Base-pairing, tautomerism, and mismatch discrimination of 7-halogenated 7-deaza-2'-deoxyisoguanosine: oligonucleotide duplexes with parallel and antiparallel chain orientation

Oligonucleotides containing 2'-deoxyisoguanosine (1, iG(d)), 7-deaza-2'-deoxyisoguanosine (2, c(7)iG(d)), and its 7-halogenated derivatives 3 and 4 were synthesized on solid phase using the phosphoramidite building blocks 5-7. The hybridization properties of oligonucleotides were studied on duplexes with parallel and antiparallel chain orientation. It was found that the 7-halogenated nucleoside analogues 3 and 4 enhance the duplex stability significantly in both parallel (ps) and antiparallel (aps) DNA. Moreover, the halogenated nucleosides shift the tautomeric keto-enol equilibrium strongly toward the keto form, with K(TAUT) [keto]/[enol] approximately 10(4) coming close to that of 2'-deoxyguanosine (10(4)-10(5)), while the nonhalogenated 7-deaza-2'-deoxyisoguanosine 2 shows a K(TAUT) of around 2000 and the enol concentration of 1 is 10% in aqueous solution. Consequently, nucleosides 3 and 4 show a much better mismatch discrimination against dT than compound 1 or 2 in antiparallel as well as in parallel DNA. 3 and 4 are expected to increase the selectivity of base incorporation opposite to isoC(d) in the form of triphosphates or in the polymerase-catalyzed reaction in comparison to 1 or 2.     

Oligonucleotide duplexes containing N8-glycosylated 8-aza-7-deazaguanine and self-assembly of 8-aza-7-deazapurines on the nucleoside and the oligomeric level

The 8-aza-7-deazaguanine N8-(2'-deoxy-beta-D-ribofuranoside) (1) was synthesized, converted into the phosphoramidite 4 and incorporated into oligonucleotides. Nucleoside 1 forms stable base pairs with 2'-deoxy-5-methylisocytidine in DNA with antiparallel chain orientation (aps) and with 2'-deoxycytidine in duplexes with parallel chains (ps). According to the CD spectra self-complementary oligonucleotides d(1-m5isoC)3 and d(1-C), form autonomous DNA-structures. Neither the nucleoside 1 nor the regularly linked 8-aza-7-deaza-2'-deoxyguanosine form G-like tetrads while the regularly linked 8-aza-7-deaza-2'-deoxyisoguanosine gives higher molecular assemblies which are destroyed by bulky 7-bromo substituents. This was verified on monomeric nucleosides by ESI-MS spectrometry and on oligonucleotides by HPLC analysis.     

7-Halogenated 7-Deaza-2′-deoxyinosines

The synthesis of the 7-deaza-2′-deoxyinosine derivatives 3a – c with chloro, bromo, and iodo substituents at position 7 is described. Glycosylation of the 7-halogenated 6-chloro-7-deazapurines 4a – c or of the 7-halogenated 6-chloro-7-deaza-2-(methylthio)purines 9a – c with 2-deoxy-3,5-di-O-(4-toluoyl)-α-D -erythro-pentofuranosyl chloride ( 5 ) furnished the intermediates 7a – c and 11a – c , respectively, which gave, upon deprotection, the desired nucleosides 3a – c .     

Pyrazolo[3,4-d][1,2,3]triazine DNA: synthesis and base pairing of 7-deaza-2,8-diaza-2'-deoxyadenosine

7-deaza-2,8-diaza-2'-deoxyadenosine (4) was synthesized from 8-aza-7-deaza-2'-deoxyadenosine (1) via the 1,N(6)-etheno derivative 5. Ring opening with sodium hydroxide followed by ring closure in the presence of sodium nitrite formed the tricyclic intermediate 5 from which the transiently introduced "etheno" moiety was removed with NBS. Compound 4 was converted to the phosphoramidite 11, which was employed in solid-phase oligonucleotide synthesis. Base pairing studies on 4, incorporated in a 12-mer duplex, showed that this adenine nucleoside analogue forms a strong base pair with dG but not with dT. This novel base pair is as stable as that of the canonical dA-dT pair. As a result of the absence of nitrogen-7 compound 4 is expected to form a face to face base pair with dG.     

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

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.     

