Oligonucleotide analogues incorporating 5-aza-cytosine therein
Oligonucleotide analogues are provided that incorporate 5-aza-cytosine in the oligonucleotide sequence, e.g., in the form of 5-aza-2′-deoxycytidine (decitabine) or 5-aza-cytidine. In particular, oligonucleotide analogues rich in decitabine-deoxyguanosine islets (DpG and GpD) are provided to target the CpG islets in the human genome, especially in the promoter regions of genes susceptible to aberrant hypermethylation. Such analogues can be used for modulation of DNA methylation, such as effective inhibition of methylation of cytosine at the C-5 position. Methods for synthesizing these oligonucleotide analogues and for modulating nucleic acid methylation are provided. Also provided are phosphoramidite building blocks for synthesizing the oligonucleotide analogues, methods for synthesizing, formulating and administering these compounds or compositions to treat conditions, such as cancer and hematological disorders.
1. An isolated or synthetic dinucleotide analogue, its salt or ester, comprising one 5-aza-cytosine as a base residue in the sequence of the dinucleotide analogue, wherein the dinucleotide is linked with a phospholinker consisting of one phosphorus and wherein the dinucleotide analogue has a general formula of:
a. Z-L-G, or G-L-Z, wherein Z is 5-aza-cytosine; G is guanine; and L is a chemical linker covalently linking Z and G; or
b. 5′-DpG-3′ or 5′GpD-3′, wherein D is decitabine; p is a phospholinker; and G is deoxyguanosine.
2. The dinucleotide analogue of claim 1 , wherein the dinucleotide analogue is 5′-DpG-3′ or 5′-GpD-3′, wherein D is decitabine; p is a phospholinker; and G is deoxyguanosine.
3. The dinucleotide analogue of claim 1 , wherein the dinucleotide analogue-has a general formula of:
Z-L-G, or G-L-Z, wherein Z is 5-aza-cytosine; G is guanine; and L is a chemical linker covalently linking Z and G.
4. The dinucleotide analogue of claim 1 , wherein the linker L or the phospholinker p is a sugar phosphorodiester linker.
5. The dinucleotide analogue of claim 1 , wherein the linker L or the phospholinker p is a ribose or 2′-deoxyribose phosphorodiester linker.
6. The dinucleotide analogue of claim 1 , wherein the linker L or the phospholinker p is a ribose or 2′-deoxyribose phosphorothioate linker.
7. The dinucleotide analogue of claim 1 , wherein the linker L or the phospholinker p is a ribose or 2′-deoxyribose boranophosphate linker.
8. The dinucleotide analogue of claim 1 , wherein the linker L or the phospholinker p is a ribose or 2′-deoxyribose methylphosphonate linker.
9. The dinucleotide analogue of claim 1 , wherein the linker L or the phospholinker p is 2′-methoxy ribose phosphorodiester linker.
10. The dinucleotide analogue of claim 1 , wherein the linker L or the phospholinker p is 2′-methoxyethyl ribose phosphorodiester linker.
11. The dinucleotide analogue of claim 1 , wherein the linker L or the phospholinker p is 2′-fluoro ribose phosphorodiester linker.
12. The dinucleotide analogue of claim 1 , wherein the salt is formed with an acid selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carboxylic, sulfonic, sulfo or phospho acids, acetic acid, proprionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid, isonicotinic acid, amino acid, glutamic acid, aspartic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxy-ethansulfonic acid, ethane-1,2-disulfonic acid, benzene-sulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid, and ascorbic acid.
13. The dinucleotide analogue of claim 1 , wherein the salt is hydrochloride, mesylate, EDTA, sulfite, L-Aspartate, maleate, phosphate, L-Glutamate, (t)-L-Tartrate, citrate, L-Lactate, succinate, acetate, hexanoate, butyrate, or propionate salt.
14. The dinucleotide analogue of claim 1 , wherein the salt is a sodium, calcium, lithium, potassium, ammonium, or trialkylammonium salt.
15. The dinucleotide analogue of claim 14 , wherein the salt is a trialkylammonium salt comprising a trimethylammonium, triethylammonium or tripropylammonium salt.
16. The dinucleotide analogue of claim 1 , wherein the dinucleotide analogue has a hydroxyl group at its 5′- or 3′-terminus that is substituted with a protecting group.
17. The dinucleotide analogue of claim 1 , wherein the dinucleotide analog comprises a non-hydroxyl protecting group selected from the group consisting of dimethoxytritoxyl (DMTr-O), O-methyl (OMe), ethylene glycol, tetraethylene glycol, hexaethylene glycol, and hexaethylene glycol phosphate.
18. A pharmaceutical composition comprising a dinucleotide analogue of claim 1 , or its pharmaceutically acceptable salt or ester, and a pharmaceutically-acceptable carrier.
19. The pharmaceutical composition according to claim 18 , the dinucleotide analogue of claim 1 or its pharmaceutically acceptable salt or ester in its solid form.
20. The dinucleotide analogue of claim 1 , wherein the dinucleotide analogue is 5′-DpG-3′, wherein D is decitabine; p is a phospholinker; and G is deoxyguanosine.