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

代谢物结构阐明被认为是代谢组学/代谢组学的一个瓶颈--在组织和生物体液中发现的代谢物(最常见的是尿液，因为它是非侵入性的，而且很容易获得)可以被识别并被证明是特定表型的生物标记物，例如疾病状态。然而，人类尿液代谢组仍然只有部分图谱。因此，有必要扩展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记物的能力。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仪(MS)和核磁共振(核磁共振)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了正交的定性数据。核磁共振是表征代谢物的一种强有力的技术--样品的定量、无损和快速处理，以及产生的数据固有的重复性，可以为实验者提供与质谱学产生的数据集正交的关于生物流体的额外信息。固相萃取(SPE)可以被认为是液-固色谱，其中使用填充柱根据极性和疏水性等相互作用分离出复杂混合物中的化合物。这使得化合物可以被提取、浓缩和提纯，用于MS/核磁共振分析。硅胶和聚合物为基础的固相萃取柱使用与硅胶或聚合物“主链”相连的带有修饰的柱(如C18十八烷基链)。用不同的化学方法对样品进行不同的修饰可以得到不同的保留图谱。经固相萃取方法处理的样品可用于分离分子，随后可使用各种一维和二维核磁共振技术和质谱仪进行结构鉴定。固相萃取柱的不同化学成分(如阳离子和阴离子交换)可用于调制和简化生物体液的核磁共振谱。这种方法类似于Zhang等人之前报道的一种方法，在该方法中，将带负电荷或带正电的二氧化硅纳米颗粒添加到生物流体样本中，分别导致带正电或带负电的代谢物的核磁共振信号减弱和抑制。该方法通过去除串音来帮助消除13C-1H HSQC谱中的重叠，有助于样品的表征。该项目的目的是利用不同化学成分的墨盒，以生产基于代谢物类别的选择性分馏人类尿液的方案。固相萃取实验的变化将通过不同的溶剂系统、尿液浓度和药盒化学成分来进行。然后，可以开发可以传输到自动SPE系统的协议。LC-MS和直接输注(DIMS)也将用于辅助代谢物鉴定。鉴定过程的一个关键方面涉及将推定注释的化合物与正品标准进行比较。然而，由于需求不足、量身定做的合成成本较高，或者只是因为以前不需要相关标准，许多标准(及其光谱)不可用。有机流动化学的使用已经被史蒂文·莱伊教授的小组所普及。流动工具包可以以模块化的方式设置，使微流控实验能够用于合成天然产品。与传统的合成方法相比，这种方法有几个优点：设备的模块化性质允许根据任何给定的实验需要改变设置，整个装置可以自动化以更好地使用和控制系统，所述反应高效运行，产生的废物更少-因此成为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.