Studies on the Base-Pairing Properties of N7-(2-Deoxy-β-D-erythro-pentofuranosyl)guanine (N7Gd)

The base-pairing properties of N⁷-(2-deoxy-β-D -erythro-pentofuranosyl)guanine (N⁷G_d; 1 ) are investigated. The nucleoside 1 was obtained by nucleobase-anion glycosylation. The glycosylation reaction of various 6-alkoxy-purin-2-amines 3a - i with 2-deoxy-3,5-di-O-(4-toluoyl)-α-D -erythro-pentofuranosyl chloride ( 8 ) was studied. The N⁹/N⁷-glycosylation ratio was found to be 1:1 when 6-isopropoxypurin-2-amine ( 3d ) was used, whereas 6-(2-methoxyethoxy)purin-2-arnine ( 3i ) gave mainly the N⁹-nucleoside (2:1). Oligonucleotides containing compound 1 were prepared by solid-phase synthesis and hybridized with complementary strands having the four conventional nucleosides located opposite to N⁷G_d. According to T_m values and enthalpy data of duplex formation, a base pair between N⁷G_d and dG is suggested. From the possible N⁷G_d dG base pair motives, Hoogsteen pairing can be excluded as 7-deaza-2′-deoxyguanosine forms the same stable base pair with N⁷G_d as dG.     

Synthesis and photophysical properties of 7-deaza-2'-deoxyadenosines bearing bipyridine ligands and their Ru(II)-complexes in position 7

The synthesis of the title 7-deazaadenine 2'-deoxyribonucleosides bearing bipyridine, phenanthroline or terpyridine ligands linked to position 7 via an acetylene or phenylene spacer is reported based on aqueous cross-coupling reactions of unprotected 7-iodo-7-deaza-2'-deoxyadenosine with ligand-functionalized acetylenes or boronic acids. The aqueous cross-coupling with acetylene or boronate building blocks containing the Ru(bpy)(3)-type of complex gave the corresponding Ru-containing nucleosides. Photophysical and electrochemical properties were studied and the most efficient type of complex was selected for future luminescent and redox labelling of DNA. The title nucleosides also showed some cytostatic and anti-HCV activities.     

Regioselective syntheses of 7-nitro-7-deazapurine nucleosides and nucleotides for efficient PCR incorporation

Substitution at the C(7) position of purine nucleotides by a potent electron-withdrawing nitro group facilitates the cleavage of glycosidic bonds under alkaline conditions. This property is useful for sequence-specific cleavage of DNA containing these analogues. Here we describe the preparation of 7-deaza-7-NO(2)-dA and 7-deaza-7-NO(2)-dG using two different approaches, starting from 2'-deoxy-adenosine and 6-chloro-7-deaza-guanine, respectively. These modified nucleosides were converted to nucleotide triphosphates, each of which can replace the corresponding, naturally occurring triphosphate to support PCR amplification. [structure: see text]     

The Base-Pairing Properties of 7-Deaza-2′-deoxyisoguanosine and 2′-Deoxyisoguanosine in Oligonucleotide Duplexes with Parallel and Antiparallel Chain Orientation

Oligonucleotides with parallel (ps) or antiparallel (aps) chain orientation containing 7-deaza-2′-deoxyisoguanosine ( 1 ) or 2′-deoxyisoguanosine ( 2 ) were prepared. The phosphoramidite and phosphonate building blocks 3 – 6 were synthesized and used in solid-phase synthesis. The diphenylcarbamoyl (dpc) residue was used for the 2-oxo group protection and the isobutyryl (iBu=ib) residue for the amino function. Hybridization experiments were performed with oligonucleotides containing 7-deazaisoguanine or isoguanine. Regarding 7-deazapurine-containing oligonucleotides, the 7-deazaisoguanine⋅cytosine base pair was the strongest in ps-duplexes, while that of 7-deazaisoguanine⋅5-methylisocytosine was the most stable one in aps-DNA. Ambiguous base pairing of 7-deazaisoguanine with cytosine, 5-methylisocytosine, thymine, and guanine was observed in the case of aps-duplexes, whereas in ps-duplexes, the ambiguity was extended to adenine. The 7-deazaisoguanine-containing duplexes showed almost identical base-pair stabilities as those containing isoguanine. According to this, various base-pair motifs are proposed. The 7-deaza-2′-deoxyisoguanosine was found to be an effective substitute of 2′-deoxyisoguanosine.     

