5-Hydroxymethylcytosine(5-HMC) is a newly confirmed stable nucleic base in certain cells, and is believed to be a biomarker for certain biological process, such as methylcytosine(MC) demethylation. A sensitive and reliable detection method for 5-HMC is important to study its biological function. But the most widely used bisulfite method cannot differentiate 5-HMC and 5-MC. It is reported that glucosyltransferases can selectively transfer glucose from UDP-Glc to 5-HMC. We synthesized a series of modified UDP-Glc derivatives, which have different functional groups at either 2' or 6' replacing 6'-OH for bioconjugation reactions. These UDP-Glc derivatives were tested with wild-type a-glucosyltransferase(AGT), wild-type �-glucosyltransferase(BGT) and BGT mutant Y261L. Experiments showed that UDP-6'-Azido-Glc can be transferred to 5-HMC by wild-type BGT only with similar activity as natural UDP-Glc. Surprisingly, UDP-2'-Azido-Glc acts as a competitive inhibitor to wild-type BGT and BGT mutant Y261L. A fluorophore or biotin can be coupled to Azido-Glc modified 5-HMC via copper (I) catalyzed or copper-free click chemistry efficiently.

Synthesis of UDP-Glc derivatives for 5-hydroxymethylcytosine detection

243rd National Meeting (2012)

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