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，经常被复杂的碳水化合物链高度修饰。这些碳水化合物参与许多过程，如细胞-细胞粘附和病原体识别。然而，这些相互作用中涉及的碳水化合物结构的类型通常不容易通过标准的合成途径获得。这主要是由于它们结构的复杂性和多样性。此外，对这些复杂而多样的糖类物种的分析本身也提出了许多分析挑战，并且通常也由于缺乏可用材料而受到阻碍。虽然这些糖的化学合成具有挑战性，但可以直接从生物分子中分离出大的碳水化合物结构，并且可以用荧光标记来帮助它们分离。这种技术被商业化地应用于生物药品的分析，（截至2014年）生物药品占欧洲十大畅销药物中的八个。了解修饰这些治疗药物表面的糖是质量控制的一个重要方面，因为表面糖的变化会影响生物药物的储存稳定性、有效性和安全性。尽管在分析领域使用了这种技术，但在该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。分离足够的用于生物分析的未标记物质通常需要重复的“盲制备”，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物质，然后标记该分离物的样品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。该项目描述了一种替代荧光标签的制备，在分离系统中表现出类似的性能，同时具有额外的功能，可以直接用于促进下游使用。该标签的目的是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分层到表面以制造人造细胞膜，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，我们可以创建一个系统，在这个系统中，糖以一种模仿正确方向的方式显示在表面上，密切模仿它们在自然系统中的显示方式。通过使用具有不同荧光标记和携带不同定义的碳水化合物的合成糖脂的不同组合，设想这些人工膜可用于研究蛋白质和致病性物种如何与细胞表面显示的糖结合。以这种方式制造人造膜不仅可以让我们研究不同的碳水化合物如何与蛋白质伴侣结合，还可以研究携带不同糖的糖脂如何被病原体吸收。这将使我们能够研究当这些识别事件发生时，这些物种如何安排自己以最大限度地提高结合效率。更深入地了解这些事件的本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，以提供迄今为止难以获得的试剂，这些试剂可以直接用于各种不同的测定、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.