Nucleosides and Oligonucleotides with Diynyl Side Chains: Base Pairing and Functionalization of 2′‐Deoxyuridine Derivatives by the Copper(I)‐Catalyzed Alkyne?Azide ‘Click’ Cycloaddition

Oligonucleotides containing the 5‐substituted 2′‐deoxyuridines 1b or 1d bearing side chains with terminal C?C bonds are described, and their duplex stability is compared with oligonucleotides containing the 5‐alkynyl compounds 1a or 1c with only one nonterminal C?C bond in the side chain. For this, 5‐iodo‐2′‐deoxyuridine ( 3 ) and diynes or alkynes were employed as starting materials in the Sonogashira cross‐coupling reaction (Scheme 1). Phosphoramidites 2b – d were prepared (Scheme 3) and used as building blocks in solid‐phase synthesis. T_m Measurements demonstrated that DNA duplexes containing the octa‐1,7‐diynyl side chain or a diprop‐2‐ynyl ether residue, i.e., containing 1b or 1d , are more stable than those containing only one triple bond, i.e., 1a or 1c (Table 3). The diyne‐modified nucleosides were employed in further functionalization reactions by using the protocol of the Cu^I‐catalyzed Huisgen–Meldal–Sharpless [2+3] cycloaddition (‘click chemistry’) (Scheme 2). An aliphatic azide, i. e., 3′‐azido‐3′‐deoxythymidine (AZT; 4 ), as well as the aromatic azido compound 5 were linked to the terminal alkyne group resulting in 1H‐1,2,3‐triazole‐modified derivatives 6 and 7 , respectively (Scheme 2), of which 6 forms a stable duplex DNA (Table 3). The Husigen–Meldal–Sharpless cycloaddition was also performed with oligonucleotides (Schemes 4 and 5).     

Oligonucleotide Analogues with Integrated Bases and Backbone. Part 17

The formation of cyclic duplexes (pairing) of known oxymethylene‐linked self‐complementary U*[o]A⁽*⁾ dinucleosides contrasts with the absence of pairing of the ethylene‐linked U*[c_a]A⁽*⁾ analogues. The origin of this difference, and the expected association of U*[x]A⁽*⁾ and A*[x]U⁽*⁾ dinucleosides with x=CH₂, O, or S was analysed. According to this analysis, pairing occurs via constitutionally isomeric Watson–Crick, reverse Watson–Crick, Hoogsteen, or reverse Hoogsteen H‐bonded linear duplexes. Each one of them may give rise to three diastereoisomeric cyclic duplexes, and each one of them can adopt three main conformations. The relative stability of all conformers with x=CH₂, O, or S were analysed. U*[x]A⁽*⁾ dinucleosides with x=CH₂ do not form stable cyclic duplexes, dinucleosides with x=O may form cyclic duplexes with a gg‐conformation about the C(4′)? C(5′) bond, and dinucleosides with x=S may form cyclic duplexes with a gt‐conformation about this bond. The temperature dependence of the chemical shift of H? N(3) of the self‐complementary, oxymethylene‐linked U*[o]A⁽*⁾ dinucleosides 1 – 6 in CDCl₃ in the concentration range of 0.4–50 mM evidences equilibria between the monoplex, mainly linear duplexes, and higher associates for 3 , between the monoplex and cyclic duplexes for 6 , and between the monoplex, linear, and cyclic duplexes as well as higher associates for 1, 2, 4 , and 5 . The self‐complementary, thiomethylene‐linked U*[s]A⁽*⁾ dinucleosides 27 – 32 and the sequence isomeric A*[s]U⁽*⁾ analogues 33 – 38 were prepared by S‐alkylation of the 6‐(mesyloxymethyl)uridine 12 and the 8‐(bromomethyl)adenosine 22 . The required thiolates were prepared in situ from the C(5′)‐acetylthio derivatives 9, 15, 19 , and 25 . The association in CHCl₃ of the thiomethylene‐linked dinucleoside analogues was studied by ¹H‐NMR and CD spectroscopy, and by vapour‐pressure osmometric determination of the apparent molecular mass. The U*[s]A⁽*⁾ alcohols 28, 30 , and 31 form cyclic duplexes connected by Watson–Crick H‐bonds, while the fully protected dimers 27 and 29 form mainly linear duplexes and higher associates. The diol 32 forms mainly cyclic duplexes in solution and corrugated ribbons in the solid state. The nucleobases of crystalline 32 form reverse Hoogsteen H‐bonds, and the resulting ribbons are cross‐linked by H‐bonds between HOCH₂? C(8/I) and N(3/I). Among the A*[s]U⁽*⁾ dimers, only the C(8/I)‐hydroxymethylated 37 forms (mainly) a cyclic duplex, characterized by reverse Hoogsteen base pairing. The dimers 34 – 36 form mainly linear duplexes and higher associates. Dimers 34 and particularly 38 gelate CHCl₃. Temperature‐dependent CD spectra of 28, 30, 31 , and 37 evidence π‐stacking in the cyclic duplexes. Base stacking in the particularly strongly associating diol 32 in CHCl₃ solution is evidenced by a melting temperature of ca. 2°.     

