Mechanisms and Designs in Biological Organic Chemistry

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We recently discovered catalytic pathways for decarboxylation that are important for our overall understanding of the formation of CO2 in biological and chemical systems. We observed that internal return of CO2 can slow or block decarboxylation. This can be overcome by trapping the nascent carbanion by protonation or oxidation. We also observed that decarboxylation is promoted by prior protonation of unsaturated carboxylic acids leading to formation of protonated carbonic acid, which decomposes to give CO2, thus avoiding internal return. In another route, bicarbonate forms initially from hydration followed by general base catalysis. We noted cases where enzymes are likely to follow similar pathways. These discoveries provide a major change in how we think about the formation and release of CO2. We will investigate the implications and extension of these mechanisms in order to understand the conditions where the various outcomes apply. We will also design catalysts for decarboxylation that use the associative mechanisms as their basis.  We will extend our recent computational approaches to discover the energy surfaces that control the reaction pathways that overcome the limitations of reactions with CO2 by alternative pathways through the hydrated carboxyl group. The mechanistic and structural similarities of decarboxylases and hydrolases suggest that hydrolytic routes can be found in other decarboxylases where the similarities have not been recognized. We will examine the potential reactivity alterations that can be introduced to test the generality of the observation. For example, we will use mutagenesis and expression to investigate the origins of the differential pathways in thiamin diphosphate-dependent decarboxylases. ***Our other main effort will devise new catalytic approaches to the aminoacylation of tRNA, facilitating the formation of proteins with non-coded amino acids. We have discovered that lanthanide ions promote the aminoacylation of cis-1,2-diols, as occurs at the 3'-terminus of tRNA. However, aminoacylation of intact tRNA is inefficient and difficult to detect, whereas mononucleotides undergo the reaction with high efficiency. We will now develop aminoacylation of dinucleotides; the resulting aminoacylated species can then be attached using a ligase with truncated tRNA.  We have tested the reaction in one model case and find that it provides a satisfactory outcome. We now will generalize the process with different aminoacyl phosphates using a variety of reaction schemes and protecting groups seeking optimal combinations.