Di(azacrown) Conjugates of 2‘-O-Methyl Oligoribonucleotides as Sequence-Selective Artificial Ribonucleases

Abstract

Functionalized 2'-O-methyl oligoribonucleotides bearing two 3-(3-hydroxypropyl)-1,5,9-triazacyclododecane ligands attached via a phosphodiester linkage to a single non-nucleosidic building block have been prepared on a solid-support by conventional phosphoramidite chemistry. The branching units employed for the purpose include 2,2-bis(3-hydroxypropylaminocarbonyl)propane-1,3-diol, 2-hydroxyethyl 3'-O-(2-hydroxyethyl)-beta-D-ribofuranoside, and 2-hydroxyethyl 2'-O-(2-hydroxyethyl)-beta-D-ribofuranoside. Each of these has been introduced as a phosphoramidite reagent either into the penultimate 3'-terminal site or in the middle of the oligonucleotide chain. The dinuclear Zn2+ complexes of these conjugates have been shown to exhibit enhanced catalytic activity over their monofunctionalized counterpart, the 3'-terminal conjugate derived from 2-hydroxyethyl 3'-O-(2-hydroxyethyl)-beta-D-ribofuranoside being the most efficient cleaving agent. This conjugate cleaves an oligoribonucleotide target at a single phosphodiester bond and shows turnover and 1000-fold cleaving activity compared to the free monomeric Zn2+ chelate of 1,5,9-triazacyclododecane.

Synthesis of Aminoglycoside Conjugates of 2′-O-Methyl Oligoribonucleotides

“…We recently described cleavage properties of di(azacrown) conjugated oligonucleotides with the same targets (14 and 15). Interestingly, the 3-O ribose (4) branched azacrown conjugate was even in that case superior to other closely related conjugates (42). Metalion-independent cleavage of HIV-1 TAR RNA by neamine-PNA conjugates has recently been reexamined by Chaubey et al (27).…”

“…In contrast to 1 and 2, coupling of 31 remained at a poor level and, hence, this building block was excluded from the synthesis of final conjugates. Coupling of the hydroxyalkylarmed branching units (4 and 5) has recently been shown to be as efficient as that of the commercially available 2′-O-methylribonucleoside building blocks; the couplings were quantitative using a standard RNA coupling protocol (42). The branching unit 3 behaved similarly.…”

“…However, only in one case (conjugate 11 vs target 14) was the first-order rate constant (k ) 9.0 × 10 -7 s -1 ) markedly different from that of the nonspecific cleavage by Zn 2+ complexes. Otherwise, the accelerations [k ) (1.5-7.0) × 10 -7 s -1 ] may be considered to be more or less marginal compared to the background obtained with the targets (14 and 15) in 90 µmol L -1 Zn 2+ concentration alone (k < 1 × 10 -6 s -1 ) (42,43). It is worth noting that cleavage activity of conjugate 9 was not markedly accelerated by addition of the Zn 2+ ion.…”

“…It should also be noted that the chimeric oligonucleotides 14 and 15 are relatively stable as targets. For example, the Zn 2+ complexes of the di(azacrown) conjugates of 2′-Omethyl oligoribonucleotides, which in an optimal case have equal catalytic activity to the most efficient Zn 2+ and Cu 2+ -based artificial nucleases (47,48), also have moderate cleavage ability [k ) (0.9-1.7) × 10 -6 s -1 ] to these targets (14 and 15) (42). Thus, the real cleavage potential of the aminoglycoside conjugates (6-13) may still remain obscure, and further studies with different targets are required.…”

“…The 2′-O-methyl oligoribonucleotide chains 37, and 40-42 were assembled on a 1 µmol scale following the standard RNA coupling protocol. Levulinoyl protections of the inserted branching units (40)(41)(42) were removed by hydrazinium acetate treatment (NH 2 NH 2 · H 2 O, pyridine, AcOH, 0.124/4/1, v/v/v, 1 h at 25°C), and then the aminoglycoside phosphoramidites (1 and 2) were coupled to the hydroxyalkyl arms of 43-45 or to the exposed 5′-end of 37. Two consecutive standard RNA couplings (0.1 mol L -1 solution of 1 or 2 in dry acetonitrile, 600 s coupling time, twice) were used in each case.…”