Oligonucleotides Containing 7‐Deaza‐2′‐deoxyinosine as Universal Nucleoside: Synthesis of 7‐Halogenated and 7‐Alkynylated Derivatives,Ambiguous Base Pairing,and Dye Functionalization by the Alkyne–Azide ‘Click’ Reaction

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 C?C 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.     

Oligodeoxynucleotides Containing 2′‐Deoxy‐1‐methyladenosine and Dimroth Rearrangement

2′‐Deoxy‐1‐methyladenosine was incorporated into synthetic oligonucleotides by phosphoramidite chemistry. Chloroacetyl protecting group and controlled anhydrous deprotection conditions were used to avoid Dimroth rearrangement. Hybridization studies of intramolecular duplexes showed that introduction of a modified residue into the loop region of the oligonucleotide hairpin increases the melting temperature. It was shown that modified oligonucleotides may be easily transformed into oligonucleotides containing 2′‐deoxy‐N⁶‐methyladenosine.     

Synthesis and Pairing Properties of 3′‐Deoxyribopyranose (4′→2′)‐Oligonucleotides (‘p‐DNA')

The preparation and the pairing properties of the new 3′‐deoxyribopyranose (4′→2′)‐oligonucleotide (=p‐DNA) pairing system, based on 3′‐deoxy‐β‐D ‐ribopyranose nucleosides is presented. D ‐Xylose was efficiently converted to the prefunctionalized 3‐deoxyribopyranose derivative 4‐O‐[(tert‐butyl)dimethylsilyl]‐3‐deoxy‐D ‐ribopyranose 1,2‐diacetate 8 (obtained as a 4 : 1 mixture of α‐ and β‐D ‐anomers; Scheme 1). From this sugar building block, the corresponding, appropriately protected thymine, guanine, 5‐methylcytosine, and purine‐2,6‐diamine nucleoside phosphoramidites 29 – 32 were prepared in a minimal number of steps (Schemes 2–4). These building blocks were assembled on a DNA synthesizer, and the corresponding p‐DNA oligonucleotides were obtained in good yields after a one‐step deprotection under standard conditions, followed by HPLC purification (Scheme 5 and Table 1). Qualitatively, p‐DNA shows the same pairing behavior as p‐RNA, forming antiparallel, exclusively Watson‐Crick‐paired duplexes that are much stronger than corresponding DNA duplexes. Duplex stabilities within the three related (i.e., based on ribopyranose nucleosides) oligonucleotide systems p‐RNA, p‐DNA, and 3′‐O‐Me‐p‐RNA were compared with each other (Table 2). Intrinsically, p‐RNA forms the strongest duplexes, followed by p‐DNA, and 3′‐O‐Me‐p‐RNA. However, by introducing the nucleobases purine‐2,6‐diamine (D) and 5‐methylcytosine (M) instead of adenine and cytosine, a substantial increase in stability of corresponding p‐DNA duplexes was observed.     

Oligonucleotides Containing Disaccharide Nucleosides

Disaccharide nucleosides with 2′‐O‐(D ‐arabinofuranosyl), 2′‐O‐(L ‐arabinofuranosyl), 2′‐O‐(D ‐ribopyranosyl), 2′‐O‐(D ‐erythrofuranosyl), and 2′‐O‐(5‐azido‐5‐deoxy‐D ‐ribofuranosyl) substituents were synthesized. These modified nucleosides were incorporated into oligonucleotides (see Table). Single substitution resulted in a ΔT_m of +0.5 to −1.4° for DNA/RNA and a ΔT_m of −0.8 to −4.7° for DNA/DNA duplexes. These disaccharide nucleosides can be well accommodated in RNA/DNA duplexes, and the presence of a NH₂−C(5″) group has a beneficial effect on duplex stability.     

