Major‐Groove‐Halogenated DNA: The Effects of Bromo and Iodo Substituents Replacing H−C(7) of 8‐Aza‐7‐deazapurine‐2,6‐diamine or H−C(5) of Uracil Residues

Oligonucleotides containing halogenated `purine' and pyrimidine bases were synthesized. Bromo and iodo substituents were introduced at the 7‐position of 8‐aza‐7‐deazapurine‐2,6‐diamine (see 2b , c ) or at the 5‐position of uracil residues (see 3b , c ). Phosphoramidites were synthesized after protection of 2b with the isobutyryl residue and of 2c with the benzoyl group. Duplexes containing the residues 2b or 2c gave always higher T<sub>m</sub> values than those of the nonmodified counterparts containing 2′‐deoxyadenosine, the purine‐2,6‐diamine 2′‐deoxyribonucleoside ( 1 ), or 2a at the same positions. Six 2b residues replacing dA in the duplex 5′‐d(TAGGTCAATACT)‐3′ ( 11 )⋅5′‐d(AGTATTGACCTA)‐3′ ( 12 ) raised the T<sub>m</sub> value from 48 to 75° (4.5° per modification (Table 3)). Contrary to this, incorporation of the 5‐halogenated 2′‐deoxyuridines 3b or 3c into oligonucleotide duplexes showed very little influence on the thermal stability, regardless of which `purine' nucleoside was located opposite to them (Tables 4 and 5). The positive effects on the thermal stability of duplexes observed in DNA were also found in DNA⋅RNA hybrids or in DNA with parallel chain orientation (Tables 8 and 9, resp.).     

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.     

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.     

O6‐Alkylguanine DNA Alkyltransferase Repair Activity Towards Intrastrand Cross‐Linked DNA is Influenced by the Internucleotide Linkage

Oligonucleotides containing an alkylene intrastrand cross‐link (IaCL) between the O⁶‐atoms of two consecutive 2′‐deoxyguanosines (dG) were prepared by solid‐phase synthesis. UV thermal denaturation studies of duplexes containing butylene and heptylene IaCL revealed a 20 °C reduction in stability compared to the unmodified duplexes. Circular dichroism profiles of these IaCL DNA duplexes exhibited signatures consistent with B‐form DNA. Human O⁶‐alkylguanine DNA alkyltransferase (hAGT) was capable of repairing both IaCL containing duplexes with slightly greater efficiency towards the heptylene analog. Interestingly, repair efficiencies of hAGT towards these IaCL were lower compared to O⁶‐alkylene linked IaCL lacking the 5′‐3′‐phosphodiester linkage between the connected 2′‐deoxyguanosine residues. These results demonstrate that the proficiency of hAGT activity towards IaCL at the O⁶‐atom of dG is influenced by the backbone phosphodiester linkage between the cross‐linked residues.     

2‐Phenanthrenyl–DNA: Synthesis,Pairing, and Fluorescence Properties

Three 2′‐phenanthrenyl‐C‐deoxyribonucleosides with donor (phenNH₂), acceptor (phenNO₂), or no (phenH) substitution on the phenanthrenyl core were synthesized and incorporated into oligodeoxyribonucleotides. Duplexes containing either one or three consecutive phenR residues, which were located opposite each other, were formed. Within these residues, the phenR residues are expected to recognize each other through interstrand stacking interactions, in much the same way as described previously for biphenyl DNA. The thermal, thermodynamic, and fluorescence properties of such duplexes were determined by UV melting analysis and fluorescence spectroscopy. Depending on the nature of the substituent, the thermal stability of single‐modified duplexes can vary between ?2.7 to +11.3 °C in T_m and that of triple‐modified duplexes from +7.8 to +11.1 °C. Van′t Hoff analysis suggested that the observed higher thermodynamic stability in phenH‐ and phenNO₂‐containing duplexes is of enthalpic origin. A single phenH or phenNO₂ residue in a bulge position also stabilizes a corresponding duplex. If a phenNO₂ residue is placed in a bulge position next to a base mismatch this can lead, in a sequence‐dependent manner, to duplex destabilization. The phenNO₂ residue was found to be a highly efficient (10–100‐fold) quencher of phenH and phenNH₂ fluorescence if placed in the opposite position to the fluorophores. When phenH and phenNH₂ residues were placed opposite each other, efficient quenching of phenH and enhancement of phenNH₂ fluorescence was found, which is an indicator for electron‐ or energy‐transfer processes between the aromatic units.     

