异核苷的连续流动化学合成工艺研究

Isonucleoside has the characteristic transposition of the nucleic base from C1' to C2' at furanosyl ring, which results in the overall conformation change of the nucleoside. According to our collaborated studies, a 15-mer single-strand oligonucleotides incorporated with isomeric nucleoside, which has the similar sequence with Thrombin binding aptamer (TBA), can specifically interact with thrombin. In vitro biological evaluation showed it has great potential to be developed into anticoagulation drug with Independent Intellectual Property Rights. In order to perform further experiments on living animals, it is urgent to obtain kilogram scale related isonucleosides. Compared to batch processing, which are widely used in manufacturing chemicals, Continuous Flow chemistry could provide high mixing and heat transfer rates, and narrow residence time distribution. Thus, the new enabling technology has many advantages such as secured reproducibility, facile automation, improved safety and process reliability etc. For the reasons outlined above, in present project, we will investigate the synthetic process development for preparation of kilogram scale isonucleoside using continuous flow chemistry. We will also investigate the effects of residence time, flow rate and reactor volume on the reaction yield in flow chemistry. The crucial relationship between flow, heat and pressure will be recorded in detail. Ultimately, a serial isonucleosides will be synthesized using the optimized process in kilogram scale. The obtained results will be very crucial for biological and clinical studies of isonucleosides.

异核苷是指核苷碱基从通常的呋喃糖环1'位挪动到2'位。近年来我们的合作研究发现，异核苷掺杂的寡聚核苷酸能够特异性结合凝血酶，阻止血液的凝结，因此有望开发出自主知识产权的抗凝血药物。为了进一步进行动物试验和临床研究，在公斤级规模上实现异核苷的制备迫在眉睫。连续流动化学(Continuous Flow chemistry)相对于传统的间歇反应器,具有物料混合速度快、传热效率高和停留时间分布窄等优点，从而合成工艺重现性好、有利于自动化控制、几乎无放大效应，很容易在实验室合成公斤级精细化学品。基于上述理由，本项目将研究异核苷的连续流动合成工艺，解决异核苷的合成难题，同时系统研究管路保留时间、流速和反应器容积等关键因素对连续流动化学反应的影响，获得反应液流动、加热和压力之间相互作用的系统数据，制备公斤级异核苷产品。该项目的研究结果对于推进异核苷的生物学和临床研究具有非常重要的意义。

近年来我们通过合作研究发现，核苷碱基从通常的呋喃糖环1'位挪动到2'位的异核苷掺杂的寡聚核苷酸能够特异性结合凝血酶，阻止血液的凝结，因此有望开发出新的抗凝血药物。为进行动物试验和临床研究提供足够的样品，本项目系统研究了异核苷的合成工艺，对可能的合成工艺进行了全面优化，开发了一条通用的异核苷合成路线。该工艺具有原料易得，合成操作简单和容易纯化等优点，并在此基础上，完成了iso-T, iso-U，iso-C和iso-A的百克级合成，完成了iso-G和iso-7-去氮腺苷的10克级合成。同时对反应中的副产物进行了详细分析鉴定，相关数据对于类似糖苷键的合成具有重要的借鉴作用，对相应核苷合成化学的理解具有积极的意义。在此基础上，本项目进一步系统研究了连续流式化学在异核苷制备工艺中的应用，积累了丰富的经验，为下一步自行设计定制流动合成仪器鉴定了基础。最后，为了研究异核苷在体内的代谢途径，设计合成了一类全新的磷杂色原酮骨架结构，该类化合物具有优异的荧光性质，表现出双荧光发射的特点，在核酸荧光标记上具有潜在的重要用途。