A highly effective and convenient “bis-click” strategy was developed for the template-independent circularization of single-stranded oligonucleotides by employing copper(I)-assisted azide–alkyne cycloaddition. Terminal triple bonds were incorporated at both ends of linear oligonucleotides. Alkynylated 7-deaza-2′-deoxyadenosine and 2′-deoxyuridine residues with different side chains were used in solid-phase synthesis with phosphoramidite chemistry. The bis-click ligation of linear 9- to 36-mer oligonucleotides with 1,4-bis(azidomethyl)benzene afforded circular DNA in a simple and selective way; azido modification of the oligonucleotide was not necessary. Short ethynyl side chains were compatible with the circularization of longer oligonucleotides, whereas octadiynyl residues were used for short 9-mers. Compared with linear duplexes, circular bis-click constructs exhibit a significantly increased duplex stability over their linear counterparts. The intramolecular bis-click ligation protocol is not limited to DNA, but may also be suitable for the construction of other macrocycles, such as circular RNAs, peptides, or polysaccharides.

Reverse Watson–Crick DNA with parallel-strand orientation (ps DNA) has been constructed. Pyrrolo-dC (PyrdC) nucleosides with phenyl and pyridinyl residues linked to the 6 position of the pyrrolo[2,3-d]pyrimidine base have been incorporated in 12- and 25-mer oligonucleotide duplexes and utilized as silver-ion binding sites. Thermal-stability studies on the parallel DNA strands demonstrated extremely strong silver-ion binding and strongly enhanced duplex stability. Stoichiometric UV and fluorescence titration experiments verified that a single ^2pyPyrdC–^2pyPyrdC pair captures two silver ions in ps DNA. A structure for the PyrdC silver-ion base pair that aligns 7-deazapurine bases head-to-tail instead of head-to-head, as suggested for canonical DNA, is proposed. The silver DNA double helix represents the first example of a ps DNA structure built up of bidentate and tridentate reverse Watson–Crick base pairs stabilized by a dinuclear silver-mediated PyrdC pair.

The title compound {systematic name: 4-amino-5-cyclopropyl-7-(2-deoxy-β-D-erythro-pentofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine}, C₁₄H₁₈N₄O₃, exhibits an anti glycosylic bond conformation, with the torsion angle χ = −108.7 (2)°. The furanose group shows a twisted C1′-exo sugar pucker (S-type), with P = 120.0 (2)° and τ_m = 40.4 (1)°. The orientation of the exocyclic C4′—C5′ bond is -ap (trans), with the torsion angle γ = −167.1 (2)°. The cyclopropyl substituent points away from the nucleobase (anti orientation). Within the three-dimensional extended crystal structure, the individual molecules are stacked and arranged into layers, which are highly ordered and stabilized by hydrogen bonding. The O atom of the exocyclic 5′-hydroxy group of the sugar residue acts as an acceptor, forming a bifurcated hydrogen bond to the amino groups of two different neighbouring molecules. By this means, four neighbouring molecules form a rhomboidal arrangement of two bifurcated hydrogen bonds involving two amino groups and two O5′ atoms of the sugar residues.

Tuneable silver arrays inside DNA duplexes were constructed by multiple incorporations of pyrrolo-dC–pyrrolo-dC base pairs. A short array with six silver ions was built within a DNA template. The size and positioning of the array is programmable and depends on the incorporation site of the modified base pairs. Always two silver ions were captured by one pyrrolo-dC–pyrrolo-dC pair as confirmed by melting experiments, UV and fluorescence titration experiments as well as ESI mass spectra. The silver-mediated pyrrolo-dC–pyrrolo-dC base pairs with phenyl residues in the 6-position were significantly more stable than those containing methyl substitutions. The caged silver ions are released from duplex DNA by sodium iodide but not by chloride or bromide anions, thus indicating very high stability of the metal–DNA complex.

8-Phenylimidazolo-dC (^phImidC, 2) forms metal-mediated DNA base pairs by entrapping two silver ions. To this end, the fluorescent “purine” 2′-deoxyribonucleoside 2 has been synthesised and converted into the phosphoramidite 6. Owing to the ease of nucleobase deprotonation, the new Ag⁺-mediated base pair containing a “purine” skeleton is much stronger than that derived from the pyrrolo- [3,4-d]pyrimidine system (^phPyrdC, 1). The silver-mediated ^phImidC–^phImidC base pair fits well into the DNA double helix and has the stability of a covalent cross-link. The formation of such artificial metal base pairs might not be limited to DNA but may be applicable to other nucleic acids such as RNA, PNA and GNA as well as other biopolymers.

