Evaluation of UDP‐GlcN Derivatives for Selective Labeling of 5‐(Hydroxymethyl)cytosine

5‐(hydroxymethyl)cytosine (5‐hmC) is a newly identified oxidative product of 5‐methylcytosine (5‐mC) in the mammalian genome, and is believed to be an important epigenetic marker influencing a variety of biological processes. In addition to its relatively low abundance, the fluctuation of 5‐hmC levels over time during cell development poses a formidable challenge for its accurate mapping and quantification. Here we describe a specific chemoenzymatic approach to 5‐hmC detection in DNA samples by using new uridine 5′‐diphosphoglucosamine (UDP‐GlcN) probes. Our approach requires modification of the glucose moiety of UDP‐Glc with small amino groups and transfer of these glucose derivatives to the hydroxy moiety of 5‐hmC by using T4 phage glucosyltransferases. We evaluated the transfer efficiencies of three glucosyltransferases (wild‐type α‐ and β‐GTs and a Y261L mutant β‐GT) with five different UDP‐Glc derivatives containing functionalized groups for subsequent bioconjugation and detection. Our results indicate that UDP‐6‐N 3 ‐Glc, UDP‐6‐GlcN, and UDP‐2‐GlcN can be transferred by β‐GT with efficiencies similar to that seen with the native UDP‐Glc cofactor. 6‐N 3 ‐Glc‐ and 6‐GlcN‐containing oligonucleotides were selectively labeled with reactive fluorescent probes. In addition, a 2 kb DNA fragment modified with 2‐GlcN groups was specifically detected by use of a commercially available antiglucosamine antibody. Alternative substrates for β‐GT and correlated glycosyltransferases might prove useful for the study of the function and dynamics of 5‐hmC and other modified nucleotides, as well as for multiplex analysis.