7-Deaza-2'-deoxyisoguanosine, a Noncanonical Nucleoside for Nucleic Acid Code Expansion and New DNA Constructs: Nucleobase Functionalization of Inverse Watson-Crick and Purine-Purine Base Pairs.

7-Deaza-2'-deoxyisoguanosine forms stable inverse Watson-Crick base pairs with 5-methyl-2'-deoxyisocytidine and purine-purine base pairs with 2'-deoxyguanosine or 5-aza-7-deaza-2'-deoxyguanosine. Both base pairs expand the genetic coding system. The manuscript reports on the functionalization of these base pairs with halogen atoms and clickable side chains introduced at 7-position of the 7-deazapurine base. Oligonucleotides containing the functionalized base pairs were prepared by solid-phase synthesis. To this end, a series of phosphoramidites were synthesized and clickable side chains with short and long linkers were incorporated in oligonucleotides. Fluorescent pyrene conjugates were obtained by postmodification. Functionalization of DNA with a single inverse Watson-Crick base pair by halogens or clickable residues has only a minor impact on duplex stability. Pyrene click adducts increase (long linker) or decrease (short linker) the double helix stability. Stable hybrid duplexes were constructed containing three consecutive purine-purine pairs of 7-functionalized 7-deaza-2'-deoxyisoguanine with guanine or 5-aza-7-deazaguanine in the center and Watson-Crick pairs at both ends. The incorporation of a hybrid base pair tract of 7-deaza-2'-deoxyisoguanosine/5-aza-7-deaza-2'-deoxyguanosine pairs stabilizes the double helix strongly. Fluorescence intensity of pyrene short linker adducts increased when the 7-deazapurine base was positioned opposite to 5-methylisocytosine (inverse base pair) compared to purine-purine base pairs with guanine or 5-aza-7-deazaguanine in opposite positions. For long liker adducts, the situation is more complex. Circular dichroism (CD) spectra of purine DNA differ to those of Watson-Crick double helices and are indicative for the new DNA constructs. The impact of 7-deaza-2'-deoxyisoguanine base pair functionalization is studied for the first time and all experimental details are reported to prepare DNA functionalized at the 7-deazaisoguanine site. The influence of single and multiple incorporations on DNA structure and stability is shown. Clickable residues introduced at the 7-position of the 7-deazaisoguanine base provide handles for Huisgen-Sharpless-Meldal click cycloadditions without harming the stability of purine-pyrimidine and purine-purine base pairs. Other chemistries might be used for bioconjugation. Our investigation paves the way for the functionalization of a new DNA related recognition system expanding the common Watson-Crick regime.

7-Deazapurine and Pyrimidine Nucleoside and Oligonucleotide Cycloadducts Formed by Inverse Diels-Alder Reactions with 3,6-Di(pyrid-2-yl)-1,2,4,5-tetrazine: Ethynylated and Vinylated Nucleobases for Functionalization and Impact of Pyridazine Adducts on DNA Base Pair Stability and Mismatch Discrimination.

The manuscript reports on 7-deazapurine and pyrimidine nucleoside and oligonucleotide cycloadducts formed by the inverse electron demand Diels-Alder (iEDDA) reaction with 3,6-di(pyrid-2-yl)-1,2,4,5-tetrazine. Cycloadducts were constructed from ethynylated and vinylated nucleobases. Oligonucleotides were synthesized containing iEDDA modifications, and the impact on duplex stability was investigated. iEDDA reactions were performed on nucleoside triple bond side chains. Oxidation was not required in these cases as dihydropyridazine intermediates are not formed. In contrast, oxidation is necessary for reactions performed on alkenyl compounds. This was verified on 5-vinyl-2'-deoxyuridine. A diastereomeric mixture of 1,2-dihydropyridazine cycloadduct intermediates was isolated, characterized, and later oxidized. 12-mer oligonucleotides containing 1,2-pyridazine inverse Diels-Alder cycloadducts and their precursors were hybridized to short DNA duplexes. For that, a series of phosphoramidites was prepared. DNA duplexes with 7-functionalized 7-deazaadenines and 5-functionalized pyrimidines display high duplex stability when spacer units are present between nucleobases and pyridazine cycloadducts. A direct connectivity of the pyridazine moiety to nucleobases as reported for metabolic labeling of vinyl nucleosides reduced duplex stability strongly. Oligonucleotides bearing linkers with and without pyridazine cycloadducts attached to the 7-deazaadenine nucleobase significantly reduced mismatch formation with dC and dG.