New pyrrolo-dC click adducts (4 and 5) tethered with a 1,2,3-triazole skeleton were synthesized and oligonucleotides were prepared. The triazole system was either directly linked to the pyrrolo moiety (5) or connected via an n-butyl linker (4). The quantum yield of nucleoside 5 (Φ=0.32), which is 10 times higher than those of 8-methylpyrrolo-dC (1 b, Φ=0.026) or the long linker derivative 4 (Φ=0.03), is maintained in oligonucleotides. Compound 5 was used as a nucleobase-discriminating fluorescence sensor in duplex DNA. Excellent mismatch discrimination was observed when 5 was positioned opposite the four canonical nucleosides. Compound 5 has the potential to be used for SNP detection in long DNA targets when conventional techniques such as high resolution melt analysis fail.

This review deals with 2-azapurine (imidazo[4,5-d] [1,2,3]triazine) nucleosides and closely related analogs. Different routes are described to yield the desired target compounds, including a sequence of ring-opening and ring-closure reactions performed on purine nucleosides or direct glycosylation of a 2-azapurine nucleobase with a sugar halide. Further, physical and spectroscopic properties of 2-azapurine nucleosides are discussed, including fluorescence, ¹³C-NMR data, single-crystal X-ray analyses, and conformation studies on selected compounds; new biological data are presented. The second part of this review is dedicated to oligonucleotides containing 2-azapurines, including building-block (phosphoramidite) preparation and their use in solid-phase oligonucleotide synthesis. Base-pairing properties of 2-azapurine nucleosides as surrogates of canonical constituents of DNA were evaluated.

The naturally occurring tRNA nucleoside preQ₀, 7-cyano-7-deazaguanosine, which is a central intermediate for other natural occurring 7-deazapurine nucleosides was synthesized via a copper(I)-ion-mediated iodocarbonitrile exchange. The reaction was performed on the easily accessible 7-iodo-7-deazaguanosine under microwave conditions. The overall reaction yield was 30% starting with the glycosylation reaction of the nucleobase. Corresponding 2′-deoxyribonucleosides were prepared following the same route.

Nucleobase-directed spin-labeling by the azide-alkyne ‘click’ (CuAAC) reaction has been performed for the first time with oligonucleotides. 7-Deaza-7-ethynyl-2′-deoxyadenosine (1) and 5-ethynyl-2′-deoxyuridine (2) were chosen to incorporate terminal triple bonds into DNA. Oligonucleotides containing 1 or 2 were synthesized on a solid phase and spin labeling with 4-azido-2,2,6,6-tetramethylpiperidine 1-oxyl (4-azido-TEMPO, 3) was performed by post-modification in solution. Two spin labels (3) were incorporated with high efficiency into the DNA duplex at spatially separated positions or into a ‘dA-dT’ base pair. Modification at the 5-position of the pyrimidine base or at the 7-position of the 7-deazapurine residue gave steric freedom to the spin label in the major groove of duplex DNA. By applying cw and pulse EPR spectroscopy, very accurate distances between spin labels, within the range of 1–2 nm, were measured. The spin–spin distance was 1.8±0.2 nm for DNA duplex 17(dA*^7,10)⋅11 containing two spin labels that are separated by two nucleotides within one individual strand. A distance of 1.4±0.2 nm was found for the spin-labeled ‘dA-dT’ base pair 15(dA*⁷)⋅16(dT*⁶). The ‘click’ approach has the potential to be applied to all four constituents of DNA, which indicates the universal applicability of the method. New insights into the structural changes of canonical or modified DNA are expected to provide additional information on novel DNA structures, protein interaction, DNA architecture, and synthetic biology.

The fluorescent 8-aza-2′-deoxyisoguanosine (4) as well as the parent 2′-deoxyisoguanosine (1) were used as protonated dCH⁺ surrogates in the third strand of oligonucleotide triplexes. Stable triplexes were formed by Hoogsteen base pairing. In contrast to dC, triplexes containing nucleoside 1 or 4 in place of dCH⁺ are already formed under neutral conditions or even at alkaline pH values. Triplex melting can be monitored separately from duplex dissociation in cases in which the third strand contains the fluorescent nucleoside 4. Third-strand binding of oligonucleotides with 4, opposite to dG, was selective as demonstrated by hybridisation experiments studying mismatch discrimination. Third-strand binding is more efficient when the stability of the DNA duplex is reduced by mismatches, giving third-strand binding more flexibility.

