pH-Independent triplex formation: hairpin DNA containing isoguanine or 9-deaza-9-propynylguanine in place of protonated cytosine

Abstract

Triplex-forming oligonucleotides (TFOs) containing 2'-deoxyisoguanosine (2), 7-bromo-7-deaza-2'-deoxyisoguanosine (2) as well as the propynylated 9-deazaguanine N7-(2'-deoxyribonucleoside) were prepared. For this the phosphoramidites 9a, b of the nucleoside 1 and, the phosphoramidites 19, 20 of compound 3b were synthesized. They were employed in solid-phase oligonucleotide synthesis to yield the protected 31-mer oligonucleotides. The deblocking of the allyl-protected oligonucleotides containing 1 was carried out by Pd(0)[PPh3]4-PPh3 followed by 25% aq. NH3. Formation of the 31-mer single-stranded intramolecular triplexes was studied by UV-melting curve analysis. In the single-stranded 31-mer oligonucleotides the protonated dC in the dCH(+)-dG-dC base triad was replaced by 2'-deoxyisoguanosine (1), 7-bromo-7-deaza-2'-deoxyisoguanosine (2) and, 9-deaza-9-propynylguanine N7-(2'-deoxyribonucleoside) (3b). The replacement of protonated dC by compounds 1 and 3b resulted in intramolecular triplexes which are formed pH-independently and are stable under neutral conditions. These triplexes contain "purine" nucleosides in the third pyrimidine rich strand of the oligonucleotide hairpin.

Synthesis of 2′-Deoxy-9-deaza Nucleosides Using Heck Methodology

“…1 H NMR (400 MHz, CDCl 3 ): δ 2.05 (dd, 1H, J = 5.5, J = 13.3 Hz), 2.77−2.84 (m, 1H), 3.74−3.79 (m, 1H), 3.95 (dd, 1H, J = 2.3 Hz, J = 12.38 Hz), 4.13 (br s, 1H), 4.36 (s, 2H), 4.69 (d, 1H, J = 5.04 Hz), 5.35 (dd, 1H, J = 5.26 Hz, 11.22 Hz), 5.43 (s, 2H), 5.52 (s, 2H), 5.59 (s, 2H), 5.84−5.87 (br d, 1H), 7.12−7.15 (m, 2H), 7.22−7.36 (m, 12H), 7.45−7.47 (m, 2H). 13 (14). From 11 (24 mg, 53.9 μmol), 14 was obtained as a yellow syrup (8 mg, 17.8 μmol, 33%).…”

“…In the course of our efforts to introduce base modifications to nucleosides − for use in studies of modified DNA, a series of 2′-deoxy-9-deaza nucleosides were envisioned wherein the nucleobase is connected to the 2′-deoxyribofuranosyl moiety via a carbon–carbon bond, rendering them C - nucleosides. − In addition to imparting resistance to hydrolytic and enzymatic cleavage resulting from the more stable carbon–carbon glycosidic bond, ,, this change also results in altering H-bond accepting N7 to NH, an H-bond donor, for 2′-deoxy-9-deaza nucleosides such as 1 and 2 (Figure ). This allows these compounds to engage in alternative hydrogen-bonding patterns such as Hoogsteen base pairing, which may find use in complex oligonucleotide structures or in the investigation of polymerases. − Similarly, Hamm et al have evaluated alternate hydrogen-bonding patterns for 2′-deoxy-9-deazaguanosine to study the promutagenic characteristics of 8-oxo-2′-deoxyguanosine …”

A concise synthesis of isoguanine 2ʹ-deoxyriboside and its adenine-like triplex formation when incorporated into DNA

“…Although not a DNA base, isoguanine (isoG) occurs naturally and various methods have been reported for the preparation of derivatives [1][2][3][4] including its riboside, 5 2'-deoxyriboside [6][7][8][9] and other analogues. 10,11 Isoguanine has been widely studied and when incorporated into nucleic acids, [12][13][14][15][16][17][18][19][20][21][22][23] has been shown to form stable base pair 7,[24][25][26][27][28] (Figure 1 left) and triplex motifs 29 (Figure 1 middle) as its N1-H tautomer. These experimental observations are supported by theoretical calculations.…”

“…33 Figure 1. Representative motifs in oligonucleotides where the N1-H tautomer of isoguanine (isoG) is targeted to isocytosine 7 (isoC), to a guanine.cytosine (G.C) base pair 29 and as the N3-H tautomer, targeted to guanine 32 (G).…”

“…At the nucleoside level, hydrogenolysis of the 2-benzyloxy group 34 would give a concise route to 2'-deoxyisoguanosine from non-nucleoside precursors. At the DNA level, removal of the 2-allyloxy group using Noyori conditions 35 would give isoguanine 29 (Figure 2 right) for the study of its triplex-forming properties and also allow comparison between A, isoG, B and L. As the starting point for synthesis of the nucleoside intermediate 5, 36 we selected the well-known and widely used 6,29,[37][38][39][40][41] Hoffer sugar 2 (2-deoxy-3,5-di-O-(p-toluoyl)-α-D-erythro-pentofuranosyl chloride). 42 Recently, we reported an improved synthesis of the Hoffer sugar that was achieved without need for distillation or chromatographic separation of intermediates, or the use of gaseous HCl.…”