(2′‐O‐Methyl‐RNA)‐3′‐PNA Chimeras: A New Class of Mixed Backbone Oligonucleotide Analogues with High Binding Affinity to RNA

The automated on‐line synthesis of DNA‐3′‐PNA chimeras 1 – 4 and (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras 5 – 8 is described, in which the 3′‐terminal part of the oligonucleotide is linked to the N‐terminal part of the PNA via N‐(ω‐hydroxyalkyl)‐N‐[(thymin‐1‐yl)acetyl]glycine units (alkyl=Et, Ph, Bu, and pentyl). By means of UV thermal denaturation, the binding affinities of all chimeras were directly compared by determining their T_m values in the duplex with complementary DNA and RNA. All investigated DNA‐3′‐PNA chimeras and (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras form more‐stable duplexes with complementary DNA and RNA than the corresponding unmodified DNA. Interestingly, a N‐(3‐hydroxypropyl)glycine linker resulted in the highest binding affinity for DNA‐3′‐PNA chimeras, whereas the (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras showed optimal binding with the homologous N‐(4‐hydroxybutyl)glycine linker. The duplexes of (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras and RNA were significantly more stable than those containing the corresponding DNA‐3′‐PNA chimeras. Surprisingly, we found that the charged (2′‐O‐methyl‐RNA)‐3′‐PNA chimera with a N‐(4‐hydroxybutyl)glycine‐based unit at the junction to the PNA part shows the same binding affinity to RNA as uncharged PNA. Potential applications of (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras include their use as antisense agents acting by a RNase‐independent mechanism of action, a prerequisite for antisense‐oligonucleotide‐mediated correction of aberrant splicing of pre‐mRNA.     

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.     

5‐Aza‐7‐deazaguanine DNA: Recognition and Strand Orientation of Oligonucleotides Incorporating Anomeric Imidazo[1,2‐a]‐1,3,5‐triazine Nucleosides

The base‐pairing properties of oligonucleotides containing the anomeric 5‐aza‐7‐deazaguanine 2′‐deoxyribonucleosides 1 and 5 are described. The oligonucleotides were prepared by solid‐phase synthesis, employing phosphoramidite or phosphonate chemistry. Stable `purine'⋅purine duplexes with antiparallel (aps) chain orientation are formed, when the α‐D ‐anomer 5 alternates with the β‐D ‐anomeric 2′‐deoxyguanosine ( 2 ) within the same oligonucleotide chain. Parallel (ps) oligonucleotide duplexes are observed, when the β‐D anomer 1 alternates with 2 . A renewed reversal of the chain orientation (ps→aps) occurs when compound 1 pairs with 2′‐deoxyisoguanosine ( 6 ). In all cases, it is unnecessary to change the orientation within a single strand when α‐D units alternate with their β‐D counterparts. Heterochiral base pairs of 5 (α‐D ) with 2′‐deoxyisoguanosine (β‐D ) are well accommodated in duplexes with random base composition and parallel chain orientation. Base pairs of 5 (α‐D ) with 2′‐deoxyguanosine (β‐D ) destabilize duplexes with antiparallel chains.     

Oligonucleotides Containing Novel 4′‐C‐ or 3′‐C‐(Aminoalkyl)‐Branched Thymidines

The synthesis of four novel 3′‐C‐branched and 4′‐C‐branched nucleosides and their transformation into the corresponding 3′‐O‐phosphoramidite building blocks for automated oligonucleotide synthesis is reported. The 4′‐C‐branched key intermediate 11 was synthesized by a convergent strategy and converted to its 2′‐O‐methyl and 2′‐deoxy‐2′‐fluoro derivatives, leading to the preparation of novel oligonucleotide analogues containing 4′‐C‐(aminomethyl)‐2′‐O‐methyl monomer X and 4′‐C‐(aminomethyl)‐2′‐deoxy‐2′‐fluoro monomer Y (Schemes 2 and 3). In general, increased binding affinity towards complementary single‐stranded DNA and RNA was obtained with these analogues compared to the unmodified references (Table 1). The presence of monomer X or monomer Y in a 2′‐O‐methyl‐RNA oligonucleotide had a negative effect on the binding affinity of the 2′‐O‐methyl‐RNA oligonucleotide towards DNA and RNA. Starting from the 3′‐C‐allyl derivative 28 , 3′‐C‐(3‐aminopropyl)‐protected nucleosides and 3′‐O‐phosphoramidite derivatives were synthesized, leading to novel oligonucleotide analogues containing 3′‐C‐(3‐aminopropyl)thymidine monomer Z or the corresponding 3′‐C‐(3‐aminopropyl)‐2′‐O,5‐dimethyluridine monomer W (Schemes 4 and 5). Incorporation of the 2′‐deoxy monomer Z induced no significant changes in the binding affinity towards DNA but decreased binding affinity towards RNA, while the 2′‐O‐methyl monomer Z induced decreased binding affinity towards DNA as well as RNA complements (Table 2).     

