An Improved Approach for Practical Synthesis of 5-Hydroxymethyl-2′-deoxycytidine (5hmdC) Phosphoramidite and Triphosphate

Abstract

5-Hydroxymethyl-2′-deoxycytidine (5hmdC) phosphoramidite and triphosphate are important building blocks in 5hmdC-containing DNA synthesis for epigenetic studies. However, efficient and practical methods for the synthesis of these compounds are still limited. The current research provides an intensively improved synthetic method that enables the preparation of commercially available cyanoethyl-protected 5hmdC phosphoramidite with an overall yield of 39% on 5 g scale. On the basis of facile and efficient accesses to cyanoethyl protected-5hmdU and 5hmdC intermediates, two efficient synthetic routes for 5hmdC triphosphate were also developed.

Preparation of 5‐Hydroxymethyl‐pyrimidine Based Nucleosides: A Reinvestigation

“…[39] For example, the cyanoethylprotected 5hmdU 2 was obtained in an excellent 90 % yield compared to previously procedures reporting only 50 % yields. [27,39,40] This two-step procedure therefore provides an easy access to a precursor of phosphoramidite building block for 5hmdU or 5hmdC-containing oligonucleotide synthesis.…”

“…In a recent paper, Pu and Sun described a procedure in which they converted the 5BrmdU intermediate to cyanoethyl ether via simultaneous alcoholysis with cyanoethanol and cleavage of TBDMS by HBr generated in situ at ambient temperature affording 2 in 78 % yield. [27] Finally, we turned to another way to obtain the 5hydroxymethyl moiety onto the pyrimidine nucleobase. In Nature, the reaction is catalyzed by the 2-oxoglutarate-and Fe(II)-dependent dioxygenases TET or JBP to introduce the 5hydroxymethyl moiety onto the carbon of the pseudo-benzylic position of thymine or methylcytosine within DNA.…”

“…Van Boom improved his precedent base J phosphoramidite synthesis by considering thymidine as a starting product through radical bromination of a bis-silylated thymi-dine derivative and substitution of the resulting crude allylic bromide with cesium acetate, thus affording fully protected 5hmdU (Figure 1B). [25] Radical bromination was also used to prepare the 5hmdC triphosphate derivative, [26,27] the cyanoethylprotected 5hmdC phosphoramidite, [28,27] the Glc-5hmdU and Glc-5hmdC triphosphate derivatives [29] as well as photolabile 2nitrobenzyl alkylated 5hmdU triphosphates [30,31,32] and photolabile 2-nitrobenzyl alkylated 5hmdC/U phosphoramidites (Figure 1B). [33] This pathway has several drawbacks such as the modest reported yields, the need to engage the bromination product in the following reactions without purification to avoid its degradation, and the use of toxic carcinogenic solvents.…”

Chemical Synthesis of Modified RNA

“…[52] The solid-phase synthesis of 5hmrC RNA (and 5hmdC DNA) is particularly challenging because of the benzylic nature of 5hmrC and the protecting groups can be substituted by nucleophiles such as methylamine, ammonia, or fluoride if inadequate deprotection conditions are chosen. Several protection concepts have been published; while N 4benzoyl and 5-CH 2 OCH 2 CH 2 CN are too stable and should be avoided, [53] N 4 -Ac in combination with 5-CH 2 OAc or 5-CH 2 OTBDMS minimizes the above mentioned side reactions during deprotection. [54] Several strategies have been described to introduce the hydroxymethyl group for the building block synthesis, the most convenient starting with bromination of 5-methyluridine (Figure 10).…”