Amyloglucosidase has been obtained from Rhizopus delemar and purified by ammonium sulphate fractionation; ethanolic precipitation; chromatography on DEAE-cellulose, DEAE-sephadex A-50 and Sephadex G-50/ Amberlite IRC-50. Ethanolic precipation and chromatography on DEAE-cellulose by stepwise elution have been proved to be the most simple and rapid procedure to remove traces of alpha-amylase from amyloglucosidase preparations. The apparent kinetic perameters of amyloglucosidase free of traces of alpha-amylase has been shown to be altered after inclusion of alpha-amylase. The increased apparent Km and V for the alpha-1,4-glucan/ amyloglucosidase/alpha-amylase system compared with those parameters for the system in the absence of alpha-amylase have been explained on the basis of the decrease in the size of the substrate molecule, which is caused by the action alpha-amylase (endo-attack pattern). The apparent kinetic perameters of amyloglucosidase oxidized traces of alpha-amylase has also substrates., Amyloglucosidase action has been established to be unable of by passing oxidation points introduced in the alpha-1,4-glucan by periodate oxidation. A theory has been developed to describe the action of amyloglucosidase on partially oxidized amylases, which predicts the decrease in the apparent Km and V with the increase in the degree of oxidation of the amylase, as it has been demonstrated experimentally. A direct recording method of assaying amyloglucosidase has been developed involving ascorbic acid as the oxygen acceptor in a glucose oxiddase/peroxidase assay system for glucose. This method permits amyloglucosidase activity to be followed by ultraviolet spectrophotometry. The merits and limitations of this procedure have been discussed. An immobilized cellulose derivative of amyloglucosidase in which the prosthetic group is involved in the covalent linkage between the enzyme end the support has been investigated and compared with the soluble enzyme. The kinetics, pH profile and the thermal stability of this immobilized enzyme and their comparison with these characteristics for the free enzyme have been investigated.

M.Sc. (Chemistry) / Please refer to full text to view abstract

D.Phil. / The topic of this thesis centres around the construction of carbocycles from carbohydrates involving the use of SmI2 as reagent. Carbocycles, in particular functionalised cyclopentanes, form important substructures in a plethora of natural compounds, many of which are biologically active. The conversion of carbohydrates into stereodefined cyclopentanoids represents an attractive means of generating this type of compounds. The SmI2-mediated cyclisation of hex-5-enals, derived from suitably substituted carbohydrate precursors via a zinc-mediated Grob-fragmentation reaction, was found to be a facile reaction generating stereodefined cyclopentanols in moderate yields. The effect of varying stereochemistry and substitution pattern on the outcome of the cyclisation was also investigated. In order to achieve a more efficient route to the cyclic products, a Grobfragmentation based on SmI2 was investigated. The substituent at the anomeric position of the carbohydrate was found to be crucial to the success of the fragmentation. This protocol gave rise to conditions under which fragmentation and cyclisation could be effected in a tandem fashion mediated by SmI2. Various attempts at intermolecular trapping of the organosamarium species generated after cyclisation proved fruitless. In order to circumvent these obstacles a tandem radical bicyclisation reaction was investigated for the construction of highly functionalised bicyclic ring systems. This approach proved to be a successful alternative for incorporating functionality at the exocyclic carbon atom which is otherwise difficult to achieve. The second part of this work is directed at the synthesis of stereodefined alkoxyaminocyclopentanes from carbohydrates promoted by SmI2. The intramolecular trapping of a radical by an oxime ether represents an attractive means of generating these compounds. The preparation of the iodo oxime ether starting materials from suitably substituted carbohydrates is discussed. The radical cyclisation under the action of SmI2 was found to be a facile process providing a rapid route to aminocyclopentanes which are used as key intermediates for the preparation of carbocyclic glycosidase inhibitors and analogues. In the third part of this work, the construction of functionalised cyclobutanols from carbohydrate precursors is discussed. The 4-exo-trig cyclisation holds promise as a general route for the stereoselective synthesis of highly functionalised cyclobutanols. Initial attempts at the 4-exo-trig cyclisation proved fruitless. It was subsequently established that the radical acceptor plays a crucial role in the success of the transformation. This led to the isolation of the first carbohydrate derived cyclobutanol. The final part of this thesis involves the development of a new approach for the conversion of suitably functionalised pentose derivatives into chiral cyclopentanols. Cyclisation of a ribose derived hemiacetal via its in situ formed hex-5-ynal is discussed. This approach opens new possibilities for the construction of complex carbocycles from readily available carbohydrate starting materials.

