Synthesis and characterization of oligonucleotides containing conformationally constrained bicyclo[3.1.0]hexane pseudosugar analogs

Abstract

Oligodeoxyribonucleotides containing pseudorotationally locked sites derived from bicyclo[3.1.0]hexane pseudosugars have been synthesized using adenosine, thymidine and abasic versions of North- and South-methanocarba nucleosides. The reaction conditions for coupling and oxidation steps of oligonucleotide synthesis have been investigated and optimized to allow efficient and facile solid-phase synthesis using phosphoramidite chemistry. Our studies demonstrate that the use of iodine for P(III) to P(V) oxidation leads to strand cleavage at the sites where the pseudosugar is North. In contrast, the same cleavage reaction was not observed in the case of South pseudosugars. Iodine oxidation generates a 5'-phosphate oligonucleotide fragment on the resin and releases the North pseudosugar into the solution. This side reaction, which is responsible for the extremely low yields observed for the incorporation of the North pseudosugar analogs, has been studied in detail and can be easily overcome by replacing iodine with t-butylhydroperoxide as oxidant.

Figure 1

Fixed location of the bicyclo[3.1.0]hexane templates in the pseudorotational cycle.

Figure 2

Schematic representation of coupled protons (arrows) in 1b and in 7 (inverted stereochemistry).

Scheme 1. Reagents and conditions: (a) 1.3 equiv. bis(N,N-diisopropyl)phosphoramidite, 0.7 equiv. tetrazole (0.45 M solution in acetonitrile), r.t., 2–6 h (60–89%).

Scheme 1. Reagents and conditions: (a) 1.3 equiv. bis(N,N-diisopropyl)phosphoramidite, 0.7 equiv. tetrazole (0.45 M solution in acetonitrile), r.t., 2–6 h (60–89%).

Scheme 2. Reagents and conditions: (a) 20 mg (0.023 mmol) 2b in CH₃CN, 1.5 equiv. 3′-O-acetylthymidine, 5 equiv. tetrazole (0.5 M in CH₃CN); (b-i) 5 equiv. I₂ (0.02 M in pyridine/H₂O), (b-ii) 0.2 ml of 10% t-BuOOH in CH₃CN/H₂O (6:1), (b-iii) 5 equiv. Beaucage's Reagent (0.2 M in CH₃CN).

Scheme 2. Reagents and conditions: (a) 20 mg (0.023 mmol) 2b in CH₃CN, 1.5 equiv. 3′-O-acetylthymidine, 5 equiv. tetrazole (0.5 M in CH₃CN); (b-i) 5 equiv. I₂ (0.02 M in pyridine/H₂O), (b-ii) 0.2 ml of 10% t-BuOOH in CH₃CN/H₂O (6:1), (b-iii) 5 equiv. Beaucage's Reagent (0.2 M in CH₃CN).

Scheme 3.

Scheme 3.

Scheme 4. Reagents and conditions: (a-i) (CH₃)₃SiCl, pyridine; (a-ii) PhCOCl, pyridine; (a-iii) conc. NH₄OH; (b) DMTCl, pyridine, DMAP; (c-i) dibenzyl diisopropylphosphoramidite, tetrazole, THF; (c-ii) t-BuOOH (Luperox® 98%); (d-i) dibenzyl diisopropylphosphoramidite, tetrazole, CH₃CN; and (d-ii) 0.02 M of I₂ in pyridine/H₂O (75:25).

Scheme 4. Reagents and conditions: (a-i) (CH₃)₃SiCl, pyridine; (a-ii) PhCOCl, pyridine; (a-iii) conc. NH₄OH; (b) DMTCl, pyridine, DMAP; (c-i) dibenzyl diisopropylphosphoramidite, tetrazole, THF; (c-ii) t-BuOOH (Luperox® 98%); (d-i) dibenzyl diisopropylphosphoramidite, tetrazole, CH₃CN; and (d-ii) 0.02 M of I₂ in pyridine/H₂O (75:25).

Scheme 5.