1,7-Dideaza-2'-deoxy-6-nitronebularine: a pyrrolo[2,3-b]pyridine nucleoside with an intramolecular hydrogen bond stabilizing the syn conformation.

The title compound [systematic name: 1-(2-deoxy-ß-D-erythro-pentofuranosyl)-4-nitro-1H-pyrrolo[2,3-b]pyridine], C12H13N3O5, forms an intramolecular hydrogen bond between the pyridine N atom as acceptor and the 5'-hydroxy group of the sugar residue as donor. Consequently, the N-glycosylic bond exhibits a syn conformation, with a χ torsion angle of 61.6 (2)°, and the pentofuranosyl residue adopts a C2'-endo envelope conformation (²E, S-type), with P = 162.1 (1)° and τm = 36.2 (1)°. The orientation of the exocyclic C4'-C5' bond is +sc (gauche, gauche), with a torsion angle γ = 49.1 (2)°. The title nucleoside forms an ordered and stacked three-dimensional network. The pyrrole ring of one layer faces the pyridine ring of an adjacent layer. Additionally, intermolecular O-H∙∙∙O and C-H∙∙∙O hydrogen bonds stabilize the crystal structure.

Construction and assembly of chimeric DNA: oligonucleotide hybrid molecules composed of parallel or antiparallel duplexes and tetrameric i-motifs.

Chimeric DNA containing parallel (ps) and antiparallel (aps) duplex elements as well as poly-dC tracts were designed and synthesized. Oligonucleotide duplexes with ps chain orientation containing reverse Watson-Crick dA-dT base pairs and short d(C)2 tails are stabilized under slightly acidic conditions by hemiprotonated dCH+-dC base pairs ("clamp" effect). Corresponding molecules with aps orientation containing Watson-Crick dA-dT base pairs do not show this phenomenon. Chimeric DNA with ps duplex elements and long d(C)5 tails at one or at both ends assemble to tetrameric i-motif structures. Molecules with two terminal d(C)5 tails form multimeric assemblies which have the potential to form nanoscopic scaffolds. A preorganization of the ps duplex chains stabilizes the i-motif assemblies up to almost neutral conditions as evidenced by thermal melting and gel electrophoresis. Although, ps DNA is generally less stable than aps DNA, the aps duplexes contribute less to the stability of the i-motif than ps DNA.

5-Ethynyl-2'-deoxycytidine: a DNA building block with a 'clickable' side chain.

The title compound [systematic name: 4-amino-1-(2-deoxy-ß-D-erythro-pentofuranosyl)-5-ethynylpyrimidin-2(1H)-one], C(11)H(13)N(3)O(4), shows two conformations in the crystalline state. The N-glycosylic bonds of both conformers adopt similar conformations, with χ = -149.2 (1)° for conformer (I-1) and -151.4 (1)° for conformer (I-2), both in the anti range. The sugar residue of (I-1) shows a C2'-endo envelope conformation ((2)E, S-type), with P = 164.7 (1)° and τ(m) = 36.9 (1)°, while (I-2) shows a major C3'-exo sugar pucker (C3'-exo-C2'-endo, (3)T(2), S-type), with P = 189.2 (1)° and τ(m) = 33.3 (1)°. Both conformers participate in the formation of a layered three-dimensional crystal structure with a chain-like arrangement of the conformers. The ethynyl groups do not participate in hydrogen bonding, but are arranged in proximal positions.

A 2'-deoxycytidine long-linker click adduct forming two conformers in the asymmetric unit.