An investigation of the reaction of the sulphuryl chloride-N, N-dimethyl formamide reagent with several carbohydrate compounds, containing free hydroxyl groups, was undertaken, mainly with the view of looking at substitution of the hydroxyl groups with chlorine atoms. The reaction was found to lead to substitution of both primary and secondary hydroxyl groups with chlorine, with inversion of configuration in the latter case. The reagent was further found to effect formylation and chlorosulphation of secondary hydroxyl groups, where nucleophilic substitution by a chlorine was not favourable. Studies involving the methyl pentopyranosides, showed that the reagent was particularly useful in the chlorosulphation and chlorination of sugars, compared with the hexopyranosides.

M.Sc. (Chemistry) / Please refer to full text to view abstract

M.Sc. (Chemistry) / Please refer to full text to view abstract

3,4,6-Tri-0-acetyl-D-glucal (1) reacted with carbon monoxide and hydrogen in the presence of dicobalt octacarbonyl to yield a mixture of two epimeric anhydrodeoxyheptitols, namely, 4,5,7-tri-0-acetyl-2,6-anhydro-3-deoxy-D-manno-heptitol (2) and 4,5,7-tri-0-acetyl-2,6-anhydro-3-deoxy-D-gluco-heptitol (3). De-0-acetylation of the mixture, followed by chromatographic separation, yielded 2,6-anhydro-3-deoxy-D-manno-heptitol (4) and 2,6-anhydro-3-deoxy-D-gluco-heptitol (5). Compounds (4) and (5) were oxidised with periodate to yield dialdehydes which on reduction with sodium borohydride afforded enantiomeric tetrol ethers. Reaction of 3,4,6-tri-0-acetyl-D-glucal (1) with carbon monoxide and deuterium, followed by de-0-acetylation and chromatographic separation gave 2,6-anhydro-5-deoxy-D-manno-heptitol-1,1,3-²H3(cis)(6) and 2,6-anhydro-3-deoxy-D-gluco-heptitol-1, 1,3-²H3(cis) (7) . Examination of the proton magnetic resonance spectra of the normal (4,5) and deuterated anhydrodeoxy heptitols (6,7) revealed their structures and showed that cis-addition of carbon monoxide and hydrogen to the double bond of (1) had taken place. Reaction of the mixture of partially acetylated heptitols (2) and (3) with p-toluenesulphonyl chloride followed by fractional crystallisation of the products gave pure 4,5,7-tri-0-acetyl-2,6-anhydro-3-deoxy-1-0-(p-toluenesul-phonyl)-D-gluco-heptitol (8). Similarly,, the mixture of (2) and (3) reacted with p-bromobenzenesulphonyl bromide to give 4,5, 7-tri-0-acetyl-2,6-anhydro-1-0-(p-bromobenzenesulphonyl)-3-deoxy-D-gluco-heptitol (11), the structure of which was confirmed by X-ray structure analysis by A. Camerman and J. Trotter. Therefore, the