RNA monomers containing 0-acetal levulinyl ester groups and their use in RNA microarrays
The present invention is directed to RNA monomers comprising O-acetal levulinyl protecting groups at the 2′ and/or the 5′-hydroxy functionalities of the ribose moiety. Said monomers may be incorporated into oligoribonucleotides or RNA polynucleotides. Furthermore, the invention is directed to methods for the synthesis of said RNA monomers, oligoribonucleotides and RNA polynucleotides, as well as methods for their deprotection and methods for the use of said compounds and compositions comprising said compounds. In particular, such compounds and compositions comprising them are used in methods for light-directed synthesis of RNA microarrays.
1. A method of synthesizing an oligoribonucleotide on a solid substrate, the method comprising:
a) immobilizing on the solid substrate a first ribonucleotide monomer comprising the compound of formula (I) to create an immobilized ribonucleotide monomer;
b) removing a protecting group so as to create a deprotected immobilized ribonucleotide monomer;
c) contacting the deprotected immobilized ribonucleotide monomer with a second ribonucleotide monomer comprising the compound of formula (I), under conditions such that the second ribonucleotide monomer couples with the deprotected immobilized ribonucleotide monomer to produce, in situ, the oligoribonucleotide on the solid substrate,
wherein the compound of formula (I) is:
wherein
B is a member selected from the group consisting of 6-amino-9H-purin-9-yl, 2-amino-1H-purin-6(9H)-on-9-yl, 4-aminopyrimidin-2(1H)-on-1-yl, pyrimidine-2,4(1H,3H)-dion-1-yl, N4-levulinylcytosine, N2-levulinylguanine, N2-(dimethylformamidine)guanine, N2-phenoxyacetylguanine, N6-(tert-butylphenoxyacetypadenine, N6-(9-fluorenylmethoxycarbonyl)adenine, N2-(9-fluorenylmethoxycarbonyl)guanine, N4-benzoylcytosine, N6-benzoyladenine, N4-isobutyrylcytosine, N4-acetylcytosine, and N2-isobutyrylgua nine;
P 1 is hydrogen or —CH 2 OC(O)CH 2 CH 2 C(O)CH 3 ;
P 2 is hydrogen or
or salts thereof, wherein R is methyl, 2-cyanoethyl, 2-chlorophenyl, or 4-chlorophenyl;
or
P 3 is hydrogen or an O-protecting group, which is a member selected from the group consisting of:
a. a base labile group selected from —CH 2 OC(O)CH 2 CH 2 C(O)CH 3 and —C(O)CH 2 CH 2 C(O)CH 3 ;
b. an acid labile group selected from 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl, 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl, 4-(N-dichloroacetyl-N-methylamino)benzyloxymethyl, dimethoxytrityl and monomethoxytrityl;
c. a reduction labile group selected from 2-tert-butyldithiomethyl and allyl;
d. a fluoride labile group selected from tert-butyldimethylsilane, 2′-O-triisopropylsilyloxymethyl, cyanoethylmethyl, and 2-(4-tolylsulfonyl)ethoxymethyl; and
e. a photolabile group selected from 2-(2-nitrophenyl)propoxycarbonyl, α-methylnitorpiperonyloxycarbonyl, and 5′-O-dimethoxybenzoincarbonate; and
wherein at least one of P 1 and P 3 is —CH 2 OC(O)CH 2 CH 2 C(O)CH 3 as the O-protecting group.
2. The method of claim 1 , wherein the first ribonucleotide monomer has a 2′-O-acetal levulinyl ester protecting group and a 5′-2-(2-nitrophenyl)propoxycarbonyl protecting group and the immobilized ribonucleotide monomer is irradiated to remove the 5′-2-(2-nitrophenyl)propoxycarbonyl protecting group.
3. The method of claim 1 , wherein the immobilized ribonucleotide monomer is irradiated with light.
4. The method of claim 1 , wherein the first ribonucleotide monomer is immobilized to the solid substrate via a linker.