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Tet enzymes oxidize 5-methyl-deoxycytidine (mdC) to 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC) and 5-carboxy-dC (cadC) in DNA. It was proposed that fdC and cadC deformylate and decarboxylate, respectively, to dC over the course of an active demethylation process. This would re-install canonical dC bases at previously methylated sites. However, whether such direct C-C bond cleavage reactions at fdC and cadC occur in vivo remains an unanswered question. Here we report the incorporation of synthetic isotope- and (R)-2'-fluorine-labeled dC and fdC derivatives into the genome of cultured mammalian cells. Following the fate of these probe molecules using UHPLC-MS/MS provided quantitative data about the formed reaction products. The data show that the labeled fdC probe is efficiently converted into the corresponding labeled dC, most likely after its incorporation into the genome. Therefore, we conclude that fdC undergoes C-C bond cleavage in stem cells, leading to the direct re-installation of unmodified dC.

A 5-formyl-2'-deoxycytidine (fdC) phosphoramidite building block that enables the synthesis of fdC-containing DNA with excellent purity and yield has been developed. In combination with phosphoramidites for 5-methyl-dC, 5-hydroxymethyl-dC, and carboxy-dC, it was possible to prepare a segment of the OCT-4 promoter that contains all four epigenetic bases. Because of the enormous interest in these new epigenetic bases, the ability to insert all four of them into DNA should be of great value for the scientific community., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ein 5 ‐ Formyl ‐ 2′ ‐ desoxycytidin(fdC) ‐ Phosphoramidit ‐ Baustein wurde entwickelt, der die Synthese fdC ‐ haltiger DNA in ausgezeichneter Reinheit und Ausbeute ermöglicht. In Kombination mit Phosphoramiditen für 5 ‐ Methyl ‐ dC, 5 ‐ Hydroxymethyl ‐ dC und 5 ‐ Carboxy ‐ dC gelang die Herstellung eines Segments des OCT4 ‐ Promotors, das alle vier epigenetischen Basen enthält. Aufgrund des großen Interesses an diesen neuen epigenetischen Basen sollte die Fähigkeit, alle vier von ihnen in DNA einzubringen, von besonderer Bedeutung sein. Biologisch relevante Promotorsegmente mit allen vier epigenetischen Nukleosiden sind nun durch Festphasensynthese zugänglich. Mit dem hier vorgestellten fdC ‐ Phosphoramidit werden keine Nebenreaktionen an 5 ‐ Formylcytosin beobachtet. Zusätzlich vermeiden milde Entschützungsbedingungen Oxidationen oder Reduktionen, was mit einer detaillierten massenspektrometrischen Analyse nachgewiesen werden konnte. [ABSTRACT FROM AUTHOR]

A 5-formyl-2′-deoxycytidine (fdC) phosphoramidite building block that enables the synthesis of fdC-containing DNA with excellent purity and yield has been developed. In combination with phosphoramidites for 5-methyl-dC, 5-hydroxymethyl-dC, and carboxy-dC, it was possible to prepare a segment of the OCT-4 promoter that contains all four epigenetic bases. Because of the enormous interest in these new epigenetic bases, the ability to insert all four of them into DNA should be of great value for the scientific community. [ABSTRACT FROM AUTHOR]