absolute structures of compounds (4) and (5) were ascertained. Compounds (8) and (11) were converted to (5) by a series of reactions. Comparison of the exchange reaction of sodium iodide with 4,5,7-tri- 0-acetyl-2,6-anhydro-3-deoxy-1-0-(p-toluenesulphonyl)-D-gluco-heptitol (8) and with 4,5,7-tri-0-acetyl-2,6-anhydro-3-deoxy-1-0-(p-toluenesulphonyl)-D-manno-heptitol (14) revealed that the equatorial primary p-toluenesulphonoxy group of (8) was exchanged more readily than that of (14). The hydroformylation of (1) yielded two enantiomeric aldehydes (16a, 16b) which were separated chromatographically via their 2,4-dinitrophenyl-hydrazones (16b) and (17b). Both (16b) and (17b) were degraded to (4) and (5), respectively. 3,4-Di-0-acetyl-D-arabinal (18) reacted with carbon monoxide and hydrogen in the presence of dicobalt octacarbonyl to yield, upon de-0-acetylation and chromatographic separation, a mixture of two epimeric anhydro-deoxyhexitols, namely, 1,5-anhydro-4-deoxy-L-ribo-hexitol (21) and 1,5-anhydro-4-deoxy-D-lyxo-hexitol (22). Compounds (21) and (22) were converted into enantiomeric 2-deoxy-3-0-(2-hydroxyethyl)-L-glycero-tetritol (23) and 2-deoxy-3-0-(2-hydroxyethyl)-D-glycero-tetritol (24). Compound (23) was identical to an authentic sample of 2-deoxy-3-0-(2-hydroxyethyl)-L-glycero-tetritol. 1,2,4,6-Tetra-0-acetyl-3-deoxy-α-D-erythro-hex-2-enopyranose (29) reacted with carbon monoxide and hydrogen in the presence of dicobalt octacarbonyl to yield 1,2,3¹,4,6-penta-0-acetyl-3-deoxy-3-C-(hydroxymethyl)-α-D-gluco-pyranose (31) besides hydrogenolysed hydrohydroxymethylated products, a similar reaction of (29) with deuterium instead of hydrogen gave 1,2,3¹,4,6-penta-0-acetyl-3-deoxy-3-C- (hydroxymethyl) -α-D-gluco-pyranose-2,3¹,3¹ -²H3 (cis) (33). The structures of (31) and (33) were established by p.m.r. spectroscopy. 2,3,4,6-Tetra-0-acetyl-α-D-glucosyl bromide (26) reacted with sodium tetracarbonylcobaltate under compressed carbon monoxide followed by treatment with triphenylphosphine to afford 2,3,4,6-tetra-0-acetyl-β-D-glucosyl tri-carbonyl triphenylphosphine cobaltate (39) and 3,4,5,7-tetra-0-acetyl-2,6- anhydro-D-glycero-D-gulo-heptosoyl tricarbonyl triphenylphosphine cobaltate (41). Reduction of. (39) and (41) with sodium borohydride followed by acetylation gave 2,3,4,6-tetra-0-acetyl-1,5-anhydro-D-glucitol (40) and 1,3,4,5,7-penta-0-acetyl-2,6-anhydro-D-glycero-D-gulo-heptitol (42). Both (40) and (42) were compared with authentic samples and shown to be the same. / Science, Faculty of / Chemistry, Department of / Graduate

