2'-Deoxy-2'-fluoro-beta-D-arabinonucleosides and oligonucleotides (2'F-ANA): synthesis and physicochemical studies

Abstract

Recently, hybrids of RNA and D-arabinonucleic acids (ANA) as well as the 2'-deoxy-2'-fluoro-D-arabinonucleic acid analog (2'F-ANA) were shown to be substrates of RNase H. This enzyme is believed to be involved in the primary mechanism by which antisense oligonucleotides cause a reduction in target RNA levels in vivo. To gain a better understanding of the properties of arabinose based oligonucleotides, we have prepared a series of 2'F-ANA sequences of homopolymeric (A and T) and mixed base composition (A, T, G and C). UV thermal melting and circular dichroic (CD) studies were used to ascertain the thermodynamic stability and helical conformation of 2'F-ANA/RNA and 2'F-ANA/DNA hybrids. It is shown that 2'F-ANA has enhanced RNA affinity relative to that of DNA and phosphorothioate DNA. The 2'-fluoroarabino modification showed favorable pairing to single-stranded DNA also. This is in sharp contrast to ANA, which forms weak ANA/DNA hybrids at best. According to the measured thermodynamic parameters for duplex formation, the increased stability of hybrids formed by 2'F-ANA (e.g., 2'F-ANA/RNA) appears to originate from conformational pre-organization of the fluorinated sugars and a favorable enthalpy of hybridization. In addition, NMR spectroscopy revealed a five-bond coupling between the 2'F and the base protons (H6/H8) of 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides. This observation is suggestive of a through-space interaction between 2'F and H6/H8 atoms. CD experiments indicate that 2'F-ANA/RNA hybrids adopt an 'A-like' structure and show more resemblance to DNA/RNA hybrids than to the pure RNA/RNA duplex. This feature is believed to be an important factor in the mechanism that allows RNase H to discriminate between 2'F-ANA/RNA (or DNA/RNA) and RNA/RNA duplexes.

Figure 1

Structures of ANA and 2′F-ANA.

Figure 2

¹H NOESY spectrum of araF-C in DMSO-d6. Arrows in the structure show the NOEs detected. The region in which the H1′/H″ and H1′/H4′ NOEs appear is shown.

Figure 3

³¹P and ¹⁹F NMR spectra of araF-A amidite. The structure shows the ‘W’ pathway between the 2′-fluorine and the 3′-phosphorus that leads to the long range ³¹P-¹⁹F coupling detected (compare ¹⁹F spectrum to the ¹⁹F/³¹P decoupled spectrum).

Figure 4

Energy minimized structure of araF-A. (A) Space filling model. Fluorine at C2′ is shown in yellow. (B) Chemical structure of araF-A showing the proposed through-space 2′F–H8 interaction.

Figure 5

Melting curves of 18-bp duplexes (sequence III, Table 1). Oligonucleotides were hybridized to (top) single stranded RNA and (bottom) ssDNA. Buffer: 140 mM KCl, 1 mM MgCl₂, 5 mM Na₂HPO₄, pH 7.2.

Figure 6

CD spectra of 18-bp duplexes at 5°C (sequence IV, Table 1). Buffer: 140 mM KCl, 1 mM MgCl₂, 5 mM Na₂HPO₄, pH 7.2.

Scheme 1. (a) DAST, CH₂Cl₂, 40°C, 16 h; (b) HBr/AcOH, 16 h; (c) bis-silyted cytosine or thymine, CCl₄, reflux, 60 h; (d) conc. NH₄OH/MeOH, 16 h; (e) MMTr-Cl, pyridine, 4-DMAP, 16 h; (f) TMS-Cl, pyridine followed by BzCl, followed by aqueaous ammonia; (g) Cl-P(OCE)N(i-Pr)₂, EtN(i-Pr)₂; THF; CEO, 2-cyanoethoxy.