Mechanisms and Designs in Biological Organic Chemistry

生物有机化学的机理和设计

• 批准号: RGPIN-2015-04993
• 负责人: Kluger, Ronald
• 金额: $ 4.3万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=613295
• 关键词: Mechanisms; Designs; Biological; Organic; Chemistry

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.

我们最近发现了脱羧化的催化途径，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，二氧化碳的内部返回可以减缓或阻止脱羧化。这可以通过质子化或氧化来捕获新生的碳离子来克服。我们还观察到，不饱和羧酸的质子化导致质子化碳酸的形成，质子化碳酸分解生成二氧化碳，从而避免了内部返回，从而促进了脱羧基。在另一种途径中，小苏打最初是由水合作用形成的，然后是一般的碱催化。我们注意到一些情况下，酶很可能遵循类似的途径。这些发现极大地改变了我们对二氧化碳形成和释放的看法。我们还将调查这些机制的影响和延伸，以了解各种结果适用的条件。我们还将设计以缔合机制为基础的脱羧化催化剂。我们将扩展我们最近的计算方法，以发现控制反应路径的能量面，这些反应路径通过水合羧基的替代路径克服与二氧化碳反应的限制。脱羧酶和水解酶的机理和结构上的相似性表明，在其他脱羧酶中也可以找到这种相似性尚未被认识到的水解途径。我们将检查可以引入的潜在反应性变化，以测试观察的一般性。例如，我们将使用诱变技术和表达技术来研究硫胺素二磷酸依赖脱羧酶的差异途径的起源。 我们的另一个主要工作是设计新的催化方法来实现tRNA的氨基酰化，促进含有非编码氨基酸的蛋白质的形成。我们发现，稀土离子促进了顺-1，2-二醇的氨酰化反应，就像发生在tRNA的3‘末端一样。然而，完整tRNA的氨酰化反应效率低且难以检测，而单核苷酸进行这一反应效率很高。我们现在将开发二核苷酸的氨酰化反应；然后可以使用带有截短tRNA的连接酶来连接得到的氨基酰化物种。我们已经在一个模型案例中测试了反应，并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寻求最佳组合的保护基团来推广不同氨基酰基磷酸盐的工艺。