Continuing research on this project is concerned with development of methods which can be used for the synthesis and semi-synthesis of peptides under mild conditions, particularly in aqueous solutions within "physiological" range of pH and temperature. Methods are being developed to specifically and reversibly protect amino groups, carboxyl groups, and several amino acid side-chain functional groups in preformed peptide fragments in aqueous soluton. Specific methods for removal of such blocking groups in aqueous solution include both mild chemical treatments and enzyme catalyzed hydrolyses. Bifunctional reagents are being developed which can be attached to polymers or other supporting media through one functional group of the molecule. The second functional group of the reagent is a specific ligand which will form a reversible covalent linkage to the side-chain of a particular amino acid residue in aqueous solutions. Supporting materials compatible with both aqueous and organic media are being evaluated to find materials suitable for carrying the side-chain specific ligands described above, which are also compatible with conventional peptide synthetic conditions in organic media, and which will allow the free approach of enzymes to attach peptides in aqueous solutions so that enzyme-labile blocking groups can be removed from synthetic intermediates on the support. All of the techniques and reagents being developed will be tested in model synthetic applications. We are focusing on bovine insulin, bovine insulin chains, and insulin chain fragments as model compounds for our semi-synthetic work.