A series of 7-fluorinated 7-deazapurine 2′-deoxyribonucleosides related to 2′-deoxyadenosine, 2′-deoxyxanthosine, and 2′-deoxyisoguanosine as well as intermediates 4b–7b, 8, 9b, 10b, and 17b were synthesized. The 7-fluoro substituent was introduced in 2,6-dichloro-7-deaza-9H-purine (11a) with Selectfluor (Scheme 1). Apart from 2,6-dichloro-7-fluoro-7-deaza-9H-purine (11b), the 7-chloro compound 11c was formed as by-product. The mixture 11b/11c was used for the glycosylation reaction; the separation of the 7-fluoro from the 7-chloro compound was performed on the level of the unprotected nucleosides. Other halogen substituents were introduced with N-halogenosuccinimides (11a11c–11e). Nucleobase-anion glycosylation afforded the nucleoside intermediates 13a–13e (Scheme 2). The 7-fluoro- and the 7-chloro-7-deaza-2′-deoxyxanthosines, 5b and 5c, respectively, were obtained from the corresponding MeO compounds 17b and 17c, or 18 (Scheme 6). The 2′-deoxyisoguanosine derivative 4b was prepared from 2-chloro-7-fluoro-7-deaza-2′-deoxyadenosine 6bvia a photochemically induced nucleophilic displacement reaction (Scheme 5). The pK_a values of the halogenated nucleosides were determined (Table 3). ¹³C-NMR Chemical-shift dependencies of C(7), C(5), and C(8) were related to the electronegativity of the 7-halogen substituents (Fig. 3). In aqueous solution, 7-halogenated 2′-deoxyribonucleosides show an approximately 70% S population (Fig. 2 and Table 1).

Oligonucleotides containing 7-deaza-2′-deoxyinosine derivatives bearing 7-halogen substituents or 7-alkynyl groups were prepared. For this, the phosphoramidites 2b–2g containing 7-substituted 7-deaza-2′-deoxyinosine analogues 1b–1g were synthesized (Scheme 2). Hybridization experiments with modified oligonucleotides demonstrate that all 2′-deoxyinosine derivatives show ambiguous base pairing, as 2′-deoxyinosine does. The duplex stability decreases in the order C_d>A_d>T_d>G_d when 2b–2g pair with these canonical nucleosides (Table 6). The self-complementary duplexes 5′-d(F⁷c⁷I-C)₆, d(Br⁷c⁷I-C)₆, and d(I⁷c⁷I-C)₆ are more stable than the parent duplex d(c⁷I-C)₆ (Table 7). An oligonucleotide containing the octa-1,7-diyn-1-yl derivative 1g, i.e., 27, was functionalized with the nonfluorescent 3-azido-7-hydroxycoumarin (28) by the Huisgen–Sharpless–Meldal cycloaddition ‘click’ reaction to afford the highly fluorescent oligonucleotide conjugate 29 (Scheme 3). Consequently, oligonucleotides incorporating the derivative 1g bearing a terminal CC bond show a number of favorable properties: i) it is possible to activate them by labeling with reporter molecules employing the ‘click’ chemistry. ii) Space demanding residues introduced in the 7-position of the 7-deazapurine base does not interfere with duplex structure and stability (Table 8). iii) The ambiguous pairing character of the nucleobase makes them universal probes for numerous applications in oligonucleotide chemistry, molecular biology, and nanobiotechnology.

5-Tripropargylamine-2′-deoxyuridine (1 a) containing two terminal triple bonds was synthesized by a Pd-assisted Sonogashira cross-coupling reaction and was subsequently converted into the corresponding phosphoramidite building block (9) and employed in solid-phase oligonucleotide synthesis. T_m experiments demonstrate that the presence of covalently attached branched tripropargylamine residues has a positive effect on the base pair stability. The two terminal CC bonds of modified DNA were functionalized by means of Cu^I-mediated 1,3-dipolar cycloaddition reactions (click chemistry) with azides such as 3-azido-7-hydroxycoumarin or 3′-azido-3′-deoxythymidine (AZT) both in solution and on solid support. In particular, with the nonfluorescent 3-azido-7-hydroxycoumarin a strongly fluorescent oligonucleotide bis-dye conjugate was generated. For comparison, the N(3)-propargylated 2′-deoxyuridine 2 was prepared from 2′-deoxyuridine and propargyl bromide and incorporated into DNA. The two terminal triple bonds of 1 a allow the simultaneous post-modification of DNA by two reporter molecules and can be applied to almost any azido derivatives (oligonucleotides, proteins, polysaccharides etc.) including those forming dendrimeric side chains.