Harald Debelak

Different conformations of 1-deazaadenosine and its 2′-deoxyribonucleoside in the solid state and in solution

Abstract Crystallization of 1-deaza-2′-deoxyadenosine (c1Ad, 1) from propanol-2 gives two forms of crystals: type A formed firstly as plates, then, on the surface of the plates, type B appeared as needles. Single crystal X-ray analyses shows that the crystals A and B differ mainly in the sugar ring conformation: A adopts the S-type (P= 179.8°; C-2′-endo-C-3′-exo; 2T3) conformation associated with an high-anti base orientation (χ=−90.7°) and the γ = 152.3° across the exocyclic C(4′)C(5′) bond; B shows N-type (P = 21.2; C-3′-endo; 3E) conformation accompanied by a somewhat different anti base orientation (χ = −116.5°) an eclipsed orientation of the exocyclic C(4′)C(5′) bond (γ = 84.5°). No intramolecular hydrogen bonds in both types of crystals can be detected. Unlike c1Ad, the ribonucleoside (c1A, 2) is in the syn conformation (χ = 56.1°) in the solid state which is caused by an intramolecular (5′)CH2OH…N(3) hydrogen bond. The compounds 1 and 2 display very similar conformation in D2O solution with a strong preference for the S-type conformation of the furanose ring accompanied by the intramolecular (5′)CH2OH…N(3) hydrogen bond.

Purification of Small Interfering RNA Using Nondenaturing Anion-Exchange Chromatography

A manufacturing and purification process for duplex oligonucleotides was established, which shortens and simplifies currently used procedures, yielding a product of higher purity. The reported procedure is based on nondenaturing anion-exchange (AEX) chromatography, which is performed on the annealed duplex rather than the individual single strands. The duplex is formed early in the process by annealing of the crude single strands directly after solid-phase synthesis. Two 30 μmol manufacturing runs using duplex purification were performed on 2 different AEX resins and compared with a manufacturing run of the same scale using conventional single-strand chromatography. The same pooling strategy was employed for all purifications. Content of optimal duplex (duplex exclusively comprising full-length single strands) was 90.5% and 90.2% for the batches obtained by duplex purification and 86.1% for the batch obtained by single-strand purification. Maximum chromatographic recoveries were 67% for the duplex purification and 68% for the single-strand purification. Hence, the manufacture of small interfering RNA (siRNA) using duplex purification was simpler and faster than conventional single-strand purification and provided better purity and similar yield of final siRNA.

1-DEAZA-3'-O-METHYLADENOSINE : A NUCLEOSIDE WITH THE SYN-CONFORMATION IN THE SOLID STATE AND IN SOLUTION

Abstract The 2′-O-methyl (2) and the 3′-O-methyl (3) derivatives of 1-deazaadenosine (1) were prepared. Single crystal X-ray analysis as well as 1H and 13C NMR studies were performed on the 3′-O-methyl-1-deazaadenosine 3. In the solid state, the glycosyl torsion angle (χ = 64.7°) is in the syn-range which is caused by an intramolecular (5′)CH2OH…N(3) hydrogen bond. The ribofuranose moiety adopts a 2 E (C-3′-exo; S) conformation and the orientation of the exocyclic C(4′)-C(5′) bond is + sc (γ(+)g). The conformation in solution was found to be very similar to that in solid state. Whereas the 2′-O-methyl derivative of 1 is a strong inhibitor of adenosine deaminase the 3′-O-methyl derivative is neither inhibitor nor substrate.

Using NMR and molecular dynamics to link structure and dynamics effects of the universal base 8-aza, 7-deaza, N8 linked adenosine analog.

A truly universal nucleobase enables a host of novel applications such as simplified templates for PCR primers, randomized sequencing and DNA based devices. A universal base must pair indiscriminately to each of the canonical bases with little or preferably no destabilization of the overall duplex. In reality, many candidates either destabilize the duplex or do not base pair indiscriminatingly. The novel base 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin- 4-amine) N8-(2′deoxyribonucleoside), a deoxyadenosine analog (UB), pairs with each of the natural DNA bases with little sequence preference. We have utilized NMR complemented with molecular dynamic calculations to characterize the structure and dynamics of a UB incorporated into a DNA duplex. The UB participates in base stacking with little to no perturbation of the local structure yet forms an unusual base pair that samples multiple conformations. These local dynamics result in the complete disappearance of a single UB proton resonance under native conditions. Accommodation of the UB is additionally stabilized via heightened backbone conformational sampling. NMR combined with various computational techniques has allowed for a comprehensive characterization of both structural and dynamic effects of the UB in a DNA duplex and underlines that the UB as a strong candidate for universal base applications.

Hoogsteen-Duplex DNA: Synthesis and Base Pairing of Oligodeoxynucleotides Containing 1-Deaza-2′-deoxyadenosine

Abstract Solid-phase synthesis of oligonucleotides containing 1-deazaadenine was carried out employing phosphonate and phosphoramidite chemistry. Hoogsteen base pairing was established for the duplex d(c1A20)·d(T20).