1,N-Etheno-2′-deoxytubercidin and pyrrolo-C: synthesis, base pairing, and fluorescence properties of 7-deazapurine nucleosides and oligonucleotides

The synthesis of 1,N⁶-etheno-7-deaza-2′-deoxyadenosine (12b) which was prepared from 7-deaza-2′-deoxyadenosine (5a) with chloroacetaldehyde is described. Also the regioselective glycosylation of the 7-deazapurine-2-one at nitrogen-1 (19) furnishing the pyrrolo-C nucleoside 7a is reported and a side chain derivative with a terminal triple bond (7d) is prepared. The fluorescence properties of these nucleosides and related compounds were determined. The etheno nucleoside 12b is strongly fluorescent showing a Stokes shift of 134 nm and a quantum yield of Φ=0.53. It proved to be stable, both in acidic and in alkaline medium while the parent purine compound 10b is labile under both conditions. Compound 12b was converted into its phosphoramidite 14 and was incorporated into oligonucleotides. Compound 12b destabilizes oligonucleotide duplexes when it is located in the center of the molecule; it stabilizes when it is incorporated in the terminal base pair or acts as an overhanging nucleoside. Temperature-dependent fluorescent measurements yielded sigmoidal melting profiles when compound 12b is stacked to the terminal base pair while a linear decrease of the fluorescence is observed when the molecule is located opposite to the four canonical nucleosides in the center of the duplex.     

Duplex Stabilization of DNA: Oligonucleotides containing 7-substituted 7-deazaadenines

The oligonucleotide building blocks 4b–d derived from 7-bromo-, 7-chloro-, and 7-methyl-substituted 7-deaza-2′-deoxyadenosines 3b–d were prepared. They were employed in the solid-phase synthesis of the oligonucleotides 7–25 . The dA residues of the homomer d(A₁₂), the alternating d[(A-T)₆], and the palindromic d(G-T-A-G-A-A-T-T-C-T-A-C) were replaced by 3b–d as well as by the parent 7-deaza-2′-deoxyadenosine ( 3a ). The melting profiles and CD spectra of oligonucleotide duplexes, showing this major groove modification, were measured, and the T_m values as well as the thermodynamic data were determined. It was found that small substituents such as Br, Cl, or Me introduced in the 7-position of a 7-deazaadenine residue increase the duplex stability compared to oligonucleotides containing adenine.     

Halogenated 7-deazapurine nucleosides: stereoselective synthesis and conformation of 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides

The stereoselective syntheses of 5-halogenated 7-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine nucleosides 3b-d, 4a-c as well as 7-deaza-2'-deoxyisoguanosine are described. Nucleobase anion glycosylation of 2-amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (5) with 3,5-di-O-benzoyl-2-deoxy-2-fluoro-alpha-D-arabinofuranosyl bromide (6) exclusively gave the beta-D-anomer, which was deblocked (--> 8), aminated at C4 (--> 3a) and selectively deaminated at C2 to yield 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl 7-deazaisoguanine (2). Condensation of the 5-halogenated 4-chloro-2-pivaloylamino-7H-pyrrolo[2,3-d]pyrimidines 9a-c with 6 furnished the N7-nucleosides 10a-c together with N2,N7-bisglycosylated compounds 11a-c. The former was converted to the corresponding 2,4-diamino-compounds 3b-d, and the latter was deblocked by NaOMe/MeOH to yield the 4-methoxy-nucleosides 4a-c. Conformational analysis of the sugar moiety of the nucleosides 2 and 3a-d was performed on the basis of vicinal [1H,1H] coupling constants. The fluorine atom in the sugar moiety shifts the sugar conformation from S towards N by about 10%, while the halogen substituents in the base moiety increase the hydrophobicity and polarizability of the nucleobases.     