The title compound {systematic name: 4-amino-1-(2-deoxy-ß-D-erythro-pentofuranosyl)-5-[6-(1-benzyl-1H-1,2,3-triazol-4-yl)hex-1-ynyl]pyrimidin-2(1H)-one}, C(24)H(28)N(6)O(4), shows two conformations in the crystalline state, viz. (I-1) and (I-2). The pyrimidine groups and side chains of the two conformers are almost superimposable, while the greatest differences between them are observed for the sugar groups. The N-glycosylic bonds of both conformers adopt similar anti conformations, with χ = -168.02 (12)° for conformer (I-1) and χ = -159.08 (12)° for conformer (I-2). The sugar residue of (I-1) shows an N-type (C3'-endo) conformation, with P = 33.1 (2)° and τ(m) = 29.5 (1)°, while the conformation of the 2'-deoxyribofuranosyl group of (I-2) is S-type (C3'-exo), with P = 204.5 (2)° and τ(m) = 33.8 (1)°. Both conformers participate in hydrogen-bond formation and exhibit identical patterns resulting in three-dimensional networks. Intermolecular hydrogen bonds are formed with neighbouring molecules of different and identical conformations (N-H...N, N-H... O, O-H...N and O-H...O).

Parallel-stranded DNA: enhancing duplex stability by the 'G-clamp' and a pyrrolo-dC derivative.

The new pyrrolo-dC derivative 4 tethered with an alkylamino side chain via a triazole linker was synthesized. Oligonucleotides containing the G-clamp 3 or the pyrrolo-dC derivative 4 were prepared. Oligonucleotide synthesis and deprotection under standard conditions led to unwanted side product formation. The side product was identified as an acrylonitrile adduct of the aminoalkyl side chain. Changing the synthesis and work-up conditions to fast-deprotection chemistry and performing ß-elimination of the cyanoethyl group on the solid support yielded pure oligonucleotides. Oligonucleotide duplexes with parallel chain orientation were constructed incorporating dA·dT and isoG(d)·dC base pairs. Replacement of dC-residues by the G-clamp 3 led to extraordinarily stable duplexes (ΔT(m) = +11 °C for two incorporations) in ps DNA, while the pyrrolo-dC derivative 4 behaved like dC. Surprisingly, the G-clamp 3 forms an even more stable base pair with 2'-deoxyisoguanosine in DNA with parallel chain orientation than with 2'-deoxyguanosine in aps DNA.

DNA gold nanoparticle conjugates incorporating thiooxonucleosides: 7-deaza-6-thio-2'-deoxyguanosine as gold surface anchor.

A new protocol for the covalent attachment of oligonucleotides to gold nanoparticles was developed. Base-modified nucleosides with thiooxo groups were acting as molecular surface anchor. Compared to already existing conjugation protocols, the new linker strategy simplifies the synthesis of DNA gold nanoparticle conjugates. The phosphoramidite of 7-deaza-6-thio-2'-deoxyguanosine (6) was used in solid-phase synthesis. Incorporation of the sulfur-containing nucleosides can be performed at any position of an oligonucleotide; even multiple incorporations are feasible, which will increase the binding stability of the corresponding oligonucleotides to the gold nanoparticles. Oligonucleotide strands immobilized at the end of a chain were easily accessible during hybridization leading to DNA gold nanoparticle network formation. On the contrary, oligonucleotides immobilized via a central position could not form a DNA-AuNP network. Melting studies of the DNA gold nanoparticle assemblies revealed sharp melting profiles with a very narrow melting transition.

7-Amino-1-(2-deoxy-ß-erythro-pentofuranosyl)-1H-1,2,3-triazolo[4,5-d]pyrimidine: an 8-azaadenine nucleoside with the nucleobase in a syn conformation.

The title compound, C(9)H(12)N(6)O(3), shows a syn-glycosylic bond orientation [χ = 64.17 (16)°]. The 2'-deoxyfuranosyl moiety exhibits an unusual C1'-exo-O4'-endo ((1)T(0); S-type) sugar pucker, with P = 111.5 (1)° and τ(m) = 40.3 (1)°. The conformation at the exocyclic C4'-C5' bond is +sc (gauche), with γ = 64.4 (1)°. The two-dimensional hydrogen-bonded network is built from intermolecular N-H...O and O-H...N hydrogen bonds. An intramolecular bifurcated hydrogen bond, with an amino N-H group as hydrogen-bond donor and the ring and hydroxymethyl O atoms of the sugar moiety as acceptors, constrains the overall conformation of the nucleoside.