Nucleobase-Functionalized 7-Deazaisoguanine and 7-Deazapurin-2,6-diamine Nucleosides: Halogenation, Cross-Coupling, and Cycloaddition.

The functionalization in position-7 of 7-deazaisoguanine and 7-deazapurin-2,6-diamine ribo- and 2'-deoxyribonucleosides by halogen atoms (chloro, bromo, iodo), and clickable alkynyl and vinyl side chains for copper-catalyzed and copper-free cycloadditions is described. Problems arising during the synthesis of the 7-iodinated isoguanine ribo- and 2'-deoxyribonucleosides were solved by the action of acetone. The impact of side chains and halogen atoms on the pKa values and hydrophobicity of nucleosides was investigated. Halogenated substituents increase the lipophilic character of nucleosides in the order Cl < Br < I and decrease the pK values of protonation. Photophysical properties (fluorescence, solvatochromism, and quantum yields) of azide-alkyne click adducts bearing pyrene as sensor groups were determined. Pyrene fluorescence was solvent-dependent and changed according to the linker lengths. Excimer emission was observed in dioxane for the long linker adduct. Bioorthogonal inverse-electron-demanding Diels-Alder cycloadditions (iEDDA) were conducted on the electron-rich vinyl groups of 7-deazaisoguanine and 7-deazapurin-2,6-diamine nucleosides as dienophiles and 3,6-dipyridyl-1,2,4,5-tetrazine as diene. The initially formed complex reaction mixture of isomers could be easily oxidized with iodine in tetrahydrofuran (THF)/pyridine leading to single aromatic tetrazine adducts within a short time and in excellent yields.

α-D-2'-Deoxyadenosine, an irradiation product of canonical DNA and a component of anomeric nucleic acids: crystal structure, packing and Hirshfeld surface analysis.

α-D-2'-Deoxyribonucleosides are products of the γ-irradiation of DNA under oxygen-free conditions and are constituents of anomeric DNA. They are not found as natural building blocks of canonical DNA. Reports on their conformational properties are limited. Herein, the single-crystal X-ray structure of α-D-2'-deoxyadenosine (α-dA), C10H13N5O3, and its conformational parameters were determined. In the crystalline state, α-dA forms two conformers in the asymmetric unit which are connected by hydrogen bonds. The sugar moiety of each conformer is arranged in a `clamp'-like fashion with respect to the other conformer, forming hydrogen bonds to its nucleobase and sugar residue. For both conformers, a syn conformation of the nucleobase with respect to the sugar moiety was found. This is contrary to the anti conformation usually preferred by α-nucleosides. The sugar conformation of both conformers is C2'-endo, and the 5'-hydroxyl groups are in a +sc orientation, probably due to the hydrogen bonds formed by the conformers. The formation of the supramolecular assembly of α-dA is controlled by hydrogen bonding and stacking interactions, which was verified by a Hirshfeld and curvedness surface analysis. Chains of hydrogen-bonded nucleobases extend parallel to the b direction and are linked to equivalent chains by hydrogen bonds involving the sugar moieties to form a sheet. A comparison of the solid-state structures of the anomeric 2'-deoxyadenosines revealed significant differences of their conformational parameters.

Purine DNA Constructs Designed to Expand the Genetic Code: Functionalization, Impact of Ionic Forms, and Molecular Recognition of 7-Deazaxanthine-7-Deazapurine-2,6-diamine Base Pairs and Their Purine Counterparts.

