Isotopomics in Chemical Biology & Shaanxi Key Laboratory of Chemical Additives for Industry, School of Chemistry & Chemical Engineering , Shaanxi University of Science & Technology , Xi'an 710021 , China.
SOA Third Institute of Oceanography , Xiamen 361005 , China.
Anal Chem. 2018 Sep 4;90(17):10293-10301. doi: 10.1021/acs.analchem.8b02022. Epub 2018 Aug 17.
The O/O ratio at both molecular and positional levels in the carbohydrates of higher plants is a reliable proxy for the plant growth environment, and a potential indicator of the plant photosynthetic carbon assimilation mode, and its physiological, biochemical and metabolic status. The lack of exploitable nuclear resonance in O and O and the extremely low O abundance make the NMR-based PSIA (position-specific isotopic analysis) a significant challenge. In this Article, an alternative three-step wet chemistry based method for accessing the O/O of glucose O-3 is presented. The O atoms (OH groups) at positions 1, 2, 5, and 6 were first protected by acetonation (converting glucose to 1,2;5,6-di- O-isopropylidene-glucofuranose). The protected glucose was then esterified at O-3 by thionoformylation. Subsequent Barton-McCombie deoxygenation quantitatively removed the O-3 from the protected sugar. Mass balance was then applied to calculate the O/O of O-3 using the isotopic values of the protected sugar before and after the deoxygenation step. The method is innovative in that (i) isolation and purification of individual compounds for O by EA/Pyrolysis/IRMS analysis is unnecessary as the reaction mixture can be analyzed on a GC/Pyrolysis/IRMS; (ii) sample quantity is dramatically reduced; and (iii) the approach to access the O-3 isotopic signal can be easily expanded to other positions within glucose and other sugars. It was shown that O-3 is enriched by 12 mUr relative to the molecular average (O-2-O-6) for a glucose of C photosynthetic origin. We highlighted the potential applications of the intramolecular O isotopic heterogeneity of glucose this method revealed.
高等植物碳水化合物在分子和位置水平上的 O/O 比是植物生长环境的可靠替代物，也是植物光合作用碳同化模式及其生理、生化和代谢状态的潜在指标。O 和 O 中缺乏可利用的核共振以及 O 的极低丰度使得基于 NMR 的 PSIA（位置特异性同位素分析）成为一项重大挑战。在本文中，提出了一种替代的三步湿化学方法，用于获取葡萄糖 O-3 的 O/O。首先通过乙酰化（将葡萄糖转化为 1,2；5,6-二-O-异亚丙基-D-呋喃葡萄糖）保护位置 1、2、5 和 6 的 O 原子（OH 基团）。然后，用硫代甲酰化将保护的葡萄糖酯化在 O-3 上。随后的 Barton-McCombie 脱氧定量地从保护的糖中去除 O-3。然后应用质量平衡法，使用脱氧步骤前后保护糖的同位素值计算 O-3 的 O/O。该方法的创新性在于：(i) 不需要通过 EA/Pyrolysis/IRMS 分析对 O 进行单独化合物的分离和纯化，因为可以在 GC/Pyrolysis/IRMS 上分析反应混合物；(ii) 样品量大大减少；以及 (iii) 访问 O-3 同位素信号的方法可以很容易地扩展到葡萄糖和其他糖中的其他位置。结果表明，相对于 C 光合作用起源的葡萄糖的分子平均值（O-2-O-6），O-3 富集了 12 mUr。我们强调了这种方法揭示的葡萄糖分子内 O 同位素异质性的潜在应用。