2-aza-2'-deoxyadenosine: synthesis, base-pairing selectivity, and stacking properties of oligonucleotides

2-Aza-2'-deoxyadenosine (2, z2Ad) is synthesized via its 1,N6-etheno derivative 7 and enzymatically deaminated to 2-aza-2'-deoxyinosine (3). Compound 2 is converted into the phosphoramidite building block 10b. This is employed in solid-phase oligonucleotide synthesis. The 2-azapurine base forms a strong base pair with guanine, but a much weaker one with adenine, thymine, and cytosine. Oligonucleotide duplexes with dangling nucleotide residues, such as 2-aza-2'-deoxyadenosine and 7-deaza-2'-deoxyadenosine (4, c7Ad), either on one or both termini, are synthesized, and the thermal stability of the duplexes is correlated with the hydrophobic properties of the dangling nucleotide residues.     

7-Substituted 7-Deaza-2′-deoxyguanosines: Regioselective Halogenation of Pyrrolo[2,3-d]pyrimidine Nucleosides

The synthesis of 7-chloro-, 7-bromo-, and 7-iodo-substituted 7-deaza-2′-deoxyguanosine derivatives 2b – d is described. The regioselective 7-halogenation with N-halogenosuccinimides was accomplished using 7-[2-deoxy-3,5-O-di(2-methylpropanoyl)-β-D -erythro- pentofuranosyl]-2-(formylamino)-4-methoxy-7H-pyrrolo[2,3-d]- pyrimidine ( 4 ) as the common precursor. A one-pot reaction (2N aq. NaOH) of the halogenated intermediates 5a – c furnished the desired compounds. Also the 7-hexynyl derivative 2e of 7-deaza-2′-deoxyguanosine is described.     

7-Deaza-2,8-diazaadenine containing oligonucleotides: synthesis, ring opening and base pairing of 7-halogenated nucleosides

Oligonucleotides containing 7-bromo-7-deaza-2,8-diaza-2′-deoxyadenosine (3) and 5-amino-3-bromo-4-carbamoyl-1-(2′-deoxy-β-d-erythro-pentofuranosyl)pyrazole (4) were synthesized. Compound 3 was prepared from 7-bromo-8-aza-7-deaza-2′-deoxyadenosine (5) via the 1,N⁶-etheno derivative 6 and was converted into the phosphoramidite 11. The 7-bromo substituent of 3 increases oligonucleotide duplex stability compared to the non-halogenated nucleoside. Oligonucleotides incorporating 3 are transformed to those containing 4 during long time deprotection at elevated temperature (25% aq ammonia, 60 °C, 30 h). Compound 3 forms a strong base pair with dG. The base pair stability decreases in the order dG>dT>dA>dC. Similar recognition selectivity is observed for the pyrazole nucleoside 4, however, due to decreased stacking and higher flexibility of the pyrazole moiety, duplexes are less stable than those containing 3.     

Methylated DNA: The influence of 7-deaza-7-methylguanine on the structure and stability of oligonucleotides

The 7-deaza-2′-deoxy-7-methylguanosine ( 2b ) [9], which is the glycosylic-bond-stable, noncharged analogue of 2′-deoxy-7-methylguanosine ( 1b ), was incorporated in DNA by solid-phase synthesis. As building blocks, the protected phosphonatc 3a and the phosphoramidite 3b were prepared. The 7-methyl group of 2b stabilizes the B-DNA duplex compared to 7-deaza-2′-deoxyguanosine but does not induce a B-Z transition as it is known from compound 1b . The stabilization by the 7-deaza-7-methylguanine moiety is sequence-dependent, and the nearest-neighbor influence is different from that of 7-deazaguanine. Homooligonucleotides of 2b show sigmoidal melting indicating a highly ordered single-stranded structure. In general, Oligonucleotides containing 2b are very stable against hydrolysis with calf-spleen phosphodiesterase (CS-PDE, 5′ → 3′ exonuclease), while phosphodiester hydrolysis with snake-venom phosphodiesterase (SV-PDE, 3′ → 5′ exonuclease) is only slightly reduced.     

Synthesis of 8-halogenated-7-deaza-2′-deoxyguanosine as an 8-oxo-2′-deoxyguanosine analogue and evaluation of its base pairing properties

8-Halogenated-7-deaza-2′-deoxyguanosines (8-halo-7-deaza-dG) were designed to structurally mimic 8-oxo-2′-deoxyguanosine (8-oxo-dG), which is representative of an oxidized nucleoside. It has been shown by NMR that the conformation around the N-glycosidic bond of (8-halo-7-deaza-dG) is preferably syn, similar to 8-oxo-dG. The base pairing properties of 8-halo-7-deaza-dG were studied by measuring the thermal denaturation temperature of the duplexes, showing that their base pair with dC is destabilized compared with natural dG. These results also support their preference for syn conformation. Unlike 8-oxo-dG, 8-halo-7-deaza-dG did not form a stable base pair with dA, most likely due to the lack of N7-H hydrogen bonding with dA. In conclusion, the newly-designed 8-halo-7-deaza-dG analogs resemble 8-oxo-dG in its shape and preference for syn conformation, but they do not form Hoogsteen base pair with the opposing dA.     