2-azapurine nucleosides: synthesis, properties, and base pairing of oligonucleotides.

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, (13)C-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.

Triplexes with 8-Aza-2'-deoxyisoguanosine replacing protonated dC: probing third strand stability with a fluorescent nucleobase targeting duplex DNA.

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.

2-Amino-8-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)imidazo[1,2-a][1,3,5]triazin-4(8H)-one monohydrate, a 2'-deoxyguanosine analogue with an altered Watson-Crick recognition site.

The title compound, C(10)H(12)FN(5)O(4).H(2)O, shows an anti glycosyl orientation [chi = -123.1 (2) degrees]. The 2-deoxy-2-fluoroarabinofuranosyl moiety exhibits a major C2'-endo sugar puckering (S-type, C2'-endo-C1'-exo, (2)T(1)), with P = 156.9 (2) degrees and tau(m) = 36.8 (1) degrees , while in solution a predominantly N conformation of the sugar moiety is observed. The conformation around the exocyclic C4'-C5' bond is -sc (trans, gauche), with gamma = -78.3 (2) degrees. Both nucleoside and solvent molecules participate in the formation of a three-dimensional hydrogen-bonding pattern via intermolecular N-H...O and O-H...O hydrogen bonds; the N atoms of the heterocyclic moiety and the F substituent do not take part in hydrogen bonding.

2'-Deoxy-5-propynyluridine: a nucleoside with two conformations in the asymmetric unit.

The title compound, 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-5-(prop-1-ynyl)pyrimidin-2,4(1H,3H)-dione, C(12)H(14)N(2)O(5), shows two conformations in the crystalline state: conformer 1 adopts a C2'-endo (close to (2)E; S-type) sugar pucker and an anti nucleobase orientation [chi = -134.04 (19) degrees], while conformer 2 shows an S sugar pucker (twisted C2'-endo-C3'-exo), which is accompanied by a different anti base orientation [chi = -162.79 (17) degrees]. Both molecules show a +sc (gauche, gauche) conformation at the exocyclic C4'-C5' bond and a coplanar orientation of the propynyl group with respect to the pyrimidine ring. The extended structure is a three-dimensional hydrogen-bond network involving intermolecular N-H...O and O-H...O hydrogen bonds. Only O atoms function as H-atom acceptor sites.

The N(1)-(2'-deoxyribofuranoside) of 3-iodo-5-nitroindole: a universal nucleoside forming nitro-iodo interactions.

The title compound [systematic name: 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-3-iodo-5-nitro-1H-indole], C(13)H(13)IN(2)O(5), exhibits an anti glycosylic bond conformation with a chi torsion angle of -114.9 (3) degrees . The furanose moiety shows a twisted C2'-endo sugar pucker (S-type), with P = 141.3 degrees and tau(m) = 40.3 degrees . The orientation of the exocyclic C4'-C5' bond is +ap (gauche, trans), with a gamma torsion angle of 177.4 (2) degrees . The extended crystal structure is stabilized by hydrogen bonding and I...O contacts, as well as by stacking interactions. The O atoms of the nitro group act as acceptors, forming bifurcated hydrogen bonds within the ac plane. Additionally, the iodo substituent forms an interplanar contact with an O atom of the nitro group, and another contact with the O atom of the 5'-hydroxy group of the sugar moiety within the ac plane is observed. These contacts can be considered as the structure-determining factors for the molecular packing in the crystal structure.

8-Aza-7-deazaguanine nucleosides and oligonucleotides with octadiynyl side chains: synthesis, functionalization by the azide-alkyne 'click' reaction and nucleobase specific fluorescence quenching of coumarin dye conjugates.

