Chemical synthesis, purification, and characterization of 3'-5'-linked canonical cyclic dinucleotides (CDNs).

Changhao Wang, Min Hao, Qianqian Qi, Yashao Chen, Jörg S Hartig

Methods in enzymology 2019

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So far, four cyclic dinucleotides (CDNs) have been discovered as important second messengers in nature, where three canonical CDNs of c-di-GMP, c-di-AMP and c-AMP-GMP were found in bacteria containing two 3'-5' phosphodiester linkages and one non-canonical CDN 2'3'-c-GMP-AMP was identified in mammals containing mixed 2'-5' and 3'-5' phosphodiester linkages. The CDNs are produced by specific cyclases and degraded by phosphodiesterases (PDEs). All of the known CDNs could bind to the stimulator of interferon genes (STING) to induce type I interferon (IFN) responses and the three bacterial CDNs are sensed by specific riboswitches to regulate gene expression. The emerging physiological functions of bacterial CDNs lead the motivation to investigate other possible canonical CDNs. In recent years, many endeavors have been devoted to develop fast, convenient and cheap strategies for chemically synthesizing CDNs and their analogues. The phosphoramidite approach using commercial starting materials has attracted much attention. Herein, we describe an adapted one-pot strategy that enables fast synthesis of crude 3'-5'-linked canonical CDNs followed by purification of the obtained CDNs using reversed phase high-performance of liquid chromatography (HPLC). Furthermore, we report the full characterization of CDNs by mass spectrometry (MS) and nuclear magnetic resonance (NMR) techniques. © 2019 Elsevier Inc. All rights reserved.

Citation

Changhao Wang, Min Hao, Qianqian Qi, Yashao Chen, Jörg S Hartig. Chemical synthesis, purification, and characterization of 3'-5'-linked canonical cyclic dinucleotides (CDNs). Methods in enzymology. 2019;625:41-59