Purine DNA represents an alternative pairing system formed by two purines in the base pair with the recognition elements of Watson-Crick DNA. Base functionalization of 7-deaza-2'-deoxyxanthosine with ethynyl and octadiynyl residues led to clickable side chain derivatives with short and long linker arms. As complementary bases, purine-2,6-diamine or 7-deazapurine-2,6-diamine 2'-deoxyribonucleosides were used. 7-Deaza-7-iodo-2'-deoxyxanthosine served as a starting material for Sonogashira cross-coupling and the p-nitrophenylethyl group for base protection. Phosphoramidite building blocks for DNA synthesis were prepared. Oligonucleotides containing single modifications or runs of three purine base pairs embedded in 12-mer Watson-Crick DNA were synthesized and hybridized with complementary strands with purine- or 7-deazapurine-2,6-diamine located opposite to the xanthine derivatives. The stability of base pairs was evaluated in a comparative study on the basis of DNA melting experiments and Tm values. As 7-deazaxanthine and xanthine nucleosides form anionic forms at neutral pH, duplex stability became pK-dependent, and the system with 7-deazapurine displayed a significant higher stability as that containing xanthine. Alkynyl side chains are well accommodated in the purine-purine helix. Click adducts with pyrene showed that short linker arms destabilize duplexes, whereas long linkers increase duplex stability. CD and fluorescence measurements provide further insights into purine-purine base pairing.

Watson-Crick Base Pairs with Protecting Groups: The 2-Amino Groups of Purine- and 7-Deazapurine-2,6-Diamine as Target Sites for DNA Functionalization by Selective Nucleobase Acylation.

The recognition of Watson-Crick base pairs carrying nucleobase protecting groups is reported as a new approach for DNA functionalization. The 2-amino groups of purine- and 7-deazapurine-2,6-diamine 2'-deoxyribonucleosides served as molecular targets for this functionalization. The 2-amino group withstands oligonucleotide deprotection with ammonia, whereas all other protecting groups are released after chemical DNA synthesis. On this basis, a method was developed for the selective functionalization of oligonucleotides at the 2-position of purines and 7-deazapurines. Melting experiments and Tm values obtained from hybridization studies revealed that duplexes with protected (2-amino-dA) and (2-amino-7-deaza-dA)-dT base pairs are as stable as their nonprotected counterparts. Mismatch discrimination of protected purine- and 7-deazapurine-2,6-diamine DNA was superior to that of nonprotected DNA. Click functionalization in the minor groove of the DNA double helix became accessible via introduction of heptynoyl protecting groups bearing a terminal triple bond. Click reactions with pyrene azide validated the usability. DNA conjugates with bulky pyrene residues at the 2-position (minor groove) developed the same high stability as those functionalized at the 7-position (major groove). This demonstrates the potential of our new method using protected base pairs for DNA functionalization and paves the way for new DNA labeling strategies.

The Base Pairs of Isoguanine and 8-Aza-7-deazaisoguanine with 5-Methylisocytosine as Targets for DNA Functionalization.

The isoguanine-isocytosine base pair (isoG-isoC) represents an important expansion of the DNA coding system. The base pair is more stable than the canonical adenine-thymine or guanine-cytosine pairs. However, nothing is known on the functionalization of the noncanonical isoG-isoC pair at the isoguanine site. In this work, functionalization of the isoG-isoC and the isosteric base pair that contains 8-aza-7-deazaisoguanine in place of isoguanine is studied. Short ethynyl, more space demanding octadiynyl, and dendritic tripropargylamine residues attached to the isoG-isoC base pairs were introduced to oligonucleotides. 12-mer duplexes were formed by hybridization with single base pair modification. The use of the two modified nucleobases gave us the freedom to shift nucleobase substituents within the major groove of double helical DNA. Clickable side chains at position-7 stabilize the base pair, whereas 8-substituents reduce its stability strongly. The weak isoguanine-thymine or 8-aza-7-deazaisoguanine-thymine base pairs show a similar sensitivity to the position of nucleobase functionalization as base pair matches formed with 5-methylisocytosine. CD spectra of all modified duplexes display the typical shape of a B-DNA with only marginal changes. Fluorescent pyrene labeled DNA with long, short, and branched linkers was generated using click chemistry. Pyrene click adducts with long linkers are essential to maintain or to increase base pair stability. Labeled duplexes are more fluorescent than corresponding single strands. For the dendritic linker excimer emission was observed for single strands but only monomer emission in duplexes.

DNA Strand Displacement with Base Pair Stabilizers: Purine-2,6-Diamine and 8-Aza-7-Bromo-7-Deazapurine-2,6-Diamine Oligonucleotides Invade Canonical DNA and New Fluorescent Pyrene Click Sensors Monitor the Reaction.