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).     

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.     

7‐De­aza‐2′‐deoxy‐7‐propynyl­guanosine

The title compound, C₁₄H₁₆N₄O₄, adopts the anti conformation at the gly­cosylic bond [χ−117.1 (5)°]. The sugar pucker of the 2′‐deoxy­ribo­furan­osyl moiety is C2′‐endo–C3′‐exo, ²T₃ (S‐type). The orientation of the exocyclic C4′—C5′ bond is +sc (gauche). The propynyl group is linear and coplanar with the nucleobase moiety. The structure of the compound is stabilized by several hydrogen bonds (N—H⋯O and O—H⋯O), leading to the formation of a multi‐layered network. The nucleobases, as well as the propynyl groups, are stacked. This stacking might cause the extraordinary stability of DNA duplexes containing this compound.     

Functionalized 3,3′,5,5′‐Tetraaryl‐1,1′‐Biphenyls: Novel Platforms for Molecular Receptors

This paper describes the development of novel aromatic platforms for supramolecular construction. By the Suzuki cross‐coupling protocol, a variety of functionalized m‐terphenyl derivatives were prepared (Schemes 1–4). Macrolactamization of bis(ammonium salt) (S,S)‐ 6 with bis(acyl halide) 7 afforded the macrocyclic receptor (S,S)‐ 2 (Scheme 1), which was shown by ¹H‐NMR titration studies to form ‘nesting' complexes of moderate stability (K_a between 130 and 290 M ^?1, 300 K) with octyl glucosides 13 – 15 (Fig. 2) in the noncompetitive solvent CDCl₃. Suzuki cross‐coupling starting from 3,3′,5,5′‐tetrabromo‐1,1′‐biphenyl provided access to a novel series of extended aromatic platforms (Scheme 5) for cleft‐type (Fig. 1) and macrotricyclic receptors such as (S,S,S,S)‐ 1 . Although mass‐spectral evidence for the formation of (S,S,S,S)‐ 1 by macrolactamization between the two functionalized 3,3′,5,5′‐tetraaryl‐1,1′‐biphenyl derivatives (S,S)‐ 33 and 36 was obtained, the ¹H‐ and ¹³C‐NMR spectra of purified material remained rather inconclusive with respect to both purity and constitution. The versatile access to the novel, differentially functionalized 3,3′,5,5′‐tetrabromo‐1,1′‐biphenyl platforms should ensure their wide use in future supramolecular construction.     

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.     

Oligonucleotide Analogues with a Nucleobase‐Including Backbone,Part 5, 2′‐Deoxy‐8‐(hydroxymethyl)adenosine‐ and 2′‐Deoxy‐6‐(hydroxymethyl)uridine‐Derived Phosphoramidites: Synthesis and Incorporation into 14‐Mer DNA Strands

The pairing propensity of new DNA analogues with a phosphinato group between O−C(3′) and a newly introduced OCH₂ group at C(8) and C(6) of 2′‐deoxyadenosine and 2′‐deoxyuridine, respectively, was evaluated by force‐field calculations and Maruzen model studies. These studies suggest that these analogues may form autonomous pairing systems, and that the incorporation of single modified units into DNA 14mers is compatible with duplex formation. To evaluate the incorporation, we prepared the required phosphoramidites 3 and 4 from 2′‐deoxyadenosine and 2′‐deoxyuridine, respectively. The phosphoramidite 5 was similarly prepared to estimate the influence of a CH₂OH group at C(8) on the duplex stability. The modified 14‐mers 6 – 9 were prepared by solid‐phase synthesis. Pairing studies show a decrease of the melting temperature by 2.5° for the duplex 13 ⋅ 9 , and of 6 – 8° for the duplexes 10 ⋅ 6 , 11 ⋅ 6 , 13 ⋅ 7 , and 14 ⋅ 8 , as compared to the unmodified duplexes.     

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.     