3′‐Deoxyribopyranose (4′→2′)‐Oligonucleotide Duplexes (=p‐DNA Duplexes) as Substitutes of RNA Hairpin Structures

By automated synthesis, we prepared hybrid oligonucleotides consisting of covalently linked RNA and p‐DNA sequences (p‐DNA=3′‐deoxyribopyranose (4′→2′)‐oligonucleotides) (see Table 1). The pairing properties of corresponding hybrid duplexes, formed from fully complementary single strands were investigated. An uninterrupted π‐π‐stacking at the p‐DNA/RNA interface and cooperative pairing between the two systems was achieved by connecting them via a 4′‐p‐DNA‐2′→5′‐RNA‐3′ and 5′‐RNA‐2′→4′‐p‐DNA‐2′ phosphodiester linkage, respectively (see Fig. 4). The RNA 2′‐phosphoramidites 9 – 12 , required for the formation of the RNA‐2′→4′‐p‐DNA phosphodiester linkage were synthesized from the corresponding, 3′‐O‐tom‐protected ribonucleosides (tom=[(triisopropylsilyl)oxy]methyl; Scheme 1). Analogues of the flavin mononucleotide (=FMN) binding aptamer 22 and the hammerhead ribozyme 25 were prepared. Each of these analogues consisted of two p‐DNA/RNA hybrid single strands with complementary p‐DNA sequences, designed to substitute stem/loop and stem motifs within the parent compounds. By comparative binding and cleavage studies, it was found that mixing of the two complementary p‐DNA/RNA hybrid sequences resulted in the formation of the fully functional analogues 23 ⋅ 24 and 27 ⋅ 28 of the FMN‐binding aptamer and of the hammerhead ribozyme, respectively.     

Synthesis and Properties of Isobicyclo‐DNA

We present the synthesis of the isobicyclo‐DNA building blocks with the nucleobases A, C, G and T, as well as biophysical and biological properties of oligonucleotides derived thereof. The synthesis of the sugar part was achieved in 5 steps starting from a known intermediate of the tricyclo‐DNA synthesis. Dodecamers containing single isobicyclo‐thymidine incorporations, fully modified A‐ and T‐containing sequences, and fully modified oligonucleotides containing all four bases were synthesized and characterized. Isobicyclo‐DNA forms stable duplexes with natural nucleic acids with a pronounced preference for DNA over RNA as complements. The most stable duplexes, however, arise by self‐pairing. Isobicyclo‐DNA forms preferentially B‐DNA‐like duplexes with DNA and A‐like duplexes with complementary RNA as determined by circular dichroism (CD) spectroscopy. Self‐paired duplexes show a yet unknown structure, as judged from CD spectroscopy. Biochemical tests revealed that isobicyclo‐DNA is stable in fetal bovine serum and does not elicit RNaseH activity.     

2′‐Ethynyl‐DNA: Synthesis and Pairing Properties

2‐Ethynyl‐DNA was developed as a potential DNA‐selective oligonucleotide analog. The synthesis of 2′‐arabino‐ethynyl‐modified nucleosides was achieved starting from properly protected 2′‐ketonucleosides by addition of lithium (trimethylsilyl)acetylide followed by reduction of the tertiary alcohol. After a series of protecting‐group manipulations, phosphoramidite building blocks suitable for solid‐phase synthesis were obtained. The synthesis of oligonucleotides from these building blocks was successful when a fast deprotection scheme was used. The pairing properties of 2′‐arabino‐ethynyl‐modified oligonucleotides can be summarized as follows: 1) The 2′‐arabino‐ethynyl modification of pyrimidine nucleosides leads to a strong destabilization in duplexes with DNA as well as with RNA. The likely reason is that the ethynyl group sterically influences the torsional preferences around the glycosidic bond leading to a conformation not suitable for duplex formation. 2) If the modification is introduced in purine nucleosides, no such influence is observed. The pairing properties are not or only slightly changed, and, in some cases (deoxyadenosine homo‐polymers), the desired stabilization of the pairing with a DNA complementary strand and destabilization with an RNA complement is observed. 3) In oligonucleotides of alternating deoxycytidine‐deoxyguanosine sequence, the incorporation of 2′‐arabino‐ethynyl deoxyguanosine surprisingly leads to the formation of a left‐handed double helix, irrespective of salt concentration. The rationalization for this behavior is that the ethynyl group locks such duplexes in a left‐handed conformation through steric blockade.     

