CD EXCITATION CHIRALITY METHOD AND ITS APPLICATIONS

CD激发手性方法及其应用

• 批准号: 2177469
• 负责人: KOJI NAKANISHI
• 金额: $ 36.67万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2177469
• 关键词: Echinodermata; Insecta; alternatives to animals in research; bacteriorhodopsins; beta lactam antibiotic; biphenyl compounds; bromobenzenes; carbohydrate structure; chemical addition; chemical binding; chemical structure function; circular dichroism; conformation; glycosides; hamsters; hydroxyl group; infrared spectrometry; mitomycin C; nuclear magnetic resonance spectroscopy; oligosaccharides; stereochemistry; stop flow technique; time resolved data

The proposal focuses on the development of two spectroscopic techniques that will deal with a variety of problems directly in the biomedicine. I. Time-resolved Fourier transform infrared (FTIR) difference spectroscopy. FTIR difference spectroscopy is an extremely sensitive technique which, under favorable conditions, allows one to follow the fate of a single proton in a protein of MW 28-43 kD as they undergo successive changes during a reaction. The reaction can be slowed down or quenched at a certain intermediate by taking measurements down to liquid He temperature. The current study proposes to exploit this new technique in a time-resolve manner. Thus, after mixing of reactants, the FTIR spectra are measured at suitable time intervals (as short as several seconds) under stopped-flow or continuous flow conditions; a subtraction of the two spectra will only show bands that have changed. The presence of biopolymers does not interfere because only the moiety that has undergone changes will appear. This is a technique that appears not to have ben used as a general tool. It is proposed to study the mode of binding of mitomycin to DNA to form the cross-linked adduct responsible for its therapeutically important cytotoxicity, the reaction of calichemycin (or esperamycin) (antibiotics with extraordinary activity) with DNA, the biosynthesis of penicillin, etc. The technique should have very broad applications. II. A simple micro-scale structure determination of oligosaccharides that requires no references. The dramatic progress seen in molecular biology depends to a great extent on the availability of micro-scale methods for determining protein and nucleic acid structures. The carbohydrates are far more difficult to handle, and the analysis depends on classical methods such as methylation analysis, which although dramatically improved still suffers from serious disadvantages; reference samples are required, anomeric and absolute configurations cannot by determined, etc. (sample level is sub-NMR). A totally new approach based on circular dichroism (CD) is proposed. Two approaches are possible. In one scheme, the free hydroxyls is in a sugar are tagged with p-phenylbenzyl groups, the sugar is methanolyzed in 5-15 min in a microwave oven, the products are HPLC-separated, and the CD of products are measured; the CD is characteristic of the substitution pattern. In the second scheme, the free hydroxyls are tagged with on chromophore while hydroxyls involved in glycosidic bonding are tagged with another chromophore; the CD of separated monosaccharide units are characteristic of the sugar and the substitution. Both methods disclose the stereochemistry of the anomeric center, require no reference and can be carried out at the microgram level. However, this is with model saccharides. The current proposal will focus on the application to read oligosaccharides of unknown structure, and those that are available in very limited amounts. This still requires additional new methodologies to be discovered. The outlook is quite promising. A mild method for cleaving the oligosaccharides into monomers, and an approach to sequence the sugars by MS will also be studied.

该提案着重于两个光谱镜的发展 直接处理各种问题的技术 生物医学。 I.时间分辨傅里叶变换红外（FTIR）差异 光谱法。 FTIR差异光谱是一种极其敏感的技术 在有利的条件下，它允许人们遵循命运 MW 28-43 kd的蛋白质中的单个质子的经历 反应过程中的连续变化。 反应可以放慢 通过测量在某个中间体下向下或淬火 下降到液体温度。 当前的研究提议 以时间解决方式利用这一新技术。 因此，之后 反应物的混合，在合适的时间测量FTIR光谱 间隔（短短几秒钟）停止流或 连续流动条件；这两个光谱的减法将 仅显示改变的乐队。 生物聚合物的存在 不干预，因为只有经历的部分 更改将出现。 这是一种似乎没有的技术 本用作通用工具。 建议研究 丝裂霉素与DNA结合以形成交联加合物 负责其在治疗上重要的细胞毒性， calichemycin（或杂志霉素）（抗生素与抗生素与 具有DNA的非凡活动），青霉素的生物合成， 等。该技术应该具有非常广泛的应用。 ii。 简单的微型结构确定 不需要参考的寡糖。 分子生物学中看到的巨大进步取决于一个很大的 微尺度方法的可用性范围 蛋白质和核酸结构。 碳水化合物远 更难处理，分析取决于经典 甲基化分析等方法，尽管 改善仍然遭受严重的缺点；参考 需要样本，异构和绝对配置不能 通过确定等。（样本级别为sub-nmr）。 一个全新的 提出了基于圆二色性（CD）的方法。 二 方法是可能的。 在一个方案中，游离羟基在 用糖标记了糖，糖为 在微波炉中5-15分钟内甲烷溶体，产品为 测量HPLC分离和产品CD； CD是 替代模式的特征。 在第二个方案中， 游离羟基在发色团上标记，羟基 与糖苷键合的参与用另一个发色团标记。 分离的单糖单元的CD是 糖和替代。 两种方法都披露了 异构中心的立体化学无需参考， 可以在微图水平上进行。 但是，这与 型糖。 当前的建议将重点放在 用于读取未知结构的寡糖和 那些有限的量。 这仍然 需要发现其他新方法。 这 展望是很有希望的。 裂解的一种轻度方法 寡糖成单体，一种方法来序列 也将研究MS的糖。