Matthias Zulauf

7-Deazaadenine-DNA: Bulky 7-Iodo Substituents or Hydrophobic 7-Hexynyl Chains Are Well Accommodated in the Major Groove of Oligonucleotide Duplexes

Modified oligonucleotideswithbulky iodo or lipophilic (hex-1-ynyl) substituents in the 7-position of the 7-deazaadenosine moiety (right) are found to form more stable duplexes than the unmodified oligomers (Tm values, far right). Possible practical applications include the isotopic labeling of DNA, improved delivery of antisense constructs through the cell membrane, and the identification of particular nucleobases by AFM.

7-Substituted 7-Deaza-2'-deoxyadenosines and 8-Aza-7-deaza-2'-deoxyadenosines: Fluorescence of DNA-Base Analogues Induced by the 7-Alkynyl Side Chain

7-Alkynylated 7-deazaadenine (pyrrolo[2,3-d]pyrimidin-4-amine) 2′-deoxyribonucleosides show strong fluorescence which is induced by the 7-alkynyl side chain (Table 3). A large Stokes shift with an emission around 400 nm is observed when the compound is irradiated at 280 nm. The solvent dependence indicates the formation of a charged transition state. The fluorescence appears when the triple bond is in conjugation with the heterocyclic base. Electron-donating substituents at the triple bond increase the fluorescence, while electron-withdrawing residues reduce it. In comparison, the 7-alkynylated 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) 2′-deoxyribonucleosides are rather weakly fluorescent (Table 4). Quantum yields and fluorescence decay times are measured. The synthesis of the 7-alkynylated 7-deaza-2′-deoxyadenosines and 8-aza-7-deaza-2′-deoxyadenosines was performed with 7-deaza-2′-deoxy-7-iodoadenosine (6) or 8-aza-7-deaza-2′-deoxy-7-iodoadenosine (22) as starting materials and employing the Pd0-catalyzed cross-coupling reaction with the corresponding alkynes (Schemes 1, 4, and 5). Catalytic hydrogenation of the side chain of the unsaturated nucleosides 5 and 17 afforded the 7-alkyl derivatives 18 and 19, respectively, which do not show significant fluorescence (Scheme 2).

Oligonucleotides Containing 7‐Deazaadenines: The Influence of the 7‐Substituent Chain Length and Charge on the Duplex Stability

Oligonucleotides carrying alkynyl and aminoalkynyl chains at the position 7 of 7-deazaadenine are synthesized, and the chain lengths as well as the bulkiness of the substituents are varied. The corresponding nucleosides 1a – f are prepared from 7-deaza-2′-deoxy-7-iodoadenosine and the particular alkynes by the Pd0-catalyzed cross-coupling reaction. The nucleosides are converted to the phosphoramidites 2a – f, which are used in solid-phase oligonucleotide synthesis. The stability of the duplexes is determined by the Tm values and the thermodynamic data. Compared to adenine or the unsubstituted 7-deazaadenine, the incorporation of a 7-ethynyl chain in a 7-deazaadenine moiety increases the duplex stability significantly, while a dodecynyl residue or a bulky steroid moiety leads to a duplex destabilization. A 3-aminoprop-1-ynyl residue (see 1g) or a 5-aminopent-1-ynyl residue (see 1h), which are charged under neutral conditions, lead to zwitterionic DNA. A high density of charged residues as found in homomers impairs duplex formation, most probably by counter-ion condensation.

STEREOELECTRONIC EFFECTS OF MODIFIED PURINES ON THE SUGAR CONFORMATION OF NUCLEOSIDES AND FLUORESCENCE PROPERTIES

Abstract Conformational analyses of the sugar moieties of a series base-modified purine-2′-deoxynucleosides on the basis of vicinal [1H,1H] coupling constants is presented (PSEUROT 6.2) Fluorescence data of several 7-deaza- and 8-azapurine 2′-deoxynucleosides are given.

Synthesis of oligonucleotides containing pyrazolo[3,4-d]pyrimidines: The influence of 7-substituted 8-aza-7-deazaadenines on the duplex structure and stability

The phosphoramidites 2a–d derived from 7-bromo- (1b), 7-iodo- (1c), 7-(hex-1-ynyl)- (1d) and 7-(2-phenylethynyl)- (1e) 8-aza-7-deaza-2′-deoxyadenosine were prepared. They were employed in the solid-phase synthesis of various oligonucleotides containing the 8-aza-7-deazaadenine system instead of that of adenine. Their Tm-values were measured and their thermodynamic data were determined. The replacement of adenine residues by 8-aza-7-deazaadenine 1a does not significantly influence the duplex stability, while the incorporation of 7-substituted derivatives 1b–e led to much more stable duplexes compared with their adenine-containing counterparts. From the CD spectra of the oligonucleotide duplexes it is evident that the overall structure of a parent DNA is retained when only very few modified bases are incorporated. Structural changes do occur when the number of modified residues is increased. This might be the result of the high-anti-conformation of the 7-substituted 8-aza-7-deaza-2′-deoxyadenosine residues.

Duplex Stability of Oligonucleotides Containing 7-Substituted 7-Deaza- and 8-Aza-7-Deazapurine Nucleosides

Abstract The synthesis of 7-substituted 7-deaza- and 8-aza-7-deazapurine 2′-deoxyribonucleosides, their incorporation into oligonucleotides, and the stability of corresponding duplexes is described.

8-Aza-7-deazapurine DNA: Synthesis and Duplex Stability of Oligonucleotides Containing 7-Substituted Bases

Abstract The 7-substituted 8-aza-7-deazapurine phosphoramidites 1a – 3c as well as the phosphoramidite 4a were synthesized. In comparison to the parent purine oligonucleotide duplexes, the 7-substituted 8-aza-7-deazapurine residues lead to a significant duplex stabilization.

PHOSPHORAMIDITES AND OLIGONUCLEOTIDES CONTAINING 7-DEAZAPURINES AND PYRIMIDINES CARRYING AMINOPROPARGYL SIDE CHAINS

The synthesis of phosphoramidites containing 7-deazaguanine, 7-deazaadenine, uracil and cytosine carrying aminopropargyl chains is described. The corresponding oligonucleotides are stabilized in duplexes thermally as well as against degradation by exonucleases.

Unexpected Dehalogenation of 3-Bromopyrazolo[3,4-d]pyrimidine Nucleosides During Nucleobase-Anion Glycosylation

Abstract The anion-glycosylation (KOH, MeCN, TDA-1) of 3-bromopyrazolo[3,4-d]-pyrimidines 4a and 4b with 2-deoxy-3,5-di-O-(p-toluoyl)-α-D-erythro-pentofuranosyl chloride (5) furnishes the regioisomeric N′-β-D-2′-deoxyribonucleosides 6a and 6b together with the dehalogenated N2-regioisomers 8a and 8b, stereoselectively. The dehalogenation takes place after the glycosylation and results from the sensitivity of the N-2 nucleosides toward aqueous base. An addition/elimination mechanism is suggested for the dehalogenation reaction.

Base-Modified Oligonucleotides With Increased Duplex Stability

: Oligonucleotides incorporating 8-aza-7-dazapurines (pyrazolo[3,4-d]pyrimidines) were synthesized. The corresponding nucleosides were prepared and were converted into phosphoramidites. The oligonucleotide duplex stability was studied and was compared to that of the parent compounds containing the canonical purine nucleosides. The presence of 7-halogeno or 7-alkynyl substituents increases the duplex stability significantly.