The proposal focuses on the development of two spectroscopic techniques that will deal with a variety of problems directly in the biomedicine. I. Time-resolved Fourier transform infrared (FTIR) difference spectroscopy. FTIR difference spectroscopy is an extremely sensitive technique which, under favorable conditions, allows one to follow the fate of a single proton in a protein of MW 28-43 kD as they undergo successive changes during a reaction. The reaction can be slowed down or quenched at a certain intermediate by taking measurements down to liquid He temperature. The current study proposes to exploit this new technique in a time-resolve manner. Thus, after mixing of reactants, the FTIR spectra are measured at suitable time intervals (as short as several seconds) under stopped-flow or continuous flow conditions; a subtraction of the two spectra will only show bands that have changed. The presence of biopolymers does not interfere because only the moiety that has undergone changes will appear. This is a technique that appears not to have ben used as a general tool. It is proposed to study the mode of binding of mitomycin to DNA to form the cross-linked adduct responsible for its therapeutically important cytotoxicity, the reaction of calichemycin (or esperamycin) (antibiotics with extraordinary activity) with DNA, the biosynthesis of penicillin, etc. The technique should have very broad applications. II. A simple micro-scale structure determination of oligosaccharides that requires no references. The dramatic progress seen in molecular biology depends to a great extent on the availability of micro-scale methods for determining protein and nucleic acid structures. The carbohydrates are far more difficult to handle, and the analysis depends on classical methods such as methylation analysis, which although dramatically improved still suffers from serious disadvantages; reference samples are required, anomeric and absolute configurations cannot by determined, etc. (sample level is sub-NMR). A totally new approach based on circular dichroism (CD) is proposed. Two approaches are possible. In one scheme, the free hydroxyls is in a sugar are tagged with p-phenylbenzyl groups, the sugar is methanolyzed in 5-15 min in a microwave oven, the products are HPLC-separated, and the CD of products are measured; the CD is characteristic of the substitution pattern. In the second scheme, the free hydroxyls are tagged with on chromophore while hydroxyls involved in glycosidic bonding are tagged with another chromophore; the CD of separated monosaccharide units are characteristic of the sugar and the substitution. Both methods disclose the stereochemistry of the anomeric center, require no reference and can be carried out at the microgram level. However, this is with model saccharides. The current proposal will focus on the application to read oligosaccharides of unknown structure, and those that are available in very limited amounts. This still requires additional new methodologies to be discovered. The outlook is quite promising. A mild method for cleaving the oligosaccharides into monomers, and an approach to sequence the sugars by MS will also be studied.
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