Purine-2,6-diamine and 8-aza-7-deaza-7-bromopurine-2,6-diamine 2'-deoxyribonucleosides (1 and 2) were implemented in isothermal DNA strand displacement reactions. Nucleoside 1 is a weak stabilizer of dA-dT base pairs, nucleoside 2 evokes strong stabilization. Strand displacement reactions used single-stranded invaders with single and multiple incorporations of stabilizers. Displacement is driven by negative enthalpy changes between target and displaced duplex. Toeholds are not required. Two new environmental sensitive fluorescent pyrene sensors were developed to monitor the progress of displacement reactions. Pyrene was connected to the nucleobase in the invader or to a dendritic linker in the output strand. Both new sensors were constructed by click chemistry; phosphoramidites and oligonucleotides were prepared. Sensors show monomer or excimer emission. Fluorescence intensity changes when the displacement reaction progresses. Our work demonstrates that strand displacement with base pair stabilizers is applicable to DNA, RNA and to related biopolymers with applications in chemical biology, nanotechnology and medicinal diagnostics.

Purine-Purine Base Pairs in Parallel DNA: ß-D Anomeric 8-Aza-7-deazaisoguanine and 7-Functionalized Conjugates Form Stable Base Pairs with α-D 5-Aza-7-deaza-2'-deoxyguanosine.

Anomeric purine-purine DNA represents a new recognition system with strands in parallel orientation. This work investigates the new heterochiral system and the positional impact of nucleobase functionalization. Tracts of anomeric isoguanine/8-aza-7-deazaisoguanine base pairs with 5-aza-7-deazaguanine were embedded in anomeric Watson-Crick DNA. It was discovered that stable purine-purine base pairs are formed in anomeric DNA. Nucleobase functionalization of the novel base pair system with short ethynyl and bulky octadiynyl chains showed that the position of functionalization is critical. From Tm values and thermodynamic data, it is disclosed that side chains at 7-position of the ß-D 8-aza-7-deaza-2'-deoxyisoguanosine-α-D 5-aza-7-deaza-2'-deoxyguanosine purine-purine pair are well accommodated in this new heterochiral DNA, whereas functionalization at 8-position of isoguanine hinders base pair formation. The new DNA base pair system has the potential to be applied in chemical biology, bioconjugation, and nanobiotechnology.

DNA with Purine-Purine Base Pairs: Size and Position of Isoguanine and 8-Aza-7-deazaisoguanine Clickable Residues Control the Molecular Recognition of Guanine and 5-Aza-7-deazaguanine.

Purine-purine base pairs represent an alternative recognition system to the purine-pyrimidine pairing reported by Watson and Crick. Modified purines are the source for non-canonical interactions. To mimic dG-dC interactions, 2'-deoxyisoguanosine (1a) and 8-aza-7-deaza-2'-deoxyisoguanosine (2a) are used to construct base pairs with 2'-deoxyguanosine or 5-aza-7-deaza-2'-deoxyguanosine (dZ). This work reports the chemical functionalization of 1a and its shape mimic 2a in purine-purine base pairs. Clickable rigid ethynyl and more flexible octadiynyl side chain derivatives of 1a and 2a were synthesized. They were protected and converted into phosphoramidites. Building blocks were employed in the synthesis of base-modified 12-mer oligonucleotides with clickable side chains. Pyrene azide was clicked to the linkers. After hybridization, oligonucleotides with purine-purine base pairs were constructed with linkers and pyrene adducts at position-8 of isoguanine and at position-7 of 8-aza-7-deazaisoguanine. Recognition and stability of purine-purine base pairs were explored using Tm values, thermodynamic data, and CD-spectroscopic changes. Side chains at position-7 of 8-aza-7-deazaisoguanine-guanine base pairs or with 5-aza-7-deazaguanine are well accommodated in DNA, whereas functionalization at 8-position of isoguanine makes the DNA unstable. Pyrene click adducts verified the observation. In conclusion, position-7 is the place of choice for purine-purine base pair functionalization.

Anomeric DNA Strand Displacement with α-D Oligonucleotides as Invaders and Ethidium Bromide as Fluorescence Sensor for Duplexes with α/ß-, ß/ß- and α/α-D Configuration.

