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°.     

Oligonucleotide Analogues with Integrated Bases and Backbone. Part 14

The self‐complementary tetrameric propargyl triols 8, 14, 18 , and 21 were synthesized to investigate the duplex formation of self‐complementary, ethynylene‐linked UUAA, AAUU, UAUA, and AUAU analogues with integrated bases and backbone (ONIBs). The linear synthesis is based on repetitive Sonogashira couplings and C‐desilylations (34–72% yield), starting from the monomeric propargyl alcohols 9 and 15 and the iodinated nucleosides 3, 7, 11 , and 13 . Strongly persistent intramolecular H‐bonds from the propargylic OH groups to N(3) of the adenosine units prevent the gg‐type orientation of the ethynyl groups at C(5′). As such, an orientation is required for the formation of cyclic duplexes, this H‐bond prevents the formation of duplexes connected by all four base pairs. However, the central units of the UAUA and AAUU analogues 18 and 14 associate in CDCl₃/(D₆)DMSO 10 : 1 to form a cyclic duplex characterized by reverse Hoogsteen base pairing. The UUAA tetramer 8 forms a cyclic UU homoduplex, while the AUAU tetramer 21 forms only linear associates. Duplex formation of the O‐silylated UUAA and AAUU tetramers is no longer prevented. The self‐complementary UUAA tetramer 22 forms Watson–Crick‐ and Hoogsteen‐type base‐paired cyclic duplexes more readily than the sequence‐isomeric AAUU tetramer 23 , further illustrating the sequence selectivity of duplex formation.     

Oligonucleotide Analogues with Integrated Bases and Backbone. Part 22

The tritylated and silylated self‐complementary A*[s]U*[s]A*[s]U* and U*[s]A*[s]U*[s]A* tetramers 18 and 24 , linked by thiomethylene groups (abbreviated as [s]) between a nucleobase and C(5′) of the neighbouring nucleoside unit were prepared by a linear synthesis based on S‐alkylation of 5′‐thionucleosides by 6‐(chloromethyl)uridines, 7 or 10 , or 8‐(chloromethyl)adenosines, 12 or 15 . The tetramers 18 and 24 were detritylated to the monoalcohols 19 and 25 , and these were desilylated to the diols 20 and 26 , respectively. The association of the tetramers 18 – 21 and 24 – 26 in CDCl₃ or in CDCl₃/(D₆)DMSO 95 : 5 was investigated by the concentration dependence of the chemical shifts for H? N(3) or H₂N? C(6). The formation of cyclic duplexes connected by four base pairs is favoured by the presence of one and especially of two OH groups. The diol 20 with the AUAU sequence prefers reverse‐Hoogsteen, and diol 26 with the UAUA sequence Watson–Crick base pairing. The structure of the cyclic duplex of 26 in CDCl₃ at 2° was derived by a combination of AMBER* modeling and simulated annealing with NMR‐derived distance and torsion‐angle restraints resulting in a Watson–Crick base‐paired right‐handed antiparallel helix showing large roll angles, especially between the centre base pairs, leading to a bent helix axis.     

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 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.     

Oligonucleotide Analogues with Integrated Bases and Backbone (ONIB). Part 31

The self‐complementary guanosine‐ and cytidine‐derived aminomethylene‐linked C*[n ]G dinucleoside 9 was synthesized by reductive amination of aldehyde 3 with an iminophosphorane derived from azide 7 . Deacylation of 9 gave the isopropylidene‐protected dinucleoside 10 . The sequence‐isomeric G*[n ]C dinucleoside 11 was similarly prepared from aldehyde 8 and azide 5 , and deacylated to 12 . The association of 10 and 12 in CHCl₃ or in CHCl₃/DMSO mixtures, and the structure of the associates were studied by ¹H‐NMR, ESI‐MS, CD, and vapor pressure osmometry (VPO). Broad ¹H‐NMR signals of dinucleosides 10 and 12 evidence an equilibrium between duplexes and quadruplexes (Hoogsteen base pairing between the Watson? Crick base‐paired duplexes). The quadruplex dominates for the G*[n ]C dinucleoside 12 between ?50° and room temperature. The sequence‐isomeric C*[n ]G 10 forms mostly only a cyclic duplex in CDCl₃ and in CDCl₃/(D₆)DMSO 9 : 1.     

