Alkylation of complementary ribonucleotides by 1,2‐dodecyl‐epoxide in a micellar environment: a liquid chromatography—electrospray ionization—sequential mass spectrometry investigation

Abstract

Alkylation of a pair of complementary ribonucleotides, adenosine monophosphate (AMP) and uridine monophosphate (UMP), was accomplished by 1,2-dodecyl-epoxide (DE) in a oil-in-water microemulsion based on the cationic surfactant Cetyl-trimethyl-ammonium-bromide, providing a suitable catalytic interface for the reagents. Several, often isomeric, alkylation products, bearing one or two hydroxy-dodecyl moieties on their structures, were identified in the reaction mixtures by high-performance liquid chromatography coupled to electrospray ionization ion trap mass spectrometry. In particular, mass spectrometry (MS)/MS spectra, implemented by extracted ion chromatograms obtained for peculiar MS/MS product ions, indicated alkylation to occur on uracil and on uracil/phosphate OH groups in singly and doubly alkylated UMP, respectively. Adenine NH2 group and phosphate or ribose OH groups were found to be involved as such (single alkylation) or in combination, in the case of alkylated derivatives of AMP. The reaction of both endocyclic N and C=O groups (tautomerized to C-OH groups) of uracil and the predominance of nucleophilic attack to the more accessible carbon of the DE epoxydic bridge (the only exception being the reaction by the NH2 group of adenine) were inferred from MS3 spectra with the help of extracted ion chromatograms for specific fragment ions, after their structural characterization. Interestingly, alkylation on one of the uracil C=O groups and, partially, on the adenine NH2 group, both potentially involved in AMP/UMP base pairing in the micellar environment, were found to be hindered when both ribonucleotides were present in the reaction mixtures.

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

“…. It is worth noting that similar fragmentations have been already observed also for UMP and AMP alkylated on the phosphate group under the same reaction conditions . On the other hand, a single product ion was detected in the MS/MS spectrum averaged in the 26–31 min interval.…”

“…It is worth noting that the C = O groups of uracil and cytosine can act as nucleophilic sites once a keto‐enolic tautomerization has occurred, leading to the generation of OH groups. Evidence for their involvement in the reaction with DE were obtained from liquid chromatography – electrospray – mass spectrometry LC‐ESI‐MS n measurements ( n = 1–3) in the case of UMP . On the other hand, alkylation at the level of less nucleophilic sites, such as the imidic and primary aminic nitrogens of UMP and AMP, respectively, and the OH groups of phosphate, already reported to be reactive towards alkylation, was suggested by the same MS investigation.…”

“…A systematic investigation on the alkylation of ribonucleotides with 1,2‐dodecyl epoxide (DE) has been then recently undertaken by our research group . The reactivity of DE towards standard ribonucleotides was assumed as a model process, mimicking those potentially occurring on DNA or RNA exposed to long‐chained alkylating agents.…”

“…The covalent modification induced by DE on adenosine‐ and uridine‐mono phosphate (AMP and uridine monophosphate (UMP), respectively) has been described by the following general scheme:

where: P represents the phosphate group; Base indicates an adenine or uracil moiety; Hyd is either a 2‐hydroxy‐dodec‐1‐yl or a 1‐hydroxy‐dodec‐2‐yl residue, depending on the site (C β or C α epoxide carbons, respectively) of nucleophilic attack on DE, and the n subscript indicates the number (up to three) of hydroxy‐dodecyl residues introduced. This result seemed to be supported by literature indications on the presence of several potential nucleophilic sites on the nucleic bases of (ribo)nucleotides .…”

“…Moreover, even the OH groups of ribose were found to react, although more slowly, with DE. As a result, a quite complex mixture of mono‐, bi‐ and, after long reaction times, tri‐alkylated ribonucleotides was obtained . The strong potential for structural elucidation provided by tandem (MS 2 ) and sequential (namely MS 3 ) mass spectrometry enabled also the recognition of peculiar effects on reactivity due to the contemporary presence of UMP and AMP in the reaction environment, indicating the existence of an interaction between the complementary bases …”