Oligonucleotides containing fluorescent 2′-deoxyisoinosine: solid-phase synthesis and duplex stability

Abstract

The fluorescent nucleoside 2'-deoxyisoinosine (2, isoId) has been incorporated into oligonucleotides. For this purpose the phosphonate 3a and the phosphoramidite 3b, as well as the polymer-linked 3d, have been synthesized and oligonucleotides were prepared by P(III) solid-phase chemistry. One or two isoId-residues were introduced into the oligomer d(T12), replacing dT either in the middle or at the 3'- and 5'-ends. The isoId-containing oligomers were hybridized with a modified d(A)12 containing the conventional nucleosides (dA, dT, dG and dC) opposite to isoId. The replacement of one dT by isoId in the centre of the duplex reduced the Tm value by approximately 15 degrees C and a decrease of approximately 25 degrees C was found when two isoId residues were incorporated. Thermodynamic data were determined from the melting curves. The destabilization was almost independent of the four naturally occurring nucleosides located opposite to isoId. The isoId (2) seems to be stacked in the duplex when dT-dA base pairs are the nearest neighbours; an internal loop is formed in the case of oligomers containing two consecutive isold residues.

Synthesis of Acyclic Nitroazole Nucleosides and Their Incorporation into Oligonucleotides, and Their Duplex and Triplex Formation

“…At present, a broad range of compounds that fullfill these criteria is known. The naturally occurring hypoxanthine as its ribo-or 2'-deoxyribonucleoside form such base pairs with the other natural DNA/RNA bases [4]. Similar behavior exhibit 2'-deoxyinosine analogues: 7-deaza-2'-deoxyinosine [5] and 2-aza-2'-deoxyinosine [6].…”

Synthesis and fluorescence study of 7-azaindole in DNA oligonucleotides replacing a purine base

“…The reaction formula for the synthesis of phosphoramidite 4 from the literature also had to be modified. The optimized mole ratio was 1:4:10 for compound 3: chlorophosphoramidite: diisopropylethylamine under our conditions, compared to 1:2:4 by Seela and Chen [10] for isoinosine or 1:6:4 by Cosstick et al [13] for 3-deazaadenosine. The large amount (10-equivalent) of diisopropylethylamine proved to be necessary in our case.…”

“…Compound 2 crystallized readily during the reaction and was easily isolated and purified. Tritylation of 2 with 1.5-equivalent of 4,4′-dimethoxytrityl chloride partially protected both the 5′-and 3′-hydroxyl groups of the sugar as revealed by 1 H-NMR spectrum of the isolated main product (not shown), although such procedure performed on other modified bases protected only the 5′-hydroxyl group [10,15,16,20,22]. Therefore, only 1.1-equivalent of 4,4′-dimethoxytrityl chloride was used for the reaction and a 75% yield for 3 was achieved after chromatography purification.…”

“…There are mainly two groups of 'bases' synthesized and incorporated into DNA oligomers: those made by minor modification of the natural bases or the derivatives of cyclic aromatic compounds. While the former includes 2-aminopurine [1][2][3][4][5][6][7][8], inosine [9] and isoinosine [9][10][11], 3-and 7-deazaadenine [12][13][14][15], and 3-and 7-deazaguanine [14,16], the latter includes analogs of pteridine [17][18][19] and indole [20][21][22][23][24], benzene [23][24][25][26], naphthalene and pyrene derivatives [24], The first group of compounds, when replacing a natural base in duplex DNA, can still form hydrogen bonds with the base in the opposite strand, while the second group of compounds cannot form any hydrogen bonds.…”

“…The fluorescence properties of the above base analogs have been utilized for studying DNA structure and duplex stability [1,2,10,14,15,22,26], protein-DNA interactions [4][5][6][7][8]13,18,25], or simply as universal base analogs [20,27]. Some of the unusual 'bases' are used as anti-HIV or anti-cancer drugs [28][29][30].…”