Oligonucleotides incorporating 7-(octa-1,7-diynyl) derivatives of 8-aza-7-deaza-2-deoxyguanosine (2d) were prepared by solid-phase synthesis. The side chain of 2d was introduced by the Sonogashira cross coupling reaction and phosphoramidites (3a, 3b) were synthesized. Duplexes containing 2d are more stabilized compared to those incorporating the non-functionalized 8-aza-7-deaza-2-deoxyguanosine (2a) demonstrating that these side chains have steric freedom in duplex DNA. Nucleoside 2d as well as 2d-containing oligonucleotides were conjugated to the non-fluorescent 3-azido-7-hydroxycoumarin 15 by the Huisgen-Meldal-Sharpless click reaction. Pyrazolo[3,4-d]pyrimidine nucleoside conjugate 16 shows a much higher fluorescence intensity than that of the corresponding pyrrolo[2,3-d]pyrimidine derivative 17. The quenching in the dye conjugate 17 was found to be stronger on the stage of monomeric conjugates than in single-stranded or duplex DNA. Nucleobase-dye contact complexes are suggested which are more favourable in the monomeric state than in the DNA chain when the nucleobase is part of the stack. The side chains with the bulky dye conjugates are well accommodated in DNA duplexes thereby forming hybrids which are slightly more stable than canonical DNA.

7-Fluorotubercidin: a halogenated derivative of a naturally occurring nucleoside antimetabolite.

The title compound [systematic name: 4-amino-5-fluoro-7-(beta-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine], C(11)H(13)FN(4)O(4), exhibits an anti glycosylic bond conformation, with a chi torsion angle of -124.7 (3) degrees. The furanose moiety shows a twisted C2'-endo sugar pucker (S-type), with P = 169.8 (3) degrees and tau(m) = 38.7 (2) degrees. The orientation of the exocyclic C4'-C5' bond is +sc (gauche, gauche), with a gamma torsion angle of 59.3 (3) degrees. The nucleobases are stacked head-to-head. The extended crystal structure is a three-dimensional hydrogen-bond network involving O-H...O, O-H...N and N-H...O hydrogen bonds. The crystal structure of the title nucleoside demonstrates that the C-C bonds nearest the F atom of the pyrrole system are significantly shortened by the electronegative halogen atom.

Mismatch formation in solution and on DNA microarrays: how modified nucleosides can overcome shortcomings of imperfect hybridization caused by oligonucleotide composition and base pairing.

The DNA microarray technology is a well-established and widely used technology although it has several drawbacks. The accurate molecular recognition of the canonical nucleobases of probe and target is the basis for reliable results obtained from microarray hybridization experiments. However, the great flexibility of base pairs within the DNA molecule allows the formation of various secondary structures incorporating Watson-Crick base pairs as well as non-canonical base pair motifs, thus becoming a source of inaccuracy and inconsistence. The first part of this report provides an overview of unusual base pair motifs formed during molecular DNA interaction in solution highlighting selected secondary structures employing non-Watson-Crick base pairs. The same mispairing phenomena obtained in solution are expected to occur for immobilized probe molecules as well as for target oligonucleotides employed in microarray hybridization experiments the effect of base pairing and oligonucleotide composition on hybridization is considered. The incorporation of nucleoside derivatives as close shape mimics of the four canonical nucleosides into the probe and target oligonucleotides is discussed as a chemical tool to resolve unwanted mispairing. The second part focuses non-Watson-Crick base pairing during hybridization performed on microarrays. This is exemplified for the unusual stable dG.dA base pair.

pH-dependent assembly of DNA-gold nanoparticles based on the i-motif.

Oligonucleotides containing stretches of 2 '-deoxycytidine residues were immobilized on 15 nm gold nanoparticles. Under acidic pH conditions a reversible supramolecular assembly is formed, induced by the formation of the tetrameric i-motif structure. The replacement of 2 '-deoxycytidine by 5-propynyl-2 '-deoxycytidine (dC*) leads to novel i-motif structures. Oligonucleotides incorporating multiple residues of dC* were immobilized on 15 nm gold nanoparticles and are able to aggregate into i-motif structures even at non-optimal pH values.