Synthesis,Characterization, and Repair of a Flexible O6‐2′‐Deoxyguanosine‐alkylene‐O6‐2′‐deoxyguanosine Intrastrand Cross‐Link

Oligonucleotides tethered by an alkylene linkage between the O⁶‐atoms of two consecutive 2′‐deoxyguanosines, which lack a phosphodiester linkage between these residues, have been synthesized as a model system of intrastrand cross‐linked (IaCL) DNA. UV thermal denaturation studies of duplexes formed between these butylene‐ and heptylene‐linked oligonucleotides with their complementary DNA sequences revealed about 20 °C reduction in stability relative to the unmodified duplex. Circular dichroism spectra of the model IaCL duplexes displayed a signature characteristic of B‐form DNA, suggesting minimal global perturbations are induced by the lesion. The model IaCL containing duplexes were investigated as substrates of O⁶‐alkylguanine DNA alkyltransferase (AGT) proteins from human and E. coli (Ada‐C and OGT). Human AGT was found to repair both model IaCL duplexes with greater efficiency towards the heptylene versus butylene analog adding to our knowledge of substrates this protein can repair.     

Hydrogen Bonding or Stacking Interactions in Differentiating Duplex Stability in Oligonucleotides Containing Synthetic Nucleoside Probes for Alkylated DNA

Oligonucleotides that hybridize to modified DNA are useful chemical tools to probe the noncovalent interactions that stabilize DNA duplexes. In an effort to better understand the interactions that influence the specificity of hybridization probes for O⁶‐alkylguanine lesions, we examined a series of synthetic nucleoside analogues (BIM, Benzi, and Peri) with respect to their ability to stabilize duplex DNA comprised of native or damaged DNA oligonucleotides. The base‐modified nucleoside analogues contained systematically varied hydrogen‐bonding and π‐stacking properties. The nucleoside probes were incorporated into DNA and paired opposite canonical bases (A, T, C, or G), O⁶‐methylguanine (O⁶‐MeG), O⁶‐benzylguanine (O⁶‐BnG), or a stable abasic site analogue (tetrahydrofuran, THF). On the basis of the free energy of duplex formation, the highest degree of stabilization was observed when Peri was paired opposite O⁶‐MeG. The thermodynamic data suggest that the smaller probes stabilize DNA duplexes more through hydrogen bonding, whereas the larger probes, with a greater capacity to π stack, contribute to duplex stabilization more on the basis of base stacking. These results demonstrate that increased helix stability could be achieved when BIM, Benzi, or Peri were paired opposite damage‐containing DNA rather than unmodified DNA (that is, O⁶‐MeG rather than G). This knowledge is expected to be useful in the design and development of nucleoside analogues for uses in DNA‐based technologies.     

Oligonucleotide Analogues with Integrated Bases and Backbone. Part 15

The self‐complementary (Z)‐configured U*[c_e]A⁽*⁾ dinucleotide analogues 6, 8, 10, 12, 14 , and 16 , and the A*[c_e]U⁽*⁾ dimers 19, 21, 23, 25, 27 , and 29 were prepared by partial hydrogenation of the corresponding ethynylene linked dimers. Photolysis of 14 led to the (E)‐alkene 17 . These dinucleotide analogues associate in CDCl₃ solution, as evidenced by NMR and CD spectroscopy. The thermodynamic parameters of the duplexation were determined by van't Hoff analysis. The (Z)‐configured U*[c_e]A⁽*⁾ dimers 14 and 16 form cyclic duplexes connected by Watson–Crick H‐bonds, the (E)‐configured U*[c_e]A dimer 17 forms linear duplexes, and the U*[c_e]A⁽*⁾ allyl alcohols 6, 8, 10 , and 12 form mixtures of linear and cyclic duplexes. The C(6/I)‐unsubstituted A*[c_e]U allyl alcohols 19 and 23 form linear duplexes, whereas the C(6/I)‐substituted A*[c_e]U* allyl alcohols 21 and 25 , and the C(5′/I)‐deoxy A*[c_e]U⁽*⁾ dimers 27 and 29 also form minor amounts of cyclic duplexes. The influence of intra‐ and intermolecular H‐bonding of the allyl alcohols and the influence of the base sequence upon the formation of cyclic duplexes are discussed.     