DNA strand displacement is a technique to exchange one strand of a double stranded DNA by another strand (invader). It is an isothermal, enzyme free method driven by single stranded overhangs (toeholds) and is employed in DNA amplification, mismatch detection and nanotechnology. We discovered that anomeric (α/ß) DNA can be used for heterochiral strand displacement. Homochiral DNA in ß-D configuration was transformed to heterochiral DNA in α-D/ß-D configuration and further to homochiral DNA with both strands in α-D configuration. Single stranded α-D DNA acts as invader. Herein, new anomeric displacement systems with and without toeholds were designed. Due to their resistance against enzymatic degradation, the systems are applicable to living cells. The light-up intercalator ethidium bromide is used as fluorescence sensor to follow the progress of displacement. Anomeric DNA displacement shows benefits over canonical DNA in view of toehold free displacement and simple detection by ethidium bromide.

Anomeric DNA: Functionalization of α-d Anomers of 7-Deaza-2'-deoxyadenosine and 2'-Deoxyuridine with Clickable Side Chains and Click Adducts in Homochiral and Heterochiral Double Helices.

Anomeric base pairs in heterochiral DNA with strands in the α-d and ß-d configurations and homochiral DNA with both strands in α-d configuration were functionalized. The α-d anomers of 2'-deoxyuridine and 7-deaza-2'-deoxyadenosine were synthesized and functionalized with clickable octadiynyl side chains. Nucleosides were protected and converted to phosphoramidites. Solid-phase synthesis furnished 12-mer oligonucleotides, which were hybridized. Pyrene click adducts display fluorescence, a few of them with excimer emission. Tm values and thermodynamic data revealed the following order of duplex stability α/α-d≫ß/ß-d≥α/ß-d. CD spectra disclosed that conformational changes occur during hybridization. Functionalized DNAs were modeled and energy minimized. Clickable side chains and bulky click adducts are well accommodated in the grooves of anomeric DNA. The investigation shows for the first time that anomeric DNAs can be functionalized in the same way as canonical DNA for potential applications in nucleic acid chemistry, chemical biology, and DNA material science.

Isoguanine (2-Hydroxyadenine) and 2-Aminoadenine Nucleosides with an 8-Aza-7-deazapurine Skeleton: Synthesis, Functionalization with Fluorescent and Clickable Side Chains, and Impact of 7-Substituents on Physical Properties.

7-Functionalized 8-aza-7-deaza-2'-deoxyisoguanine and 8-aza-7-deaza-2-aminoadenine 2'-deoxyribonucleosides decorated with fluorescent pyrene or benzofuran sensor tags or clickable side chains with terminal triple bonds were synthesized. 8-Aza-7-deaza-7-iodo-2-amino-2'-deoxyadenosine was used as the central intermediate and was accessible by an improved two-step glycosylation/amination protocol. Functionalization of position-7 was performed either on 8-aza-7-deaza-7-iodo-2-amino-2'-deoxyadenosine followed by selective deamination of the 2-amino group or on 7-iodinated 8-aza-7-deaza-2'-deoxyisoguanosine. Sonogashira and Suzuki-Miyaura cross-coupling reactions were employed for this purpose. Octadiynyl side chains were selected as linkers for click reactions with azido pyrenes. KTaut values calculated from H2O/dioxane mixtures revealed that side chains have a significant influence on the tautomeric equilibrium. Photophysical properties (fluorescence, solvatochromism, and quantum yields) of the new 8-aza-7-deazapurine nucleosides with fluorescent side chains were determined. Remarkably, a strong excimer fluorescence in H2O was observed for pyrene dye conjugates of 8-aza-7-deazaisoguanine and 2-aminoadenine nucleosides with a long linker. In other solvents including methanol, excimer fluorescence was negligible. The 2-aminoadenine and isoguanine nucleosides with the 8-aza-7-deazapurine skeleton expand the class of nucleosides applicable to fluorescence detection with respect to diagnostic and therapeutic purposes.

Anomeric and Enantiomeric 2'-Deoxycytidines: Base Pair Stability in the Absence and Presence of Silver Ions.

Dodecamer duplex DNA containing anomeric (α/ß-d) and enantiomeric (ß-l/ß-d) 2'-deoxycytidine mismatches was studied with respect to base pair stability in the absence and presence of silver ions. Stable duplexes with silver-mediated cytosine-cytosine pairs were formed by all anomeric and enantiomeric combinations. Stability changes were observed depending on the composition of the mismatches. Most strikingly, the new silver-mediated base pair of anomeric α-d-dC with enantiomeric ß-l-dC is superior to the well-noted ß-d/ß-d-dC pair in terms of stability. CD spectra were used to follow global helical changes of DNA structure.