2'-Deoxyimmunosine: a thiazolo[4,5-d]pyrimidine nucleoside adopting the syn conformation.

The title compound [systematic name: 5-amino-3-(2-deoxy-beta-D-erythro-pentofuranosyl)thiazolo[4,5-d]pyrimidine-2,7-(3H,6H)-dione], C(10)H(12)N(4)O(5)S, exhibits a syn glycosylic bond conformation, with a torsion angle chi of 61.0 (3) degrees. The furanose moiety adopts the N-type sugar pucker ((3)T4), with P = 33.0 (5) degrees and tau(m) = 15.1 (1) degrees. The conformation at the exocyclic C4'-C5' bond is +ap (trans), with the torsion angle gamma = 176.71 (14) degrees. The extended structure is a three-dimensional hydrogen-bond network involving O-H...O and N-H...O hydrogen bonds.

Oligonucleotides forming an i-motif: the pH-dependent assembly of individual strands and branched structures containing 2'-deoxy-5-propynylcytidine.

Non-branched and branched oligonucleotides incorporating consecutive runs of 2'-deoxy-5-propynylcytidine residues () instead of 2'-deoxycytidine () were synthesized. For this, phosphoramidite building blocks of 2'-deoxy-5-propynylcytidine () were prepared using acetyl, benzoyl or N,N-di-n-butylaminomethylidene protecting groups. The formation of the i-motif assemblies incorporating 2'-deoxy-5-propynylcytidine residues was confirmed by temperature-dependent CD- and UV-spectra as well as by ion-exchange chromatography. The low pK(a)-value of nucleoside (pK(a) = 3.3) compared to dC (pK(a) = 4.5) required strong acidic conditions for i-motif formation. Branched oligonucleotide residues with strands in a parallel orientation lead to a strong stabilization of the i-motif allowing aggregation even at non-optimal pH conditions (pH = 5). The immobilization of oligonucleotides incorporating multiple residues of on 15 nm gold nanoparticles generated DNA-gold nanoparticle conjugates which are able to aggregate into i-motif structures at pH 5.

2'-Deoxy-5-propynylcytidine: a nucleoside forming two solid-state conformations.

The title compound, 4-amino-1-(2-deoxy-beta-D-erythropentofuranosyl)-5-(prop-1-ynyl)pyrimidin-2(1H)-one, C12H15N3O4, shows two conformations in the crystalline state which differ mainly in the glycosylic bond torsion angle and the sugar pucker. Both molecules exhibit an anti glycosylic bond conformation, with torsion angles chi = -135.0 (2) and -156.4 (2) degrees for molecules 1 and 2, respectively. The sugar moieties show a twisted C2'-endo sugar pucker (S-type), with P = 173.3 and 192.5 degrees for molecules 1 and 2, respectively. The crystal structure is characterized by a three-dimensional network that is stabilized by several intermolecular hydrogen bonds between the two conformers.

2'-Deoxy-7-propynyl-7-deazaadenosine: a DNA duplex-stabilizing nucleoside.

In the title compound, 2'-deoxy-7-propynyl-7-deazaadenosine, C14H16N4O3, the torsion angle of the N-glycosylic bond is anti [chi = -130.7 (2) degrees ]. The sugar pucker of the 2'-deoxyribofuranosyl moiety is C2'-endo-C3'-exo, 2T3 (S-type), with P = 185.9 (2) degrees and tau(m) = 39.1 (1) degrees , and the orientation of the exocyclic C4'-C5' bond is -ap (trans). The 7-substituted propynyl group is nearly coplanar with the heterocyclic base moiety. Molecules of the nucleoside form a layered network in which the heterocyclic bases are stacked head-to-tail with a closest distance of 3.197 (1) A. The crystal structure of the nucleoside is stabilized by three intermolecular hydrogen bonds of types N-H... O, O-H... N and O-H... O.