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.     

Pentopyranosyl Oligonucleotide Systems,Communication No. 10 , The α‐L‐Lyxopyranosyl‐(4′→2′)‐oligonucleotide System

To determine whether the remarkable chemical properties of the pyranosyl isomer of RNA as an informational Watson‐Crick base‐pairing system are unique to the pentopyranosyl‐(4′→2′)‐oligonucleotide isomer derived from the RNA‐building block D ‐ribose, studies on the entire family of diastereoisomeric pyranosyl‐(4′→2′)‐oligonucleotide systems deriving from D ‐ribose, L ‐lyxose, D ‐xylose, and L ‐arabinose were carried out. The result of these extended studies is unambiguous: not only pyranosyl‐RNA, but all members of the pentopyranosyl‐(4′→2′)‐oligonucleotide family are highly efficient Watson‐Crick base‐pairing systems. Their synthesis and pairing properties will be described in a series of publications in this journal. The present paper describes the α‐L ‐lyxopyranosyl‐(4′→2′)‐system.     

NMR Solution Structure of the Duplex Formed by Self‐Pairing of α‐L‐Arabinopyranosyl‐(4′2′)‐(CGAATTCG)

The solution structure of the duplex formed by α‐L ‐arabinopyranosyl‐(4′→2′)‐(CGAATTCG) was studied by NMR. The resonances of all H‐, P‐ and most C‐atoms could be assigned. Dihedral angles and distance estimates derived from coupling constants and NOESY spectra were used as restraints in a simulated annealing calculation, which generated a well‐defined bundle of structures for the six innermost nucleotide pairs. The essential features of the resulting structures are an antiparallel, Watson Crick‐paired duplex with a strong backbone inclination of ca. −50° and, therefore, predominant interstrand base stacking. The very similar inclination and rise parameters of arabinopyranosyl‐(4′→2′)‐oligonucleotides and p‐RNA explain why these two pentapyranosyl isomers are able to cross‐pair.     

Pentopyranosyl Oligonucleotide Systems,Communication No.12, The β‐D‐Xylopyranosyl‐(4′→2′‐oligonucleotide System

β‐D ‐Xylopyranosyl‐(4′→2′)‐oligonucleotides containing adenine and thymine as nucleobases were synthesized as a part of a systematic study of the pairing properties of pentopyranosyl oligonucleotides. Contrary to earlier expectations based on qualitative conformational criteria, β‐D ‐xylopyranosyl‐(4′→2′)‐oligonucleotides show Watson‐Crick pairing comparable in strength to that shown by pyranosyl‐RNA.     

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 .     

Hydroacridines: Part 23. 1H and 13C NMR spectra of sym‐octahydroacridine,its 9‐(3‐pyridyl) and 9‐(4‐pyridyl) derivatives and the corresponding N(10)‐oxides. An experimental approach to the diamagnetic anisotropy of the pyridine nucleus

The complete ¹H NMR chemical shift assignments of 1,2,3,4,5,6,7,8‐octahydroacridine ( 1 ), 1,2,3,4,5,6,7,8‐octahydro‐9‐(3‐pyridyl)acridine ( 2 ), 1,2,3,4,5,6,7,8‐octahydro‐9‐(4‐pyridyl)acridine ( 3 ) and the corresponding N(10)‐oxides 1a , 2a and 3a , respectively, were achieved on the basis of 400 MHz ¹H NMR spectra and proton–proton decoupling, HMQC and NOEDIFF experiments. The spectral data for the above compounds provided the first experimental evidence of the difference in the anisotropy effect of the two non‐symmetrical moieties of the pyridine nucleus, and allowed us to ascertain that the shielding effect of the moiety defined by the C(2′)—N—C(6′) atoms is weaker than that of the C(3′)—C(4′)—C(5′) moiety. The ¹³C NMR spectra of 1 – 3 and 1a – 3a and the effect of N(10)‐oxidation on the ¹³C NMR chemical shifts are also discussed. The N‐oxidation of 2 and 3 with m‐chloroperbenzoic acid occurred regiospecifically, affording the N(10)‐oxides 2a and 3a free of N(1′)‐oxide isomers. Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis and Triple‐Helix‐Stabilization Properties of Branched Oligonucleotides Carrying 8‐Aminoadenine Moieties