5-Aza-7-deazaguanine-Isoguanine and Guanine-Isoguanine Base Pairs in Watson-Crick DNA: The Impact of Purine Tracts, Clickable Dendritic Side Chains, and Pyrene Adducts.

The Watson-Crick coding system depends on the molecular recognition of complementary purine and pyrimidine bases. Now, the construction of hybrid DNAs with Watson-Crick and purine-purine base pairs decorated with dendritic side chains was performed. Oligonucleotides with single and multiple incorporations of 5-aza-7-deaza-2'-deoxyguanosine, its tripropargylamine derivative, and 2'-deoxyisoguanosine were synthesized. Duplex stability decreased if single modified purine-purine base pairs were inserted, but increased if pyrene residues were introduced by click chemistry. A growing number of consecutive 5-aza-7-deazaguanine-isoguanine base pairs led to strong stepwise duplex stabilization, a phenomenon not observed for the guanine-isoguanine base pair. Spacious residues are well accommodated in the large groove of purine-purine DNA tracts. Changes to the global helical structure monitored by circular dichroism spectroscopy show the impact of functionalization to the global double-helix structure. This study explores new areas of molecular recognition realized by purine base pairs that are complementary in hydrogen bonding, but not in size, relative to canonical pairs.

The 2-Amino Group of 8-Aza-7-deaza-7-bromopurine-2,6-diamine and Purine-2,6-diamine as Stabilizer for the Adenine-Thymine Base Pair in Heterochiral DNA with Strands in Anomeric Configuration.

Stabilization of DNA is beneficial for many applications in the fields of DNA therapeutics, diagnostics, and materials science. Now, this phenomenon is studied on heterochiral DNA, an autonomous DNA recognition system with complementary strands in α-D and ß-D configuration showing parallel strand orientation. The 12-mer heterochiral duplexes were constructed from anomeric (α/ß-D) oligonucleotide single-strands. Purine-2,6-diamine and 8-aza-7-deaza-7-bromopurine-2,6-diamine 2'-deoxyribonucleosides having the capability to form tridentate base pairs with dT were used to strengthen the stability of the dA-dT base pair. Tm data and thermodynamic values obtained from UV melting profiles indicated that the 8-aza-7-deaza 2'-deoxyribonucleoside decorated with a bromo substituent is so far the most efficient stabilizer for heterochiral DNA. Compared with that, the stabilizing effect of the purine-2,6-diamine 2'-deoxyribonucleoside is low. Global changes of helix structures were identified by circular dichroism (CD) spectra during melting.

Heterochiral DNA with Complementary Strands with α-d and ß-d Configurations: Hydrogen-Bonded and Silver-Mediated Base Pairs with Impact of 7-Deazapurines Replacing Purines.

Heterochiral DNA with hydrogen-bonded and silver-mediated base pairs have been constructed using complementary strands with nucleosides with α-d or ß-d configuration. Anomeric phosphoramidites were employed to assemble the oligonucleotides. According to the Tm values and thermodynamic data, the duplex stability of the heterochiral duplexes was similar to that of homochiral DNA, but mismatch discrimination was better in heterochiral DNA. Replacement of purines by 7-deazapurines resulted in stable parallel duplexes, thereby confirming Watson-Crick-type base pairing. When cytosine was facing cytosine, thymine or adenine residues, duplex DNA formed silver-mediated base pairs in the presence of silver ions. Although the CD spectra of single strands with α-d configuration display mirror-like shapes to those with the ß-d configuration, the CD spectra of the hydrogen-bonded duplexes and those with a limited number of silver pairs show a B-type double helix almost indistinguishable from natural DNA. Nonmelting silver ion-DNA complexes with entirely different CD spectra were generated when the number of silver ions was equal to the number of base pairs.

Alkynylated and Dendronized 5-Aza-7-deazaguanine Nucleosides: Cross-Coupling with Tripropargylamine and Linear Alkynes, Click Functionalization, and Fluorescence of Pyrene Adducts†.

