Integrated workflows for glycan analysis: tagging strategies to facilitate structural and functional characterisation of carbohydrates

蛋白质和其他重要的生物分子，如脂类，往往高度装饰着复杂的碳水化合物链。这些碳水化合物参与许多过程，如细胞-细胞黏附和病原体识别。然而，这些相互作用涉及的碳水化合物结构类型通常不容易通过标准的合成路线获得。这在很大程度上是由于它们的结构复杂性和多样性。此外，对这些复杂多样的糖类物种的分析本身也带来了许多分析挑战，而且还常常因为缺乏可用的材料而受到阻碍。虽然这些糖的化学合成是具有挑战性的，但大的碳水化合物结构可以直接从生物分子中分离出来，并可以用荧光标记来帮助它们分离。这种技术被用于生物药物的商业分析，截至2014年，生物药物占了欧洲十大畅销药物中的八种。了解修饰这类药物表面的糖类是质量控制的一个重要方面，因为表面显示的糖类的变化会影响生物药物的储存稳定性、有效性和安全性。尽管在分析领域中使用了这项技术，但在这一过程中通常采用的标记策略导致分离的碳水化合物对下游应用几乎没有用处，并且它们通常被丢弃。分离足够的未标记物质用于生物检测通常需要重复的“盲法准备”，即从从蛋白质表面切割的糖的复杂混合物中分离出未标记材料，然后对该分离株的样品进行标记并检查其保真度和纯度。这种分离碳水化合物的方法需要相对大量的起始材料和多个耗时的后处理步骤。本项目描述了一种替代的荧光标记的制备，该标记在分离系统中具有类似的性质，同时具有附加的功能，可以直接用于下游使用。其意图是，该标签将允许通过工业碳水化合物分析中应用的程序分离的标记糖用于合成糖脂的生产。这些合成的糖脂将被层叠在表面以创建人造细胞膜，并被插入液滴中以模拟整个细胞。使用这些类型的方法来呈现糖，我们可以创建这样的系统，其中糖以一种模仿正确方向的方式显示在表面上，紧密地模拟它们在自然系统中的显示方式。通过使用带有不同荧光标记和携带不同定义碳水化合物的合成糖脂的不同组合，可以设想这些人造膜可以用于研究蛋白质和病原体如何与显示在细胞表面的糖结合。以这种方式创建人造膜将使我们不仅能够研究不同的碳水化合物如何与蛋白质伙伴结合，而且还可以研究含有不同糖的糖脂组合如何被病原体招募。这将使我们能够调查当这些识别事件发生时，这些物种是如何安排自己来最大化结合效率的。更深入地了解这些事件的性质以及这些分子的行为和相互作用可以为生物过程提供重要的见解，并有可能为诊断工具和新疗法的开发提供信息。还希望同样的标记糖可以提供给更广泛的科学界，以提供迄今难以获得的试剂，这些试剂可以直接用于各种不同的分析、分析技术和合成应用。

Proteins and other important biological molecules such as lipids are often highly decorated with complex chains of carbohydrates. These carbohydrates are involved in many processes, such as cell-cell adhesion and the recognition of pathogens.The types of carbohydrate structures involved in these interactions, however, are often not easily accessible in quantity by standard synthetic routes. This is largely due to their structural complexity and their diversity. In addition, the analysis of these complex and diverse sugars species itself poses many analytical challenges and is often also additionally hampered by the lack of available material. Although chemical synthesis of these sugars is challenging, large carbohydrate structures can be isolated directly from biological molecules and they can be tagged with a fluorescent label to aid their separation. Such technology is exploited commercially in the analysis of biopharmaceuticals, which (as of 2014) made up eight of the ten top selling drugs in Europe. An understanding of the sugars decorating the surface of such therapeutics is an important aspect of quality control, as changes in the sugars displayed on the surface can affect the storage stability, the efficacy and the safety of biologic drugs. Despite the use of this technique in the field of analysis, the labelling strategies commonly employed in this process result in the isolated carbohydrates being of little use for downstream applications and they are routinely discarded. The isolation of sufficient unlabelled material for use in biological assays often requires repeated "blind preparation" where unlabelled material is isolated from a complex mixture of sugars cleaved from the surface of a protein, and a sample of this isolate then labelled and checked for fidelity and purity. Such approaches to isolating carbohydrates require comparatively large quantities of starting material and multiple time-consuming post-processing steps.This project describes the preparation of an alternative fluorescent label exhibiting comparable properties in the separation system, whilst bearing additional functionality which can be exploited to facilitate downstream uses directly. It is the intention that this label will allow for labelled sugars isolated by procedures applied in industrial carbohydrate analysis to be utilised in the production of synthetic glycolipids. These synthetic glycolipids will be layered onto a surface to create artificial cell membranes and also inserted into droplets to mimic whole cells. Using these types of methods to present sugars, we can create systems where the sugars are displayed on a surface in such a way that they mimic the correct orientation, closely modelling how they would be displayed in a natural system.By using different combinations of synthetic glycolipids bearing different fluorescent labels and carrying different defined carbohydrates, it is envisaged that these artificial membrane can be used to investigate how proteins and pathogenic species bind to the sugars displayed on the cell surface. Creating artificial membranes in this way will allow us to investigate not only how different carbohydrates bind to protein partners, but also how combinations of glycolipids bearing different sugars are recruited by pathogens. It will allow us to investigate how such species arrange themselves to maximise binding efficiency when these recognition events take place. A deeper understanding of the nature of these events and how these molecules behave and interact can provide important insights into biological processes, and have the potential to inform the development of diagnostic tools and novel therapeutics.It is also hoped that the same tagged sugars can be made available to the wider scientific community to provide access to hitherto difficult to obtain reagents which can be used directly in a variety of different assays, analytical techniques and synthetic applications.