Oligonucleotides Containing 7-Deazaadenines: The Influence of the 7-Substituent Chain Length and Charge on the Duplex Stability

Oligonucleotides carrying alkynyl and aminoalkynyl chains at the position 7 of 7-deazaadenine are synthesized, and the chain lengths as well as the bulkiness of the substituents are varied. The corresponding nucleosides 1a – f are prepared from 7-deaza-2′-deoxy-7-iodoadenosine and the particular alkynes by the Pd⁰-catalyzed cross-coupling reaction. The nucleosides are converted to the phosphoramidites 2a – f , which are used in solid-phase oligonucleotide synthesis. The stability of the duplexes is determined by the T_m values and the thermodynamic data. Compared to adenine or the unsubstituted 7-deazaadenine, the incorporation of a 7-ethynyl chain in a 7-deazaadenine moiety increases the duplex stability significantly, while a dodecynyl residue or a bulky steroid moiety leads to a duplex destabilization. A 3-aminoprop-1-ynyl residue (see 1g ) or a 5-aminopent-1-ynyl residue (see 1h ), which are charged under neutral conditions, lead to zwitterionic DNA. A high density of charged residues as found in homomers impairs duplex formation, most probably by counter-ion condensation.     

Oligonucleotides Containing Pyrazolo[3,4-d]pyrimidines: The Influence of 7-Substituted 8-Aza-7-deaza-2′-deoxyguanosines on the Duplex Structure and Stability

Oligonucleotides containing 7-substituted 8-aza-7-deazaguanines (=6-amino-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-ones) were prepared by automated solid-phase synthesis. A series of 7-alkynylated 8-aza-7-deaza-2′-deoxyguanosines (see 4a – d ) were synthesized with the 7-iodonucleoside 3c as starting material and by the Pd⁰/Cu^I-catalyzed cross-coupling reaction with various alkynes. Phosphoramidites were prepared from the 7-substituted 8-aza-7-deaza-2′-deoxyguanosine derivatives carrying halogeno, cyano, and hexynyl substituents. From the melting profiles of oligonucleotide duplexes, the T_m values as well as the thermodynamic data were determined. A significant duplex stabilization by the 7-substituents was observed for the DNA⋅DNA duplexes, but not in the case of DNA⋅RNA hybrids.     

6-Azauracil or 8-aza-7-deazaadenine nucleosides and oligonucleotides: the effect of 2'-fluoro substituents and nucleobase nitrogens on conformation and base pairing

The stereoselective syntheses of 6-azauracil- and 8-aza-7-deazaadenine 2'-deoxy-2'-fluoro-beta-d-arabinofuranosides and employing nucleobase anion glycosylation with 3,5-di-O-benzoyl-2-deoxy-2-fluoro-alpha-d-arabinofuranosyl bromide as the sugar component are described; the 6-azauracil 2'-deoxy-2'-fluoro-beta-d-ribofuranoside was prepared from 6-azauridine via the 2,2'-anhydro intermediate and transformation of the sugar with DAST. Compounds show a preferred N-conformer population (100% N for , and 78% N for ) being rather different from nucleosides not containing the combination of a fluorine atom at the 2'-position and a nitrogen next to the glycosylation site. Oligonucleotides incorporating and were synthesized using the phosphoramidites and . Although the N-conformation is favoured in the series of 6-azauracil- and 8-aza-7-deazaadenine 2'-deoxy-2'-fluoroarabinonucleosides only the pyrimidine compound shows an unfavourable effect on duplex stability, while oligonucleotide duplexes containing the 8-aza-7-deazaadenine-2'-deoxy-2'-fluoroarabinonucleoside were as stable as those incorporating dA or 8-aza-7-deaza-2'-deoxyadenosine .