Formamidopyrimidine lesions from initial oxidation and N7-methylation of dGuo

HPLC analyses of the 5′-TT-(MeFapy-dGuo)-TTC-3′ oligonucleotide. A. Analysis of the oligonucleotide synthesis with a “short” deprotection cycle. B. Analysis of the oligonucleotide synthesis with a “long” deprotection cycle. C. Analysis of peak 1, identified as the pyranose form of the MeFapy-dGuo lesion, after initial purification. D. Analysis of interconverting peaks 2-4, identified as the furanose form of the MeFapy-dGuo lesion, after each peak was individually purified.

HPLC analysis of the enzymatic digestion of oligonucleotide 8a with phosphodiesterase I and alkaline phosphatase.

Sequencing of oligonucleotide 8a by controlled enzymatic digestion with phosphodiesterase I (top) and II (bottom) followed by MALDI-TOF-MS.

NMR analysis of the furanose (I) and pyranose (II) forms of the MeFapy-dGuo lesion in 5′-A-(MeFapy-dGuo)-C-3′ sequence. Tile plots of ³¹P-¹H COSY (top), ¹H-¹H TOCSY (middle), and ¹H-¹³C HSQC (bottom) spectra. The connectivity was traced from H3′ of dAdo to the phosphate (a) to the H5” of the MeFapy-dGuo (b) then to C5′ of the MeFapy-dGuo for the furanose form (I), and from the phosphate to the H4′ of the MeFapy-dGuo (b) then to C4′ of the MeFapy-dGuo for the pyranose form (II). The multiplicity edited HSQC confirmed that the furanose C5′ is a methylene carbon and a methine carbon for the pyranose form (negative phase peaks = red, positive phase peaks = black).

Comparison of the ¹³C chemical shifts for furanose and pyranose forms of the 2-deoxyribose units.

HPLC analysis of the deglycosylation of MeFapy-Gua base in oligonucleotide 5′-CCTCTTC-(MeFapy-dGuo)-CTCTC-3′ at pH 6.5. A. Starting 13mer oligonucleotide containing the MeFapy-dGuo nucleotide; B) partial deglycosylation after 24 h at pH 6.5; C) after 48 h; D) after addition of endonuclease V.

Reduction products of the putative C1′-N⁶ imine of MeFapy-dGuo.

Thermal melting analysis of duplex of 8a. The T_m for the unmodified duplex was 59° C.

Anomerization of Fapy derivatives is slow at neutral pH

The MeFapy-dGuo phosphoramidite (4) was prepared in four steps and 24% overall yield from dGuo (Scheme 1). Conversion of dGuo to 1 was accomplished as previously described (12). Protected dGuo derivative 1 was treated with an excess of methyl iodide in DMSO for 45 min at room temperature to produce the cationic N7-methyl-dGuo 2, which was not isolated. After removal of the excess methyl iodide, 2 was briefly exposed to 1 M NaOH to affect ring-opening of the imidazolium ion, then neutralized with 0.1 M HCl to afford 3 as a white precipitate. The protected MeFapy-dGuo nucleoside 3 was isolated by filtration and converted to the desired MeFapy-dGuo phosphoramidite 4 without further purification.