Within our continued effort aimed at developing conjugate vaccines for infectious diseases from synthetic fragments of bacterial carbohydrates we have three ongoing projects. Two are concerned with a vaccine for cholera and one with a vaccine for anthrax. Existing vaccines for these diseases are based on cellular material and, in addition to having undesirable side effects, do not provide long-term immunity. Development of the two vaccines is important from both the point of view of public health and of national interest. Development of a potent vaccine for cholera is important because of the involvement of our military in protecting US interests in developing third world countries. While anthrax does not constitute a major health problem in the civilized world, new concerns regarding anthrax have emerged because of potential use of some form of Bacillus anthracis, the etiological cause of anthrax, as a biological weapon. Our work towards a potent conjugate vaccine for cholera involves synthesis of oligosaccharides that mimic the structure of O-specific polysaccharide (O-PS) of Vibrio cholerae in the form suitable for conjugation, conjugation of these antigens to suitable carriers, and serological evaluation of the immunogenicity of the resulting neoglycoconjugates. The approach towards a vaccine for anthrax is based on preparation of a neoglycoconjugate from a suitable carrier and the tetrasaccharide side chain of the major glycoprotein of Bacillus anthracis exosporium. In the past, we have focused on improving diagnostic tools for the detection of presence of anthrax spores. Since preliminary work indicated that the anthrose-containing tetrasaccharide chain seemed to be highly specific for B. anthracis, during the period associated with this report we focused on testing the possibility of altering immunogenicity of the protective antigen (PA), which is protein in nature, by coupling it with the tetrasaccharide moiety from the BclA protein. Preliminary immunization studies suggested that this tetrasaccharide construct may enhance the immune response generated by PA. This was indicated by approximately 20% increase of protection of mice challenged with Ames spores. Unfortunately, due to lack of funds on the side of our collaborators, further work on optimizing the conjugates had to be temporarily discontinued. In the cholera project, while immunization studies are ongoing, we have focused on developing new methods which would allow localization of site of conjugation of synthetic antigens on the carrier protein by mass spectrometry. We have found that glycation sites in neoglycoglycoconjugates could be revealed, following proteolytic digest, by matrix-assisted laser desorptionionization tandem mass spectrometry. Under these conditions, high energy collision-induced fragmentation occurs, resulting in spectra which reflect presence of fragments of the carbohydrate attached to amino acid sequences. As a result, we were able to identify the conjugation sites involving three different lysine residues (Lys235, Lys437 and Lys455). Although only few points of glycation could be determined, the information obtained was very useful for the peptide sequencing. Knowing the glycation sites in neoglycoconjugate can be applied in quality control of conjugate vaccines. In addition, since the site of glycation may affect immunogenicity we expect the ability to determine the glycation sites to be helpful in optimizing the immunogenicity of future conjugate vaccines. We have also made adequate progress in the project aimed on developing therapies for Fabry and Gaucher diseases, which are caused by insufficient activity of biodegrading enzymes resulting in accumulation of toxic levels of globosylceramide (Gb3). Patients suffering from Fabry disease experience, among other symptoms, myocardial infarctions, strokes and progressive kidney failure. Developing therapies for kidney diseases is one of the missions of NIDDK. Enzyme replacement therapy is somewhat successful treatment with some patients with Gaucher disease disorder, but this approach has been largely unsuccessful with Fabry disease. Even when on ERP, Fabry disease patients still experience, among other symptoms, myocardial infarctions, strokes and progressive kidney failure. An alternative therapeutic strategy for these diseases is substrate reduction therapy, which is aimed at reducing the enzymatic formation of Gb3. In collaboration with top authorities in this area we are designing a suitable substrate for the enzyme that is involved in the synthesis of Gb3. Based on our improvement of the chemical synthesis of 4&#8242;-deoxy-lactose we have prepared a number of analogs of lactosyl ceramides whose potency as inhibitors of enzymatic synthesis of Gb3 will be tested in a suitable enzyme system that catalyzes the formation of Gb3 from UDP-galactose and lactosylceramide.