Oligonucleotide Analogues with Integrated Bases and Backbones. Part 24

Inspection of Maruzen models and force‐field calculations suggest that oligonucleotide analogues integrating backbone and bases (ONIBs) with an aminomethylene linker form similar cyclic duplexes as the analogous oxymethylene linked dinucleosides. The self‐complementary adenosine‐ and uridine‐derived aminomethylene‐linked A*[n ]U dinucleosides 15 – 17 were prepared by an aza‐Wittig reaction of the aldehyde 10 with an iminophosphorane derived from azide 6 . The sequence‐isomeric U*[n ]A dinucleosides 18 – 20 were similarly prepared from aldehyde 3 and azide 12 . The N‐ethylamine 5 , the acetamides 7 and 14 , and the amine 13 were prepared as references for the conformational analysis of the dinucleosides. In contradistinction to the results of calculations, the N‐ethylamine 5 exists as intramolecularly H‐bonded hydroxyimino tautomer. The association in CDCl₃ of these dinucleosides was studied by ¹H‐NMR and CD spectroscopy. The A*[n ]U dinucleosides 16 and 17 associate more strongly than the sequence isomers 19 and 20 ; the cyclic duplexes of 16 form preferentially Watson–Crick‐type base pairs, while 17, 19 , and 20 show both Watson–Crick‐ and Hoogsteen‐type base pairing. The cyclic duplexes of the aminomethylene‐linked dinucleosides prefer a gg‐orientation of the linker. No evidence was found for an intramolecular H‐bond of the aminomethylene group. The CD spectra of 16 and 17 show a strong, those of 19 a weak, and those of 20 almost no temperature dependence.     

Synthesis and Properties of Nonpolar DNA (Arylalkyl)phosphonates

The eight (arylalkyl)‐modified phosphoramidites (=(arylalkyl)phosphonamidites) 1 – 8 (Fig. 2) were synthesized (Schemes 1–3) and incorporated at different positions into 2′‐deoxyoligonucleotides. The [P(R)]‐ and [P(S)]‐diastereoisomers of the hexanucleotides 32 – 39 (Table 1) and of the dodecanucleotides 41 – 45 (Table 2) obtained were separated by means of reversed‐phase HPLC. UV, CD, and fluorescence spectroscopy were used to investigate the thermal stability (T_m) and the structural changes of their DNA duplexes with 5′‐d(CGCGCG)‐3′ and 5′‐d(ATGATTGACCTG)‐3′, respectively. The T_m values significantly depend on the place of modification (Table 2). A dangling‐end effect is observed when the [3‐(anthracen‐9‐yl)propyl]‐modified 8 is attached at the 5′‐terminus (see duplex with 45c ). In the case of the incorporation of aromatic moieties tethered via a methylene linker to the P‐atom (benzyl‐ and (naphthalen‐1‐ylmethyl)‐modified 1 and 6 , resp.), the duplexes with the [P(R)]‐oligonucleotides are more stable than those with the [P(S)]‐isomers, whereas in the case of longer alkyl chains at the P‐atom (see 2 – 5 ), the T_m values show the reverse tendency. The observed T_m differences are assigned to changes in base stacking (Figs. 6 and 7).     

Oligonucleotide Analogues with Integrated Bases and Backbones. Part 26

The self‐complementary aminomethylene‐linked A*[n] U* dinucleosides 23 – 26 were prepared by reductive coupling of aldehyde 10 and azide 8 . The U*[n] A* sequence isomers 19 – 21 were similarly prepared from aldehyde 14 and azide 3 . The substituents at C(6/I) of 23 – 26 and at C(8/I) of 19 – 21 strongly favour the syn‐conformation. The A*[n] U* dinucleoside 23 associates more strongly than the sequence‐isomeric U*[n] A* dinucleoside 19 . The A*[n] U* dinucleosides 23 and 24 associate more strongly than the analogues devoid of the substituent at C(6/I), while the U*[n] A* dinucleoside 19 associates less strongly than the analogue devoid of the substituent at C(8/I). While 23 and 24 form cyclic duplexes mostly by Watson–Crick‐type base pairing, 25 only forms linear associates. The U*[n] A* dinucleoside 19 forms mostly linear duplexes and higher associates, and 21 forms cyclic duplexes showing both Watson–Crick‐ and Hoogsteen‐type base pairing. The cyclic duplexes of the aminomethylene‐linked dinucleosides show both the gg‐ and gt‐orientation of the linker, with the gg‐orientation being preferred.     

