Mechanisms and Designs in Biological Organic Chemistry

我们最近发现了脱羧的催化途径，这对我们全面了解生物和化学系统中二氧化碳的形成非常重要。我们观察到CO2的内部返回可以减缓或阻止脱羧。这可以通过质子化或氧化捕获新生的碳离子来克服。我们还观察到，不饱和羧酸的预先质子化会促进脱羧，从而形成质子化的碳酸，而质子化的碳酸分解产生二氧化碳，从而避免了内部返回。在另一种途径中，碳酸氢盐最初由水合作用形成，然后是一般碱催化作用。我们注意到酶可能遵循类似途径的情况。这些发现使我们对二氧化碳形成和释放的看法发生了重大变化。我们将调查这些机制的影响和扩展，以了解各种结果适用的条件。我们还将设计以结合机制为基础的脱羧催化剂。我们将扩展我们最近的计算方法，以发现控制反应途径的能量表面，通过水合羧基的替代途径克服与二氧化碳反应的限制。脱羧酶和水解酶在机理和结构上的相似性表明，在其他脱羧酶中也可以发现水解途径，而这些途径的相似性尚未被认识到。我们将检查潜在的反应性变化，可以引入来测试观察的普遍性。例如，我们将使用诱变和表达来研究硫胺素二磷酸依赖性脱羧酶的差异途径的起源。

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.