The importance of stereochemistry in biological systems has been accentuated by rapid advances in bioscience and the pharmaceutical industry. Circular dichroic (CD) spectroscopy is becoming an indispensable tool for determining absolute configurations and conformations of molecules in solution. However, CD is one of the most underutilized techniques, primarily due to the prevalence of empirical """"""""rules"""""""" leading to ambiguous stereochemical assignments. Fortunately, recent developments in experimental, technical, computational and theoretical areas have led to rapid advances in CD methodology. In particular, exciton coupled CD, a method under development since the late 1960's, has become a versatile and powerful technique for performing studies at micro- to submicrogram scales. Based on nonempirical coupled oscillator theory, the exciton coupled method can provide an unambiguous and, in principle, nonempirical determination of absolute configuration. The focus of this proposal is to extend exciton coupled CD to unexplored areas of structural studies of complex natural products and interactions of ligand/receptor complexes. Further, we aim to develop clear and direct protocols for CD analysis on a sub-microgram scale. i) New chemical/CD strategies will be developed for the determination of absolute configurational assignments. This research will focus on challenging cases where conventional CD methods cannot be applied due to structural restrictions in the substrate. To solve such problems, we propose to utilize the employment of powerful arylmetalloporphyrin tweezers (with zinc, magnesium, iron, and tin as metal) and new classes of auxiliary chiral """"""""twisters."""""""" ii) Basic studies on fluorescence detected exciton coupled (FDCD) will be investigated. Following our first promising results obtained in 1997 showing that the detection sensitivity can be increased 50 to 100-fold under favorable conditions, further work is proposed to clarify important chemical, spectroscopic and technical issues of FDCD. iii) The exciton coupled CD method will be applied on a sub- microgram scale for direct configurational assignment of synthetic and endogenous ceramides and phytosphingosines. The determination of the absolute configuration of these three important antibiotics will clarify the importance of stereochemistry in their biological function. iv) CD and FDCD will be applied to ligand/receptor interactions. This will include CD studies of retinal/opsin in rhodopsin and retinal/bacterioopsin in bacteriorhodopsin. Vinca alkaloid/tubulin and taxoid/tubulin complexes will be analyzed by FDCD.
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