Application of novel SPE-NMR protocols and flow-chemistry synthesis for comprehensive annotation of the human urinary metabolome, metabolite identific

代谢物结构解析被认为是代谢组学/代谢组学中的瓶颈-在组织和生物流体（最常见的是尿液，因为它是非侵入性收集的并且容易获得）中发现的代谢物可以被鉴定并且被证明是特定表型（例如疾病状态）的生物标志物特征。然而，人类尿液代谢组仍然只是部分映射。因此，有必要扩大人类尿液代谢组的注释，以提高我们表征人群表型和发现与疾病和饮食相关的生物标志物的能力。目前，用于代谢物注释和鉴定的两种最强大的分析技术是质谱（MS）和核磁共振（NMR）光谱，因为它们以精确和高通量的方式在绝对和相对定量的基础上提供正交定性数据。NMR是一种用于表征代谢物的强大技术-样品的定量、非破坏性和快速处理，以及所产生数据的固有再现性，可以为实验人员提供关于生物流体的额外信息维度，这些信息与质谱法产生的数据集正交。固相萃取（SPE）可以被认为是液-固色谱法，其中填充柱用于基于诸如极性和疏水性的相互作用分离出复杂混合物中的化合物。这允许化合物被提取、富集和纯化用于MS/NMR分析。基于二氧化硅和聚合物的固相萃取利用具有附接到二氧化硅或聚合物“骨架”的修饰（例如C18十八烷基链）的柱。采用不同的化学物质进行不同的修饰，可以获得不同的保留曲线。采用SPE方法预处理的样品可以用于分子分离，随后使用各种1D和2D NMR技术和MS进行结构解析。SPE柱的不同化学物质（如阳离子和阴离子交换）可以用于调节和简化生物流体的NMR光谱。这种方法类似于Zhang等人先前报道的方法，其中将带负电荷或带正电荷的二氧化硅纳米颗粒添加到生物流体样品中，分别导致带正电荷或带负电荷的代谢物的NMR信号的减弱和抑制。该方法有助于通过去除串扰来去除13 C-1H HSQC光谱中的重叠，从而有助于样品的表征。该项目的目的是利用不同化学成分的检测盒，以便根据代谢物类别制定选择性分离人尿液的方案。SPE实验的变化将通过改变溶剂系统、尿液浓度和检测盒化学成分来完成。然后可以开发可以转移到自动SPE系统上的方案。还将使用LC-MS和直接输注（DIMS）辅助代谢物鉴别。鉴定过程的一个关键方面涉及将pupeli注释的化合物与真实标准进行比较。然而，许多标准品（及其光谱）由于缺乏需求、高定制合成成本或仅仅因为所讨论的标准品以前不需要而不可用。有机流动化学的使用已经由Steven Ley教授的小组推广。流动试剂盒可以以模块化的方式设置，使微流体实验能够用于天然产物的合成。这种方法与“传统”合成相比有几个优点：设备的模块化性质允许根据任何给定实验的需要改变设置，整个装置可以自动化，以便更好地使用和控制系统，并且所讨论的反应以高效率运行，产生更少的废物-因此是未来可持续化学的基石。

Metabolite structure elucidation is considered a bottleneck in metabonomics/metabolomics - metabolites found within tissues and biofluids (the most common being urine, as it is collected non-invasively and easily available) can be identified and proven to be biomarkers characteristic of specific phenotypes, such as disease states. However, the human urinary metabolome remains only partially mapped. There is hence a need to expand the annotation of the human urine metabolome in order to improve our capacity for characterization of population phenotypes and discovery of biomarkers related to diseases and diet. Currently, the two most powerful analytical techniques used for metabolite annotation and identification are mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy, as they provide orthogonal qualitative data, on top of absolute and relative quantification in a precise and high-throughput manner. NMR is a powerful technique in the characterisation of metabolites - the quantitative, non-destructive, and rapid processing of samples, as well as the inherent reproducibility of the data produced, can give experimenters an additional dimension of information about biofluids, which are orthogonal to the datasets produced by mass spectrometry.Solid Phase Extraction (SPE) can be thought of as liquid-solid chromatography, in which a packed column is used to separate out compounds within a complex mixture based on interactions such as polarity and hydrophobicity. This allows for the compounds to be extracted, enriched, and purified for MS/NMR analysis. Silica- and polymer-based solid phase extraction utilises columns with modifications (such as C18 octadecyl chains) attached to a silica or polymer 'backbone'. Different modifications with different chemistries allow for a different retention profile.Samples pre-treated with SPE methods can be utilized for isolation of molecules and subsequent structure elucidation using various 1D and 2D NMR techniques and MS. The different chemistries of SPE cartridges (such as cation and anion exchange) can be utilised in order to modulate and simplify NMR spectra of biofluids. This approach is similar to one previously reported by Zhang et al, in which negatively or positively charged silica nanoparticles were added to a biofluid sample, causing weakening and suppression of the NMR signals of positively or negatively charged metabolites, respectively. The approach helped to remove overlap in 13C-1H HSQC spectra by removal of crosspeaks, aiding characterisation of the sample. The aim of the project is to utilise cartridges of differing chemistries, in order to produce protocols for the selective fractionation of human urine based on metabolite class. Variations of SPE experiments will be done by varying solvent systems, urine concentrations, and cartridge chemistries. Protocols which can be transferred onto an automated SPE system can then be developed. LC-MS and direct infusion (DIMS) will also be used to aid metabolite identification. A key aspect of the identification process involves the comparison of putatively annotated compounds to authentic standards. However, many standards (and their spectra) are not available due to lack of demand, high tailored-synthetic costs, or simply because the standard in question was not previously required. The use of organic flow chemistry has been popularised by the group of Prof. Steven Ley. Flow kits can be set up in a modular fashion, enabling microfluidic experiments to be run for the synthesis of natural products. This approach has several advantages over 'traditional' synthesis: the modular nature of the equipment allows for setups to be changed as necessary for any given experiment, the entire rig can be automated for better ease of use and control over a system, and the reactions in question run at high efficiencies, creating less waste - hence being a keystone in the future of sustainable chemistry.