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The goal of our research is to build on an initial discovery by our laboratory of a simple receptor able to undergo a pH-dependent conformational shift characterized by the formation of a network of phenol-amine hydrogen bonds. This network produces a “closed”, or cup-like structure, which binds metal ions in a size-selective manner. In order to further understand and optimize this process, we are examining the structure and properties of several structural analogs of our initially described receptor. During the first year of research, we focused on two primary areas. First, we extended our initial quantum chemical (ab initio and density functional - DFT) calculations on amino acid side chains to several model receptors. This is important as a way to both understand the results of ion titration experiments, and predict the behavior of new receptors. Second, we successfully synthesized four of the proposed pH-switchable ion receptors: the 3-aminotyrosine derivative, the 3-chlorotyrosine derivative, the 3-nitrotyrosine derivative, and the 3-aminophenylalanine derivative. Unfortunately, we discovered soon after completing the initial characterization of these compounds that impurities (dicyclohexyl carbodiimide and dicyclohexyl urea) carried over from the amino acid coupling reaction could not be removed from the final products, despite extensive extraction and/or chromatographic procedures. This difficulty was not encountered in our original synthesis of the trityrosine derivative. Surprisingly, these materials remained even after deprotection of the amino acids (Boc removal with trifluoroacetic acid). Therefore, a revision of the synthetic scheme became essential. Initially, we focused on altering the number of equivalents of dicyclohexyl carbodiimide used in the coupling reaction, in the hope that the use of fewer equivalents would reduce the amount of impurities in the final product. Unfortunately, this was unsuccessful. Focusing primarily on the 3-nitrotyrosine derivative, we switched to using diisopropyl carbodiimide as the coupling reagent. This indeed provided the coupled material in reasonable yield, and we were able to successfully able to obtain the final product after deprotection in high analytical purity. Efforts to synthesize the 3-chlorotyrosine and 3-aminophenylalanine derivatives are in progress. With the 3-nitrotyrosine derivative in hand, the next step is to begin the process of analyzing its interaction with ions as a function of pH. Titration experiments will be carried out by both isothermal titration calorimetry (ITC) and UV-Vis, and will follow the same general scheme used in our published work. We anticipate that the 3-nitrotyrosine receptor will be able to form the cup-shaped structure organized by hydrogen bonds at a pH range of 7.2 to 9.4. To confirm the pKa values of the amino groups in these receptors, we have also embarked on an extension of our method for pKa determination by ITC to amines (Aim 2 of the original proposal). Originally developed for measuring thiol pKa by examining the pH-dependent rate of reaction with iodoacetamide, our original intent here was to use ninhydrin as an amino-selective reagent for analogously measuring amine pKa. Initial experiments proved promising (including a successful determination of the lysine sidechain pKa), and we began applying the method to other amines. What we have found, however, is that the rate of decomposition of ninhydrin in solution at basic pH values is sufficiently high that its use in this context is not reliable. Therefore, we are in the process of examining N-Succinimidyl S-acetylthioacetate (SATA) as an alternative reagent. If we are successful in using SATA for the measurement of the pKa's of a standard set of amines, we will apply the methodology to our newly synthesized ion receptors.