Probing cell surfaces of host and nonhost species of a mycoparasite, using FITC-labeled lectins

Cell surfaces of susceptible host species (Mortierella pusllla and Cboanepilora cucurbitarum ), resistant host (Pilascolomyces articulosus ), nonhost (Mortierella candelabrum ) and the mycoparasite (Piptocepilalis virginiana) were examined for sugar distribution patterns using light and fluorescent microscopy techniques. The susceptible host, resistant host and the mycoparasite species exhibited a similar sugar distribution profile; they all showed N-acetyl glucosamine and D-glucose on their cell wall surfaces. The nonhost cell wall surface showed a positive binding reaction to FITClectins specific for N-acetyl glucosamine and also for OI.-fucose, N-acetyl galactosamine and galactose. Treatment of these fungi with mild concentrations of proteinases (both commercial as well as the mycoparasiteproteinase) resulted in the revelation of additional sugars on the fungal cell walls. The susceptible host treated with proteinase expressed higher levels of N-acetyl glucosamine and D-glucose. The susceptible host also showed the presence of OI.-fucose, N-acetyl galactosamine and galactose. The proteinasetreated susceptible host cell walls also showed an increase in the levels of attachment with the mycoparasite. Treatment of the resistant host with proteinases revealed OI.-fucose in addition to N-acetyl glucosamine and D-glucose. Treatment of the nonhost cell wall with proteinase resulted in the exposure of low levels of D-glucose, in addition to sugars found on the untreated nonhost cell wall surface. The mycoparasite treated with proteinase revealed OI.-fucose, N-acetyl galactosamine and galactose on its cell surface in addition to the sugars N-acetyl glucosamine and D-glucose. Protoplasts were isolated from hosts and nonhost fungi and their surfaces were examined for sugar distribution patterns. The susceptible host and nonhost protoplast membranes showed all the sugars (N-acetyl glucosamine, D-glucose, (It.-fucose, N-acetyl galactosamine and galactose) tested for. The resistant host protoplast membrane however, had only N-acetyl glucosamine and D-glucose exposed. This sugar distribution profile resembles that exhibited by the untreated resistant host cell wall, as well as that shown by the untreated mycoparasite cell surface. Only susceptible host protoplasts were successful in attaching to the mycoparasite surface. Resistant host protoplasts did not show any interaction with the i mycoparasite cell surface. Both susceptible as well as resistant host protoplasts were incapable of attaching to agarose beads surface-coated with specific carbohydrates. The mycoparasite however, did attach to agarose beads surface-coated with either N-acetyl glucosamine, D-glucose/Dmannose or o:,- methyl-D-mannose. The relevance of the cell wall and the protoplast membrane in the light of the present results, in reacting appropriately to bring about either a susceptible, a resistant or a nonhost response has been discussed.

Modifications to the Suzuki reaction and mechanistic insights on the NBS mediated cleavage of benzylidene acetals /

One of the most challenging tasks for a synthetic organic chemist today, is the development of chemo, regio, and stereoselective methodologies toward the total synthesis of macromolecules. r . The objective of my thesis was to develop methodologies towards this end. The first part of my project was to develop highly functionalized chirons from D-glucose, a cheap, chiral starting material, to be utilized in this capacity. The second part of the project dealt with modifying the carbon-carbon bond forming Suzuki reaction, which is utilized quite often as a means of combining molecular sub units in total synthesis applications. As previously stated the first area of the project was to develop high value chirons from D-glucose, but the mechanism of their formation was also investigated. The free radical initiated oxidative fragmentation of benzylidene acetals was investigated through the use of several test-case substrates in order to unravel the possible mechanistic pathways. This was performed by reacting the different acetals with N-bromosuccinimide and benzoyl peroxide in chlorobenzene at 70^C in all cases. Of the three mechanistic pathways discussed in the literature, it was determined, from the various reaction products obtained, that the fragmentation of the initial benzylic radical does not occur spontaneously but rather, oxidation proceeds to give the benzyl bromide, which then fragments via a polar pathway. It was also discovered that the regioselectivity of the fragmentation step could be altered through incorporation of an allylic system into the benzylidene acetal. This allows for the acquisition of a new set of densely functionalized. chiral, valuable synthetic intermediates in only a few steps and in high yields from a-Dglucose. The second part of the project was the utilization of the phosphonium salt room temperature ionic liquid tetradecyltrihexylphosphonium chloride (THPC) as an efficient reusable medium for the palladium catalyzed Suzuki cross-coupling reaction of aryl halides, including aryl chlorides, under mild conditions. The cross-coupling reactions were found to proceed in THPC containing small amounts of water and toluene using potassium phosphate and 1% Pd2(dba)3. Variously substituted iodobenzenes, including electron rich derivatives, reacted efficiently in THPC with a variety of arylboronic acids and afforded complete conversion within 1 hour at 50 ^C. The corresponding aryl bromides also reacted under these conditions with the addition of a catalytic amount of triphenylphosphine that allowed for complete conversion and high isolated yields. The reactions involving aryl chlorides were considerably slower, although the addition of triphenylphosphine and heating at 70 ^C allowed high conversion of electron deficient derivatives. Addition of water and hexane to the reaction products results in a triphasic system in which the top hexane phase contained the biaryl products, the palladium catalyst remained fully dissolved in the central THPC layer, while the inorganic salts were extracted into the lower aqueous phase. The catalyst was then recycled by removing the top and bottom layers and adding the reagents to the ionic liquid which was heated again at 50 ^C; resulting in complete turnover of iodobenzene. Repetition of this procedure gave the biphenyl product in 82-97% yield (repeated five times) for both the initial and recycled reaction sequences.