed, which allowed for quantitative measurement of UGPase and USPase activities, down to a pmol per min level. The assay was then used to screen a chemical library (which contained 17,500 potential inhibitors) to identify several compounds affecting UGPase and USPase. Hit-optimization on one of the compounds revealed even stronger inhibitors of UGPase and USPase which also strongly inhibited Arabidopsis pollen germination, by disturbing UDP-sugar metabolism. The inhibitors may represent useful tools to study in vivo roles of the pyrophosphorylases, as a complement to previous genetics-based studies. The thesis also includes two review papers on mechanisms of synthesis of NDP-sugars. The first review covered the characterization of USPase from both prokaryotic and eukaryotic organisms, whereas the second review was a comprehensive survey of NDP-sugar producing enzymes (not only UDP-sugar producing and not only pyrophosphorylases). All these enzymes were discussed with respect to their substrate specificities and structural features (if known) and their proposed in vivo functions.