Enzymic synthesis of complex carbohydrates using evolved enzymes

哺乳动物细胞被大量的糖分子包裹，这些糖分子在从入侵的细菌和病毒感染细胞，免疫系统如何应对这些攻击，到各种细胞如何相互交流等过程中发挥着重要作用。这种交流在正常发育中起着至关重要的作用，在癌症等疾病状态下也会受到影响。人们越来越需要能够合成这样的糖分子，用于广泛领域的研究，但复杂的糖不能克隆，并且由于分子本身复杂的三维形状，它们的化学合成是复杂的。合成化学家已经开发出一些糖类似物的路线，但这些程序很困难，效率也不是很高。自然界的催化剂，即酶，以极高的精确度和效率进行这样的反应，但人类在制造复合糖中使用它们是有限的，因为酶已经进化成进行特定的反应，而这些反应并不总是化学家希望进行的反应。当我们希望制造天然糖的非天然模拟物时，这一点尤其正确。幸运的是，一种在试管中设计或进化新的、所需的酶活性的方法是可用的，本申请寻求资金来使用这种方法来改变两种天然酶的性质，使它们能够进行我们想要的反应，并使它们能够用于合成我们想要的新型复合糖。我们已经瞄准了一种叫做NAL的酶，改变它的功能来制造新的单糖，并证明了所提出的方法的基础。现在，我们将改变两种不同的酶（称为CNS和ST），以使用我们的新单糖来合成在我们细胞外部发现的复合糖的模拟物。

Mammalian cells are coated with a vast range of sugar molecules that play important roles in processes ranging from the infection of cells by invading bacteria and viruses, and how the immune system deals with these attacks, to how various cells communicate with each other. Such communication plays vital roles in normal development as well as being affected during disease states such as cancer. There is a growing need to be able to synthesise such sugar molecules for research in a wide range of areas, but complex sugars cannot be cloned, and their chemical synthesis is complicated because of the complex three-dimensional shapes of the molecules themselves. Synthetic chemists have developed routes to some sugar analogues, but these procedures are difficult and not very efficent. Nature's catalysts, the enzymes, carry out such reactions with great precision and efficiency, but their use by man in making complex sugars is limited because the enzymes have evolved to carry out specific reactions, which are not always those that the chemist wishes to carry out. This is particularly true when we wish to make unnatural mimics of the natural sugar. Fortunately, a method of engineering or evolving new, desired enzyme activities in the test-tube is available, and this application seeks funds to use this methodology to alter the properties of two natural enzymes to allow them to carry out the reactions that we want, and to allow them to be used to synthesise the novel complex sugars that we wish. We have already targetted one enzyme, called NAL, to alter its function to make new simple sugars, and have proved the basis of the methodology proposed. Now we will alter two different enzymes (called CNS and ST) to use our new simple sugars to be incoporated into mimics of the complex sugars that are found on the outsides of our cells.