Oligonucleotides incorporating 5-(octa-1,7-diynyl)-2'-deoxycytidine 1a, 5-(octa-1,7-diynyl)-2'-deoxyuridine 2a and 7-deaza-7-(octa-1,7-diynyl)-2'-deoxyguanosine 3a, 7-deaza-7-(octa-1,7-diynyl)-2'-deoxyadenosine 4a were prepared. For this, the phosphoramidites 7, 10, and 13 were synthesized and employed in solid-phase oligonucleotide synthesis. The octa-1,7-diynyl nucleosides 1a- 4a were obtained from their corresponding iodo derivatives using the palladium-assisted Sonogashira cross-coupling reaction. The Tm values demonstrated that DNA duplexes containing octa-1,7-diynyl nucleosides show a positive influence on the DNA duplex stability when they are introduced at the 5-position of pyrimidines or at the 7-position of 7-deazapurines. The terminal alkyne residue of oligonucleotides were selectively conjugated to the azide residue of the nonfluorescent 3-azido-7-hydroxycoumarin ( 38) using the protocol of copper(I)-catalyzed [3 + 2] Huisgen--Sharpless--Meldal cycloaddition "click chemistry" resulting in the formation of strongly fluorescent 1,2,3-triazole conjugates. The fluorescence properties of oligonucleotides with covalently linked coumarin--nucleobase assemblies were investigated. Among the four modified bases, the 7-deazapurines show stronger fluorescence quenching than that of pyrimidines.
Toll-like receptors (TLRs) are key targets in the design of immunomodulating agents for use as vaccine adjuvants and anticancer treatments. The imidazoquinolines, imiquimod and resiquimod, have been shown to activate TLR-7 and -8 which in turn induce cytokine production as part of the innate immune response. Herein, we report the synthesis and discovery of a C7-methoxycarbonyl derivative of imiquimod that stimulates cytokine production but is devoid of TLR-7/8 activity. Data is presented that shows this analog not only induces IL-12p40 and TNF production, similar to that of imiquimod and resiquimod, but greatly enhances the production of IL-1β, a key cytokine involved in activation of CD4 T cells. It is further demonstrated that TLR-7/8 activation can be recovered by the addition of a C2-alkyl substituent to this newly discovered analog. The results support the existence of an alternative mechanism of action by which imidazoquinolines can stimulate cytokine production.
The syntheses of 7-deaza-7-fluoro-2¢-deoxyadenosine (1a), 7-deaza-7-fluoro-2¢-deoxyinosine (3a) and 7-deaza-7-fluoro-2¢-deoxyguanosine (3c) as well as of the 2¢-deoxy-2¢-fluoroarabinofuranosyl derivatives 1b, 3b are described. Starting materials were 6-chloro-7-fluoro-7-deazapurine (4-chloro-5-fluoropyrrolo[2,3-d]pyrimidine, 4b) and 2-pivaloylamino-6-chloro-7-fluoro-7-deazapurine (2-pivaloylamino-4-chloro-5-fluoropyrrolo[2,3-d]pyrimidine, 6). Nucleobase anion glycosylation of 4b or 6 with the halogenoses 7 or 9 furnished the protected b-D-nucleosides 8, 10 and 11, which were converted to compounds 1a, 1b by ammonolysis and to 2a-c by sodium methoxide treatment. The 2¢-deoxyinosine derivatives 3a, 3b and the guanosine analogue 3c were obtained from 2a-c. Conformational analysis of the nucleosides 1a, 1b was performed in solution and in the solid state. Single-crystal X-ray analyses showed that the sugar moiety of 1a exhibits the Sconformation (P = 164.8°, t = 40.1°). In solution the 2¢-fluoro substituents shift the sugar conformation slightly towards N.
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