Oligonucleotide Analogues with Integrated Bases and Backbone. Part 13

The self‐complementary UA and AU dinucleotide analogues 41 – 45, 47, 48 , and 51 – 60 were prepared by Sonogashira coupling of 6‐iodouridines with C(5′)‐ethynylated adenosines and of 8‐iodoadenosines with C(5′)‐ethynylated uridines. The dinucleotide analogues associate in CDCl₃ solution. The C(6/I)‐unsubstituted AU dimers 51 and 54 prefer an anti‐oriented uracilyl group and form stretched linear duplexes. The UA propargyl alcohols 41 and 43 – 45 possess a persistent intramolecular O(5′/I)? H???N(3/I) H‐bond and, thus, a syn‐oriented adeninyl and a gt‐ or tg‐oriented ethynyl moiety; they form corrugated linear duplexes. All other dimers form cyclic duplexes characterized by syn‐oriented nucleobases. The preferred orientation of the ethynyl moiety (the C(4′),C(5′) torsion angle) defines a conformation between gg and one where the ethynyl group eclipses O(4′/I). The UA dimers 42, 47 , and 48 form Watson–Crick H‐bonds, the AU dimers 56 and 58 – 60 H‐bonds of the Watson–Crick‐type, the AU dimers 53 and 55 reverse‐Hoogsteen, and 57 Hoogsteen H‐bonds. The pairing mode depends on the substituent of C(5′/I) (H, OSiⁱPr₃; OH) and on the H‐bonds of HO? C(5′/I) in the AU dimers. Association constants were derived from the concentration‐dependent chemical shift for HN(3) of the uracilyl moiety; they vary from 45–104 M ^?1 for linear duplexes to 197–2307 M ^?1 for cyclic duplexes. The thermodynamic parameters were determined by van't Hoff analysis of the temperature‐dependence of the (concentration‐dependent) chemical shift for HN(3) of the uracilyl moiety. Neglecting stacking energies, one finds an average energy of 3.5–4.0 kcal/mol per intermolecular H‐bond. Base stacking is evidenced by the temperature‐dependent CD spectra. The crystal structure of 54 shows two antiparallel chains of dimers connected by Watson‐Crick H‐bonds. The chains are bridged by a strong H‐bond between the propargylic OH and O?C(4) and by weak reverse A ? A Hoogsteen H‐bonds.     

Impact of Single Basepair Mismatches on Electron‐Transfer Processes at Fc‐PNA⋅DNA Modified Gold Surfaces

Gold‐surface grafted peptide nucleic acid (PNA) strands, which carry a redox‐active ferrocene tag, present unique tools to electrochemically investigate their mechanical bending elasticity based on the kinetics of electron‐transfer (ET) processes. A comparative study of the mechanical bending properties and the thermodynamic stability of a series of 12‐mer Fc‐PNA?DNA duplexes was carried out. A single basepair mismatch was integrated at all possible strand positions to provide nanoscopic insights into the physicochemical changes provoked by the presence of a single basepair mismatch with regard to its position within the strand. The ET processes at single mismatch Fc‐PNA?DNA modified surfaces were found to proceed with increasing diffusion limitation and decreasing standard ET rate constants k⁰ when the single basepair mismatch was dislocated along the strand towards its free‐dangling Fc‐modified end. The observed ET characteristics are considered to be due to a punctual increase in the strand elasticity at the mismatch position. The kinetic mismatch discrimination with respect to the fully‐complementary duplex presents a basis for an electrochemical DNA sensing strategy based on the Fc‐PNA?DNA bending dynamics for loosely packed monolayers. In a general sense, the strand elasticity presents a further physicochemical property which is affected by a single basepair mismatch which may possibly be used as a basis for future DNA sensing concepts for the specific detection of single basepair mismatches.     