The change of the recognition face of 5-aza-7-deazaguanine bridgehead nucleosides with respect to purine nucleosides permits the construction of new purine-purine or purine-pyrimidine base pairs in DNA and RNA. Clickable derivatives of 5-aza-7-deazaguanine were synthesized by introducing ethynyl, 1,7-octadiynyl, and tripropargylamino side chains in the 7-position of the 5-aza-7-deazapurine moiety by Sonogashira cross-coupling. Click reactions were performed with 1-azidomethylpyrene by the copper-catalyzed azide-alkyne cycloaddition. The copper(I)-catalyzed click reaction on the tripropargylamino nucleoside was significantly faster and higher yielding than that for nucleosides carrying linear alkynyl chains. Also, this reaction could be performed with copper(II) as the catalyst. An autocatalyzed cycle was suggested in which the click product acts as a catalyst. Pyrene click adducts of linear alkynylated nucleosides showed pyrene monomer emission, while tripropargylamino adducts showed monomer and excimer fluorescence. The fluorescence intensities of the 5-aza-7-deazaguanine nucleosides were higher than those of their 7-deazaguanine counterparts. The reported clickable nucleosides can be utilized to functionalize or to cross-link monomeric nucleosides or DNA for diagnostic or imaging purposes and other applications in nucleic acid chemistry and biotechnology.

Silver-Mediated Homochiral and Heterochiral α-dC/ß-dC Base Pairs: Synthesis of α-dC through Glycosylation and Impact of Consecutive, Isolated, and Multiple Metal Ion Pairs on DNA Stability.

Isolated and consecutive heterochiral α-dC- base pairs have been incorporated into 12-mer oligonucleotide duplexes at various positions, thereby replacing Watson-Crick pairs. To this end, a new synthesis of the α-d anomer of dC has been developed, and oligonucleotides containing α-dC residues have been synthesized. Silver-mediated base pairs were formed upon the addition of silver ions. Furthermore, we have established that heterochiral α-dC-dC base pairs can approach the stability of a Watson-Crick pair, whereas homochiral dC-dC pairs are significantly less stable. A positional change of the silver-mediated base pairs affects the duplex stability and reveals the nearest-neighbor influence. When the number of silver ions was equivalent to the number of duplex base pairs (12), non-melting silver-rich complexes were formed. Structural changes have been supported by circular dichroism (CD) spectra, which showed that the B-DNA structure was maintained whilst the silver ion concentration was low. At high silver ion concentration, silver-rich complexes displaying different CD spectra were formed.

Nucleobase-Functionalized 5-Aza-7-deazaguanine Ribo- and 2'-Deoxyribonucleosides: Glycosylation, Pd-Assisted Cross-Coupling, and Photophysical Properties.

The special nucleobase recognition pattern of 5-aza-7-deazaguanine nucleosides makes them valuable for construction of homo purine DNA, silver-mediated base pairs, and expansion of the four letter genetic coding system. To widen the utility of 5-aza-7-deazaguanine nucleosides, side chains were introduced at position-7 of the nucleobase. As key compounds, 7-iodo nucleosides were synthesized. Nucleobase anion glycosylation of the iodo derivative of isobutyrylated 5-aza-7-deazaguanine with the bromo sugar of 2,3,5-tri-O-benzoyl-1-O-acetyl-d-ribofuranose gave the pure ß-D anomeric N-9 glycosylation product (67%), whereas one-pot Vorbrüggen conditions gave only 42% of the iodinated nucleoside. The noniodinated nucleoside was formed in 84%. For the synthesis of 2'-deoxyribonucleosides, anion glycosylation performed with Hoffer's 2'-deoxyhalogenose yielded an anomeric mixture (α-D = 33% and ß-D = 39%) of 2'-deoxyribonucleosides. Various side chain derivatives were prepared from nonprotected nucleosides by Pd-assisted Sonogashira or Suzuki-Miyaura cross-coupling. Among the functionalized ribonucleosides and anomeric 2'-deoxyribonucleosides, some of them showed strong fluorescence. Benzofuran and pyrene derivatives display high quantum yields in non-aqueous solvents and solvatochromism. Single-crystal X-ray analysis of 7-iodo-5-aza-7-deaza-2'-deoxyguanosine displayed intermolecular iodo-oxygen interactions in the crystal and channels filled with solvent molecules.