Design sysnthesis and analyisis of novel derivatives of industrially important chromogens for improved bacterial detection

在医疗和食品行业，快速准确地检测和鉴定样品中的致病菌或食品腐败菌至关重要。分子方法在这一领域显示出巨大的前景，但仍然需要在培养基上获得作为纯活实体的生物体，用于进一步鉴定或分型，并获得临床医生可用于有效治疗感染的抗生素敏感性的细节[1]。许多选择性培养基配方自50年或更早以前引入以来几乎没有变化，除了大约15年前将显色底物引入琼脂培养基之外。后者通常是以糖的形式与色原结合形成无色的糖苷。糖苷被适当的酶水解释放出色原，在细菌菌落周围出现有色晕圈。糖苷酶并不普遍，有些可能仅限于少数在特定选择性培养基上生长的物种。这允许对微生物进行推定鉴定，从而节省分析人员大量的时间和精力[2]。可以将不同的糖添加到相同的色原中以改变特异性，因为只有糖影响酶活性[3]。在这个多学科的项目中，学生将参与设计，合成和分析用于细菌检测的工业上有用的色原的新型衍生物。他们将使用计算方法来预测视觉吸收最大值，从而预测已知色原的新型衍生物的颜色，例如使用WinPPP和/或PISYTEM等软件程序。该项目中感兴趣的色原包括活性染料，如黑5和橙子G。该项目的一个特别目标是开发新的色原，其颜色补充现有的显色底物范围。这将允许在培养基中使用更大多样性的显色底物，以分离混合物中的生物体，所述混合物在存在或不存在一种或两种特定酶的情况下难以分离。另一个目的是扩大与色原连接的糖的范围，以确定目前不可商购的糖苷是否可用于区分细菌物种。糖苷将使用有机化学技术合成，学生将接受培训，以执行大型和小型反应，并处理空气敏感材料。将需要制定一些新的方法。将使用一系列技术（如色谱法和重结晶）对材料进行纯化。然后将使用最先进的分析化学技术（如NMR和UV-Vis光谱法以及质谱法）对糖苷产物进行表征。然后进行生物学分析，以确定特定糖苷酶从底物中释放色原的能力，以提供有色沉淀。然后将使用食品和临床样品中存在的全菌进行研究，以确定底物是否确实允许基于它们所含的特定糖苷酶识别特定细菌。由于糖苷酶具有严格的特异性，因此必须制备靶标的单个异构体（端基异构体），以获得有意义的生物学结果。奥斯本教授的小组在碳水化合物化学和前体药物治疗方面的相关合成专业知识将确保确实制备出纯的单个异构体。这种合成专业知识将得到Bovill博士团队的补充，该团队专门从事食源性病原体的分析和抑制。[1]奥伦加角例如，J. Microbiol.方法，2009年，出版中[2] Kiernan，J.A. biotech.组织化学，[3]巴特沃思，洛杉矶例如，应用微生物学杂志，2004年，96，170

In the medical and food sectors it is vitally important to quickly and accurately detect and identify pathogenic or food spoilage bacteria in samples. Molecular methods are showing great promise in this area but there is still a need to obtain the organism as a pure viable entity on a culture medium for further identification or typing and to obtain details of antibiotic susceptibility that may be used by the clinician for effective treatment of infection [1]. Many selective culture media formulations have changed little since their introduction 50 years or more ago with the exception of the introduction of chromogenic substrates to agar media some 15 years ago. The latter are generally in the form of a sugar joined to a chromogen to form a glycoside that is colourless. Hydrolysis of the glycoside by an appropriate enzyme releases the chromogen and a coloured halo appears around the bacterial colony. Glycosidase enzymes are not widespread and some may be restricted to only a few species that grow on a particular selective medium. This allows a presumptive identification of the organism saving the analyst a great deal of time and effort [2]. Different sugars may be added to the same chromogen to alter specificity since only the sugar affects enzyme activity [3]. In this multidisciplinary project the student will be involved in the design, synthesis and analysis of novel derivatives of industrially useful chromogens for bacterial detection. They will use computational methods to predict the visual absorption maxima and hence the colour of novel derivatives of known chromogens, for example by using software programmes such as WinPPP and/or PISYTEM. Chromogens of interest within the project include the reactive dyes such as Black 5 and Orange G. A particular aim of the project will be to develop novel chromogens with colours that complement the existing range of available chromogenic substrates. This will allow a greater multiplicity of chromogenic substrates to be used in media to separate organisms in mixtures that are difficult to separate on the presence or absence of one or two particular enzymes. A further aim will be to extend the range of sugars linked to chromogens to determine if glycosides not currently commercially available are useful for the differention of bacterial species. Glycosides will be synthesised using organic chemistry techniques and the student will be trained to perform both large and small scale reactions, and also to handle air sensitive materials. The development of some new methodology will be required. Materials will be purified using a range of techniques such as chromotography and recrystallisation. Glycoside products will then be characterised using state of the art analytical chemistry techniques, such as NMR and UV-Vis spectroscopy, and mass spectrometry. Biological analysis will then be performed to determine the ability of specific glycosidase enzymes to release the chromogen from the substrates, to afford coloured precipitates. Studies using whole bacteria present within food and clinical samples will then be performed to determine whether the substrates do indeed allow identification of particular bacteria, based on specific glycosidase enzymes that they contain. Due to the stringent specificity of glycosidase enzymes, it is essential that individual isomers (anomers) of the targets are prepared for the biological results to be meaningful. Relevant synthetic expertise in both carbohydrate chemistry and prodrug therapy within Professor Osborn's group will ensure that pure individual isomers will indeed be prepared. This synthetic expertise will be complemented by that within Dr Bovill's group, which specialises in the analysis and inhibition of growth of food borne pathogens. [1] Orenga, S. et al., J. Microbiol. Methods, 2009, in press [2] Kiernan, J.A. Biotech. Histochem., 2007, 82, 73 [3] Butterworth, L.A. et al., J. Appl. Microbiol., 2004, 96, 170