The synthesis of several branched oligonucleotides, i.e., of the parallel hairpins 5 – 8 and the Y‐shaped 9 is described, together with their use in the formation of pyrimidine?pyrimidine?purine triple helices. Special attention was paid to the optimization of the assembly of the second strand from asymmetric branching molecules. The presence of 8‐aminoadenine moieties in the Watson? Crick purine strand and 2′‐O‐methyl‐RNA in the Hoogsteen pyrimidine strand produced strong stabilization of the triplex.     

Oligonucleosides with a Nucleobase‐Including Backbone,Part 3, Synthesis of Acetyleno‐Linked Adenosine Dimers

The adenosine‐derived dimers 14a – d and 15b – d have been prepared by coupling the protected 8‐iodoadenosines 3 and 13 with the C(5′)‐ethynylated adenosine derivatives 5 , 6 , 11 , and 12 (Scheme 4). Similarly, the 5′‐epimeric dimer 16 was prepared by coupling 3 with the alkyne 8 (Scheme 5). The propargylic alcohol 4 was transformed into the N‐benzoylated alkyne 5 and into the amine 6 , while the epimeric alcohol 7 was converted to the epimeric amine 8 and the 5′‐deoxy analogues 11 and 12 (Scheme 3). Cross‐coupling of the iodoadenosine 13 with the alkyne 5 to 14a was optimised; it is influenced by the N‐benzoyl and the Et₃SiO group of the alkyne, but hardly by the N‐benzoyl group of the 8‐iodoadenosine. The alkyne is most reactive when it is O‐silylated, but not N‐benzoylated. Cross‐coupling of the 5′‐deoxyalkynes proceeded more slowly. The dimers 14a – d , 15b – d , and 16 were obtained in good yields (Table 2). Deprotection of 14d and 16 led to 18 and 20 , respectively (Scheme 5). The diols 17 and 19 and the hexols 18 and 20 prefer the syn‐conformation in (D₆)DMSO, completely for unit II and ≥80% for unit I; they exhibit partially persistent intramolecular O(5′)−H⋅⋅⋅N(3) H‐bonds. The persistence increases from 18% (unit I of 19 ), 32% (unit II of 17 and 19 ), 45% (unit I of 17 ), 52% (unit II of 18 and 20 ), and 55% (unit I of 20 ) to 82% (unit I of 18 ).     

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.     

The α‐L‐Threofuranosyl‐(3′→2′)‐oligonucleotide System (‘TNA'): Synthesis and Pairing Properties

Our studies of α‐L ‐Threofuranosyl‐(3′→2′)‐oligonucleotides (‘TNA') are part of a systematic experimental inquiry into the base‐pairing properties of potentially natural nucleic acid alternatives taken from RNA's close structural neighborhood. TNA is an efficient Watson‐Crick base‐pairing system and has the capability of informational cross‐pairing with both RNA and DNA. This property, together with the system's constitutional and (presumed) generational simplicity, warrants special scrutiny of TNA in the context of the search for chemical clues to RNA's origin.     

Solution Structure of a Hexitol Nucleic Acid Duplex with Four Consecutive T⋅T Base Pairs

The structure of the hexitol nucleic acid (HNA) h(GCGCTTTTGCGC) was determined by NMR spectroscopy. This unnatural nucleic acid was developed as a mimic for A‐RNA. In solution, the studied sequence is forming a symmetric double‐stranded structure with four central consecutive T⋅T wobble pairs flanked by G⋅C Watson‐Crick base pairs. The stem regions adopt an A‐type helical structure. Discrete changes in backbone angles are altering the course of the helix axis in the internal loop region. Two H‐bonds are formed in each wobble pair, and base stacking is preserved in the duplex, explaining the stability of the duplex. This structure elucidation provides information about the influence of a (T)₄ fragment on local helix geometries as well as on the nature of the T⋅T mismatch base pairing in a TTTT tract.     

