靶向识别官能团质谱手性试剂的合成及其在糖尿未病状态手性生物标志物筛选中的应用

Discriminating the differences of the molecular chirality remains an important issue in analyzing drug metabolism and pharmacokinetics for the pharmaceutical industry and regulatory authorities, furthermore, molecular chirality is one of the characteristics of many endogenous metabolites in body. During the recent years, determination and quantification of trace metabolites, which are from in vivo tested subjects, have become feasible, because of the development of high-throughput, high-resolution UPLC-MS and the chemometrics data processing techniques. However, there are few reports providing the method to analyze the chiral metabolites. In this study, a new chrial reagent will be synthesized to analyze the chiral metabolites with carboxyl or amino groups. This chiral reagent will be designed to contain the isotope peak in MS spectrum, should increase the detection sensitivity, and also has an easy structural inference model. Through separating and identifying the chiral metabolites in potential diabetes patients using the specifically designed and synthesized chiral reagents, we will explore the factors and disciplines, which will significantly influence the separation of enantiomers; will investigate the relationship between the structure of chiral reagent and the separation performance; and also will summarize the fragmentation regularity of the metabolites in the mass spectrometry. Through this study, we will provides an excellent ideas of using chiral derivatization reagents to separate and identify the enantiomers; give the theory evidence; and also develop a novel analysis method for promoting the development of chiral metabonomics.

分子手性的差异仍是医药工业、药品监管部门药物代谢和药代动力学研究中具有挑战性研究课题之一，而且手性又是许多内源性代谢物重要的特征。现代超高效液相色谱-质谱 (UPLC-MS)联用技术结合化学计量学较好地实现了对大量样品和微量代谢物的快速分析，极大地推动了代谢组学的发展，但是在手性代谢组学方面，至今没有文献报道。鉴于此，在本项目中将拟以具有氨基、羧基官能团的手性代谢物为分析对象，设计合成含同位素特征峰的可鉴别手性对映体，而且既能提高质谱检测灵敏度，又易于结构推断的新型质谱手性试剂。本项目将各官能团识别手性试剂应用于糖尿未病时期的潜在手性代谢生物标志物的分离及鉴定，考察影响对映体分离的因素及分离规律，研究手性试剂结构与分离效能的关系，总结在质谱中代谢物结构的裂解规律，为研究和开发手性代谢组学优良的手性衍生化试剂提供思路和理论依据，同时为推动手性代谢组学的发展提供新的分析方法。

现代色谱-质谱联用技术极大地推动了代谢组学的发展，而在手性代谢组学方面，因手性代谢物m/z比相同无法在质谱中得到鉴别。鉴于此，我们以氨基，羧基官能团的手性代谢物为分析对象，设计合成了可鉴别手性对映体、且能提高质谱检测灵敏度、易于结构推断的6种靶标氨基官能团手性质谱衍生化试剂d0/d5-BZC-TZD, TPPP-TZD, TPPP-TZD-NHS, 4-CEBTPP, 2-CEBDPS；2种靶标羧基光能团手性质谱试剂TPPP-PRN, Apy-PPZ。并考察了质谱裂解规律，评价了手性拆分效能。. 氨基手性代谢物标记d0/d5-BZC-TZD试剂对R-/S-1-苯乙胺和R-/S-1-(1-萘基)乙胺的分离度为1.90-2.21，在MS/MS裂解中得到m/z 77.05和m/z 105.05的特征碎片离子。另外，OTPTHE可对11种D, L-氨基酸（Ala，Thr，Lys，Ile，Ser，His，Pro，Met，Trp，Val，Leu）在30分钟内达到完全分离。期中D, L-Ser在人血浆添加平均回收率为99.48%-104.06%。利用10名健康志愿者的血浆进行了D, L-Ser的定量分析，结果血浆中D, L-Ser的平均含量为2.65 ± 1.31 ug/mL，20.36 ± 4.51 ug/mL。其L-Ser平均含量是D-Ser的约10倍。 靶标羧基官能团手性代谢物标记OTPA试剂应用于4种非甾体抗炎药（NSAIDs）布洛芬（IBU）、萘普生（NAP）、酮洛芬（KET）、氯索洛芬（LOX）和两种手性羧基化合物2-苯基丁酸（PBA）、2-苯基丙酸（PPA）的分离度为1.54~2.23。健康志愿者血浆添加回收率范围为88.58%~104.66%。在市售两种片剂中平均添加回收率为93.71%~95.40%。含量准确度均大于96.49%。. 本研究首次合成了三苯基膦带正电荷结构为主的靶向识别氨基、羧基官能团手性代谢物的8种新型质谱手性衍生化试剂。因本试剂可用于人体血浆等生物样品中的手性氨基、羧基代谢物的分析。为开发手性代谢组学优良的手性衍生化试剂提供新的思路和理论依据，为推动手性代谢组学的发展提供新的分析方法。