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

在医疗和食品部门，快速准确地检测和识别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，以便进一步鉴定或分型，并获得可供临床医生用于有效治疗感染的抗生素敏感性细节[1]。除了大约15年前将发色底物引入琼脂培养基外，许多选择性培养基配方自50年前或更长时间以来几乎没有变化。后者通常以糖的形式与发色原结合，形成无色的糖苷。糖苷被适当的酶水解会释放发色原，细菌菌落周围会出现一个彩色的光晕。糖苷酶并不普遍，有些酶可能仅限于生长在特定选择性培养基上的少数几种。这使得对有机体的假定识别为分析员节省了大量的时间和精力[2]。不同的糖可以添加到同一发色原中以改变特异性，因为只有糖影响酶的活性[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发色原的新型衍生品。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衍生物的颜色，例如通过使用软件程序，如WinPPP和/或PISYTEM。该项目感兴趣的色原包括活性染料，如黑5和橙色G。该项目的一个特别目标将是开发新的色原，其颜色可以补充现有的各种可供使用的显色底物。这将允许在介质中使用更多的显色底物来分离混合物中的生物，这些生物在存在或不存在一种或两种特定的酶的情况下难以分离。另一个目标将是扩大与发色原有关的糖的范围，以确定目前商业上无法获得的糖苷是否对细菌物种的差异有用。学生将使用有机化学技术合成糖苷，并将接受训练，以执行大小规模的反应，以及处理空气敏感材料。将需要开发一些新的方法。材料将使用一系列技术进行提纯，如层析和重结晶。然后，将使用最先进的分析化学技术，如核磁共振和UV-Vis光谱以及质谱学对糖苷产品进行表征。然后进行生物分析，以确定特定糖苷酶从底物中释放色原的能力，以提供有色沉淀物。然后，将使用食品和临床样本中存在的整个细菌进行研究，以确定底物是否确实可以根据特定细菌包含的糖苷酶来识别特定细菌。由于糖苷酶的严格专一性，为了使生物结果有意义，制备靶标的单个异构体(异构体)是必不可少的。奥斯本教授团队在碳水化合物化学和前药治疗方面的相关合成专业知识将确保确实将制备出纯的个体异构体。博维尔博士的团队专门从事分析和抑制食源性病原体的生长，这一综合专业知识将得到Bovill博士团队的补充。[1]Orenga，S.等人，J.Microbiol。方法，2009，出版社[2]Kiernan，J.A.Biotech。史氏，2007，82，73[3]巴特沃斯，L.A.等人，J.《微生物》，2004，96,170

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