Oligonucleosides with a Nucleobase‐Including Backbone,Part 4, A Convergent Synthesis of Ethynediyl‐Linked Adenosine Tetramers

Deprotection of the tetramer 24 , obtained by coupling the iodinated dimer 18 with the alkyne 23 gave the 8′,5‐ethynediyl‐linked adenosine‐derived tetramer 27 (Scheme 3). As direct iodination of C(5′)‐ethynylated adenosine derivatives failed, we prepared 18 via the 8‐amino derivative 17 that was available by coupling the imine 15 with the iodide 7 ; 15 , in its turn, was obtained from the 8‐chloro derivative 12 via the 4‐methoxybenzylamine 14 (Scheme 2). This method for the introduction of the 8‐iodo substituent was worked out with the N‐benzoyladenosine 1 that was transformed into the azide 2 by lithiation and treatment with tosyl azide (Scheme 1). Reduction of 2 led to the amine 3 that was transformed into 7 . 1,3‐Dipolar cycloaddition of 3 and (trimethylsilyl)acetylene gave 6 . The 8‐substituted derivatives 4a – d were prepared similarly to 2 , but could not be transformed into 7 . The known chloride 8 was transformed into the iodide 11 via the amines 9 and 10 . The amines 3 , 10 , and 16 form more or less completely persistent intramolecular C(8)N−H⋅⋅⋅O(5′) H‐bonds, while the dimeric amine 17 forms a ca. 50% persistent H‐bond. There is no UV evidence for a base‐base interaction in the protected and deprotected dimers and tetramers.     

Hoogsteen-Duplex DNA: Synthesis and base pairing of oligonucleotides containing 1-deaza-2′-deoxyadenosine

Oligodeoxyribonucleotides containing 1-deaza-2′-deoxyadenosine ( = 7-amino-3-(2-deoxy-β-D -erythro-pentofuranosyl)-3H-imidazo[4, 5-b]pyridine; 1b ) form Hoogsteen duplexes. Watson-Crick base pairs cannot be built up due to the absence of N(1). For these studies, oligonucleotide building blocks – the phosphonate 3a and the phosphoramidite 3b – were prepared from 1b via 4a and 5 , as well as the Fractosil-linked 6b , and used in solid-phase synthesis. The applicability of various N-protecting groups (see 4a – c ) was also studied. The Hoogsteen duplex d[(c¹A)₂₀] · d(T₂₀) ( 11 · 13 ; T_m 15°) is less stable than d(A₂₀) · d(T₂₀) ( 12 · 13 ; T_m 60°). The block oligomers d([c¹A)₁₀–;T₁₀] ( 14 ) and d[T₁₀–(c¹A)₁₀] ( 15 ) containing purine and pyrimidine bases in the same strand are also able to form duplexes with each other. The chain polarity was found to be parallel.     

Stability of Hoogsteen‐Type Triplexes – Electrostatic Attraction between Duplex Backbone and Triplex‐Forming Oligonucleotide (TFO) Using an Intercalating Conjugate

Syntheses are described for two novel twisted intercalating nucleic acid (TINA) monomers where the intercalator comprises a benzene ring linked to a naphthalimide moiety via an ethynediyl bridge. The intercalators Y and Z have a 2‐(dimethylamino)ethyl and a methyl residue on the naphthalimide moiety, respectively. When used as triplex‐forming oligonucleotides (TFOs), the novel naphthalimide TINAs show extraordinary high thermal stability in Hoogsteen‐type triplexes and duplexes with high discrimination of mismatch strands. DNA Strands containing the intercalator Y show higher thermal triplex stability than DNA strands containing the intercalator Z . This observation can be explained by the ionic interaction of the protonated dimethylamino group under physiological conditions, targeting the negatively charged phosphate backbone of the duplex. This interaction leads to an extra binding mode between the TFO and the duplex, in agreement with molecular‐modeling studies. We believe that this is the first example of an intercalator linking the TFO to the phosphate backbone of the duplex by an ionic interaction, which is a promising tool to achieve a higher triplex stability.