Cytosine to uracil conversion through hydrolytic deamination of cytidine monophosphate hydroxy‐alkylated on the amino group: a liquid chromatography – electrospray ionization – mass spectrometry investigation

Abstract

A novel pathway for cytosine to uracil conversion performed in a micellar environment, leading to the generation of uridine monophosphate (UMP), was evidenced during the alkylation reaction of cytidine monophosphate (CMP) by dodecyl epoxide. Liquid chromatography-electrospray ionization - ion trap - mass spectrometry was used to separate and identify the reaction products and to follow their formation over time. The detection of hydroxy-amino-dodecane, concurrently with free UMP, in the reaction mixture suggested that, among the various alkyl-derivatives formed, CMP alkylated on the amino group of cytosine could undergo tautomerization to an imine and hydrolytic deamination, generating UMP. Interestingly, no evidence for this peculiar conversion pathway was obtained when guanosine monophosphate (GMP), the complementary ribonucleotide of CMP, was also present in the reaction mixture, due to the fact that NH(2)-alkylated CMP was not formed in this case. The last finding emphasized the role played by CMP-GMP molecular interactions, mediated by a micellar environment, in hindering the alkylation reaction at the level of the cytosine amino group.

Alkylation of complementary ribonucleotides in nanoreactors

“…As far as CMP is concerned, most of the detected products (mono-and bi-alkylated species) have been described in detail in a previous study dedicated to the conversion of CMP derivatives into UMP ones. 15 Their structures and those relevant to UMP derivatives, formed through CMP deamination processes (see Paths A and B in Scheme 1 of ref. 15) and subsequent alkylations by DE, have been summarized in Tables 1 and 2.…”

“…In a very recent paper the attention has been focused on the alkylation of CMP. 15 However, the alkylation reaction pathway was significantly complicated by the presence of deamination processes, leading to the generation of UMP alkylated derivatives. Actually, DNA bases can undergo ''spontaneous'' hydrolytic deamination, 16 with pyrimidine bases being more susceptible than the purine ones.…”

“…In our systems, a confirmation was obtained for the occurrence of hydrolytic deamination of N 3 -alkylated CMP to the corresponding N 3 -alkylated UMP analogue which was further bi-alkylated on a phosphate OH group (see Scheme 1 in ref. 15). On the other hand, evidence for the presence of a different deamination pathway, involving NH 2alkylated CMP and finally leading to free UMP was obtained for the first time.…”

“…On the other hand, evidence for the presence of a different deamination pathway, involving NH 2alkylated CMP and finally leading to free UMP was obtained for the first time. 15 Such a peculiar process was rationalized in terms of enamine-imine tautomerization followed by imine hydrolysis, with release of 1-amino-2-hydroxy-dodecane or 1-hydroxy-2-amino-dodecane (AHD or HAD), arising from nucleophilic attack of the cytosine NH 2 group at the DE oxirane ring (see Path B of Scheme 1 in ref. 15).…”

“…15 Such a peculiar process was rationalized in terms of enamine-imine tautomerization followed by imine hydrolysis, with release of 1-amino-2-hydroxy-dodecane or 1-hydroxy-2-amino-dodecane (AHD or HAD), arising from nucleophilic attack of the cytosine NH 2 group at the DE oxirane ring (see Path B of Scheme 1 in ref. 15). Hydroxy-aminododecanes were actually detected in the reaction mixture by a LC-ESI-MS analysis in positive polarity.…”