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CO2的形成非常重要。我们观察到，内部返回的CO2可以减缓或阻止脱羧。这可以通过质子化或氧化捕获新生负碳离子来克服。我们还观察到，脱羧促进不饱和羧酸的质子化，导致形成质子化的碳酸，其分解得到CO2，从而避免内部返回。在另一种途径中，碳酸氢盐最初由水合作用形成，随后由一般碱催化形成。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的形成和释放的看法发生了重大变化。我们将研究这些机制的含义和扩展，以了解各种结果适用的条件。我们还将设计脱羧催化剂，使用缔合机制作为其基础。 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，这些反应途径通过水合羧基的替代途径克服与CO2反应的限制。脱羧酶和水解酶的机制和结构相似性表明，水解途径可以在其他脱羧酶中发现，其中的相似性尚未得到承认。我们将检查可以引入的潜在反应性改变，以测试观察结果的一般性。例如，我们将使用诱变和表达来研究二磷酸硫胺素依赖性脱羧酶中差异途径的起源。 我们的另一个主要工作是设计新的催化方法来氨酰化tRNA，促进非编码氨基酸蛋白质的形成。我们已经发现镧系元素离子促进顺式-1，2-二醇的氨酰化，如发生在tRNA的3 ′-末端。然而，完整的tRNA的氨酰化是低效的，并且难以检测，而单核苷酸则以高效率进行反应。我们现在将开发二核苷酸的氨酰化;然后可以使用连接酶将所产生的氨酰化物质与截短的tRNA连接。 我们已经在一个模型案例中测试了反应，发现它提供了令人满意的结果。我们现在将使用各种反应方案和保护基团来推广不同氨酰磷酸酯的方法，以寻求最佳组合。

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.