Oligodeoxynucleotide Duplexes Containing (5′S)‐5′‐C‐Alkyl‐Modified 2′‐Deoxynucleosides: Can an Alkyl Zipper across the DNA Minor‐Groove Enhance Duplex Stability?

A series of oligonucleotides containing (5′S)‐5′‐C‐butyl‐ and (5′S)‐5′‐C‐isopentyl‐substituted 2′‐deoxyribonucleosides were designed, prepared, and characterized with the intention to explore alkyl‐zipper formation between opposing alkyl chains across the minor groove of oligonucleotide duplexes as a means to modulate DNA‐duplex stability. From four possible arrangements of the alkyl groups that differ in the density of packing of the alkyl chains across the minor groove, three (duplex types I – III , Fig. 2) could experimentally be realized and their duplex‐forming properties analyzed by UV‐melting curves, CD spectroscopy, and isothermal titration calorimetry (ITC), as well as by molecular modeling. The results show that all arrangements of alkyl residues within the minor groove of DNA are thermally destabilizing by 1.5–3°/modification in T_m. We found that, within the proposed duplexes with more loosely packed alkyl groups (type‐ III duplexes), accommodation of alkyl residues without extended distorsion of the helical parameters of B‐DNA is possible but does not lead to higher thermodynamic stability. The more densely packed and more unevenly distributed arrangement (type‐ II duplexes) seems to suffer from ecliptic positioning of opposite alkyl groups, which might account for a systematic negative contribution to stability due to steric interactions. The decreased stability in the type‐ III duplexes described here may be due either to missing hydrophobic interactions of the alkyl groups (not bulky enough to make close contacts), or to an overcompensation of favorable alkyl‐zipper formation presumably by loss of structured H₂O in the minor groove.     

Nucleotides,Part LXIX,Synthesis of Phosphoramidite Building Blocks of Isoxanthopterin N8‐(2′‐Deoxy‐β‐D‐ribonucleosides): New Fluorescence Markers for Oligonucleotide Synthesis

The chemical synthesis of isoxanthopterin and 6‐phenylisoxanthopterin N⁸‐(2′‐deoxy‐β‐D ‐ribofuranosyl nucleosides) is described as well as their conversion into suitably protected 3′‐phosphoramidite building blocks to be used as marker molecules for DNA synthesis. Applying the npe/npeoc (=2‐(4‐nitrophenyl)ethyl/[2‐(4‐nitrophenyl)ethoxy]carbonyl) strategy, we used the new building blocks in the preparation of oligonucleotides by an automated solid‐support approach. The hybridization properties of a series of labelled oligomers were studied by UV‐melting techniques. It was found that the newly synthesized markers only slightly interfered with the abilities of the labelled oligomers to form stable duplexes with complementary oligonucleotides.     

Oligonucleotide Analogues with Integrated Bases and Backbone

The thiomethylene‐linked U*[s]U⁽*⁾ dimers 9 – 14 were synthesized by substitution of the 6‐[(mesyloxy)methyl]uridine 6 by the thiolate derived from the uridine‐5′‐thioacetates 7 and 8 followed by O‐deprotection. Similarly, the thiomethylene‐linked A*[s]A⁽*⁾ dimers 9 – 14 were obtained from the 8‐(bromomethyl)adenosine 15 and the adenosine‐5′‐thioacetates 16 and 17 . The concentration dependence of both H? N(3) of the U*[s]U⁽*⁾ dimers 9 – 14 evidences the formation of linear and cyclic duplexes, and of linear higher associates, C(8 or 6)CH₂OH and/or C(5′/II)OH groups favouring the formation of cyclic duplexes. The concentration dependence of the chemical shift for both H₂N? C(6) of the A*[s]A⁽*⁾ dimers 18 – 23 evidences the formation of mainly linear associates. The heteroassociation of U*[s]U⁽*⁾ to A*[s]A⁽*⁾ dimers is stronger than the homoassociation of U*[s]U⁽*⁾ dimers, as evidenced by diluting equimolar mixtures of 11 / 20 and 13 / 22 . A 1 : 1 stoichiometry of the heteroassociation is evidenced by a Job's plot for 11 / 20 , and by mole ratio plots for 9 / 18, 10 / 19, 12 / 21, 13 / 22 , and 14 / 23 .