2′‐Deoxyuridine and 2′‐Deoxyisocytidine as Constituents of DNA with Parallel Chain Orientation: The Stabilization of the iCd⋅Gd Base Pair by the 5‐Methyl Group

Parallel‐stranded oligonucleotides containing 2′‐deoxyuridine ( 2 ) and 2′‐deoxyisocytidine ( 4 ) were synthesized. The phosphoramidite 11 employed in the solid‐phase synthesis carries a (dimethylamino)methylidene residue as amino‐protecting group. This group stabilizes the acid‐labile glycosylic bond of 4 and enables the base‐catalyzed deprotection of oligonucleotides without degrading the nucleoside 4 residues. Oligonucleotide duplexes incorporating the 5‐Me derivatives of 2 (→2′‐deoxythymidine) and 4 (→2′‐deoxy‐5‐methylisocytidine), which are more stable than those containing the unmethylated nucleosides, were also compared. Depending on the nearest‐neighbor environment, Me groups provide an additional stabilization through Me/Me contacts or Me/backbone interactions.     

Oligonucleotides Containing 7‐Deaza‐2′‐deoxyxanthosine: Synthesis,Base Protection,and Base‐Pair Stability

Oligonucleotides incorporating 7‐deaza‐2′‐deoxyxanthosine ( 3 ) and 2′‐deoxyxanthosine ( 1 ) were prepared by solid‐phase synthesis using the phosphoramidites 6 – 9 and 16 which were protected with allyl, diphenylcarbamoyl, or 2‐(4‐nitrophenyl)ethyl groups. Among the various groups, only the 2‐(4‐nitrophenyl)ethyl group was applicable to 7‐deazaxanthine protection being removed with 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU) by β‐elimination, while the deprotection of the allyl residue with Pd⁰ catalyst or the diphenylcarbamoyl group with ammonia failed. Contrarily, the allyl group was found to be an excellent protecting group for 2′‐deoxyxanthosine ( 1 ). The base pairing of nucleoside 3 with the four canonical DNA constituents as well as with 3‐bromo‐1‐(2‐deoxy‐β‐D ‐erythro‐pentofuranosyl)‐1H‐pyrazolo[3,4‐d]pyrimidine‐4,6‐diamine ( 4 ) within the 12‐mer duplexes was studied, showing that 7‐deaza‐2′‐deoxyxanthosine ( 3 ) has the same universal base‐pairing properties as 2′‐deoxyxanthosine ( 1 ). Contrary to the latter, it is extremely stable at the N‐glycosylic bond, while compound 1 is easily hydrolyzed under slightly acidic conditions. Due to the pK_a values 5.7 ( 1 ) and 6.7 ( 3 ), both compounds form monoanions under neutral conditions (95% for 1 ; 65% for 3 ). Although both compounds form monoanions at pH 7.0, pH‐dependent T_m measurements showed that the base‐pair stability of 7‐deaza‐2′‐deoxyxanthosine ( 3 ) with dT is pH‐independent. This indicates that the 2‐oxo group is not involved in base‐pair formation.     

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.     

Decomposition of model alkoxyamines in simple and polymerizing systems. II. Diastereomeric N‐(2‐methylpropyl)‐N‐(1‐diethyl‐phosphono‐2,2‐dimethyl‐propyl)‐aminoxyl‐based compounds

Thermal reactions of the alkoxyamine diastereomers DEPN‐R′ [DEPN: N‐(2‐methylpropyl)‐N‐(1‐diethylphosphophono‐2,2‐dimethyl‐propyl)‐aminoxyl; R′: methoxy‐carbonylethyl and phenylethyl] with (R,R) + (S,S) and (R,S) + (S,R) configurations have been investigated by ¹H NMR at 100 °C. During the overall decay the diastereomers interconvert, and an analytical treatment of the combined processes is presented. Rate constants are obtained for the cleavage and reformation of DEPN‐R′ from NMR, electron spin resonance, and chemically induced dynamic nuclear polarization experiments also using 2,2,6,6‐tetramethylpiperidinyl‐1‐oxyl (TEMPO) as a radical scavenger. The rate constants depend on the diastereomer configuration and the residues R′. Simulations of the kinetics observed with styrene and methyl methacrylate containing solutions yielded rate constants for unimeric and polymeric alkoxyamines DEPN‐(M)_n‐R′. The results were compatible with the known DEPN mediation of living styrene and acrylate polymerizations. For methyl methacrylate the equilibrium constant of the reversible cleavage of the dormant chains DEPN‐(M)_n‐R′ is very large and renders successful living polymerizations unlikely. Mechanistic and kinetic differences of DEPN‐ and TEMPO‐mediated polymerizations are discussed. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 3264–3283, 2002