The long term objective of this project is to develop a class of well characterized, low valent early transition metal reagents that will allow one to cross couple unsaturated organic functional groups in a regio- and stereoselective manner. Furthermore, all of the reagents are to be simple coordination compounds that are either commercially available or easily prepared in one step. Placing these standards upon the reagents ensures that if interesting chemistry is uncovered, it may be immediately applied by us and others in organic synthesis. In this proposal we are continuing our investigation of how transition metal radicals can be applied in organic synthesis. During the past grant period we discovered a method for cross coupling two different, yet electronically similar aldehydes in a stereoselctive manner. The so called intermolecular pinacol cross coupling reaction is a very powerful method for constructing vicinal diols. Therefore, a significant part of this project will focus on defining the scope of this reaction. This will include exploring a wide range of potential substrates that can function as chelation accelerated (CA) aldehydes. Establishing this reaction as a useful method in organic synthesis will also be a primary concern. Our approach in this case is to apply pinacol coupling to the synthesis of several biologically relevant classes of molecules. For example, we feel intermolecular pinacol cross coupling can be used to develop new and expedient routes to several classes of sugars (e.g. amino sugars), polyhydroxylated alkaloids, phytosphingosines, aminocyclitols, Etrisubstituted allylic alcohols and a host of other interesting compounds. Intramolecular pinacol coupling is also discussed in the context of developing new routes to both small ring, polyhydroxylated compounds (e.g. cyclobutanediols) as well as large ring systems (e.g. cembranes and cembranolides). Methods for achieving enantioselective pinacol coupling are also discussed. We have pioneered the development of amperometric detection in narrow-bore capillary electrophoresis and the use of this technique to detect neurotransmitters in the cytoplasm of single nerve cells. The goals of this proposal involve the miniaturization of capillary electrophoresis and development of detection schemes for separation and detection of neurochemicals removed from the cytoplasm of single nerve cells. A major portion of this proposal deals with methods development for the neurochemical experiments. 5-mum i.d. capillaries will be used with amperometric detection and a novel injection scheme to sample the cytoplasm of single invertebrate neurons. The use of 2-mum and even 1-mum i.d. capillaries will be investigated for sampling the cytoplasm of single mammalian cells. Detection schemes based on pulsed amperometry and copper/copper oxide electrodes will be investigated for detecting removed intracellular second messengers. In addition to systems involving electrochemical detection, schemes based on laser fluorescence and electrogenerated chemiluminescence will be examined for detection of neuropeptides. Finally, lowvolume capillary electrophoresis will be coupled to flow SIMS TOF MS for acquisition, separation and detection of the major components of cell membranes. This will lead to the development of technology to sample a small segment (2-5 mum diameter) of membrane and provide analysis of localized membrane regions of single cells. The neurochemical goals of this proposal emphasize the study of neurotransmitter and intracellular second messenger levels in single cell cytoplasm. These experiments are meant to complement and expand our experiments involving dynamic monitoring of neurotransmitters and intracellular messengers in cytoplasm with voltammetry.