Sugar mercaptals were partially methylated with Purdie's reagents in tetrahydrofuran as a solvent, and the relative reactivity of hydroxyl groups was determined by estimation of the ratio of mono-0̲-methyl ethers. The highest reactivity of the 2-hydroxyl group, followed by that of the 3-hydroxyl group, was accounted for by the inductive effect of the mercaptal group and the variation of the ratios among the sugar mercaptals was further explained by the intramolecular hydrogen bonding in the zig-zag conformation of sugar mercaptals. The exceptionally high reactivity of the 3-hydroxyl group of D̳-galactose diethyl mercaptal was also shown to be explicable in terms of the hydrogen bonding. An appreciable reactivity of the primary hydroxyl groups was noted and attributed to the least steric hindrance at this position. A mechanism for the Purdie methylation was proposed. Mg values of various mono-0̲-methyl sugars were recorded and some modifications in the separation of mono-0̲-methyl sugars were also described. 5-0̲-Methyl-L̳-arabinose and two crystalline derivatives were synthesized for the first time, and 2-0̲-methyl-L̳-arabonolactone, hitherto known as a sirup, was crystallized. / Science, Faculty of / Chemistry, Department of / Graduate

It is well established that various partially methylated tetroses and pentoses occur in periodate oxidation products of methylated polysaccharides. To provide reference compounds as a means of identifying some of these sugars 2,3 di-O-methyi-L-threose, 2,3 di-O-methyi-D-erythrose, and 3,4 di-O-methyl-L-xylose have been synthesized. The threose and xylose were synthesized from ∝-methyl-D-glucoside by benzylidene condensation, methyiation, hydrolysis, reduction and periodate oxidation, whereas the erythrose was obtained from D-mannose by a similar sequence of reactions. The sugars were obtained as syrups and were characterized by the preparation of crystalline derivatives. ii. Black Spruce hemicelluloses:- By using different concentrations of alkali three different hemiceiluloses were extracted from black spruce holocellulose. Of these two fractions, the xylan and the glucomannan were purified by fractional precipitation using barium hydroxide solution and Fehling's solution. Previous workers have observed that the xylan fraction was always contaminated by small amounts of galactose. This was also true in the present instance if barium hydroxide alone was used for purification, but when this was followed by two treatments with Fehling's solution the galactose was completely eliminated. This is believed to be the first case of the isolation of such a xylan in a pure state from the hemicelluloses of a coniferous wood. The third fraction - the galactoglucomannan was found to be quite difficult to re-dissolve in alkali and as a result its purification is not quite complete. / Science, Faculty of / Chemistry, Department of / Graduate

The crystal and molecular structures of three chlorinated carbohydrates whose formal nomenclatures are methyl 2-chloro-2-deoxy-α-D-galactopyranoside methyl 4,6-dichloro-4,6-dideoxy- α -D-glucopyranoside, and methyl 4,6-diohloro-4,6-dideoxy- α -D-galactopyranoside have been determined by X-ray diffraction using various methods. A scintillation counter was used in each case to collect the data: for the first, CuK[subscript α] radiation was used; for the second and third, MoK[subscript α] radiation was used. The structure of the 2-chloro-galactoslde was solved by a combination of the Patterson method and trial and error methods. Two possible positions of the chlorine atom found from the Patterson function were differentiated by minimizing R (using h k 0 projection data only) in rotation of a model of the molecule about each chlorine position. A model was used to go from the solved two dimensional structure to three dimensions. Successive Fourier summations and block diagonal least squares refinement established the crystal to be composed of a mixture of the α and β anomers of methyl 2-chloro-2-deoxy-D-galaotopyranoside In the approximate ratio of 2 α: lβ. Both the α and β anomers are in their expected C-l (chair) conformations. Hydrogen bonding involving 0(3), 0(4) and 0(6) links molecules together into infinite sheets, two molecules thick and perpendicular to the x-axis. Mean bond distances are: C-C = 1.53 A, C-0 = 1.42 A and C-Cl - 1.75 A. The structure of the 4,6-dlchloro-glucoside was solved by a combination of the Patterson method (to locate the two chlorines), successive Fourier summations (to locate the carbons and oxygens), block diagonal least squares refinement, and a difference synthesis (to locate eight of the hydrogens). The absolute configuration was determined by the anomalous dispersion method (CuK[subscript α] radiation). The molecule is in the expected C-l conformation. Hydrogen bonding, involving 0(2) and 0(3), links molecules together into infinite chains parallel to the y-axis; the mean planes of the molecules are approximately perpendicular to the direction of these chains. Mean bond distances are C-C = 1.52 A, C-0 = 1.42 A and C-Cl = 1.78 A. The structure of the 4,6-dichloro-galactoside was solved by direct methods applied to the two dimensional data of two centrosymmetric projections. A series of programs, employing the Vand-Pepinsky method of phasing reflexions contained in Sayre relationships, was written to do this. After refinement using block diagonal least squares, the two solutions were combined into the three dimensional solution, which was further refined. A difference Fourier summation revealed the position of six hydrogens. The molecule is again in the expected C-l conformation. Hydrogen bonding Involved 0(1), 0(2), and 0(3) in a complicated network which includes a bifurcated hydrogen bond. As in the 2-chloro-galactoside, molecules are linked by hydrogen bonds into infinite sheets, two molecules thick and perpendicular to the x-axis, but whereas in that structure the baslo symmetry elements propagating the networks of bonding were unit cell translations and two-fold rotation axes, the basic elements propagating the networks in 4,6-dlchloro-galaoto side are unit cell translations and two-fold screw axes. Mean bond distances in this structure are: C-C = 1.53 A, C-0 = 1.45 A, and C-Cl =1.80 A. / Science, Faculty of / Chemistry, Department of / Graduate