Synthesis of Triazole-Linked SAM-Adenosine Conjugates: Functionalization of Adenosine at N-1 or N-6 Position without Protecting Groups

More than 150 RNA chemical modifications have been identified to date. Among them, methylation of adenosine at the N-6 position (m6A) is crucial for RNA metabolism, stability and other important biological events. In particular, this is the most abundant mark found in mRNA in mammalian cells. The presence of a methyl group at the N-1 position of adenosine (m1A) is mostly found in ncRNA and mRNA and is mainly responsible for stability and translation fidelity. These modifications are installed by m6A and m1A RNA methyltransferases (RNA MTases), respectively. In human, deregulation of m6A RNA MTases activity is associated with many diseases including cancer. To date, the molecular mechanism involved in the methyl transfer, in particular substrate recognition, remains unclear. We report the synthesis of new SAM-adenosine conjugates containing a triazole linker branched at the N-1 or N-6 position of adenosine. Our methodology does not require protecting groups for the functionalization of adenosine at these two positions. The molecules described here were designed as potential bisubstrate analogues for m6A and m1A RNA MTases that could be further employed for structural studies. This is the first report of compounds mimicking the transition state of the methylation reaction catalyzed by m1A RNA MTases.


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In this context, we recently described the synthesis of SAM-adenosine conjugates as first 67 transition state analogues for m 6 A RNA MTases and their use as tools for structural study [23,24].

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We showed that a SAM-adenosine conjugate containing a three-carbon linker tethering the analogue

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This deviation indicates that our bisubstrate analogues are not optimal. In this study, we pursue the (Scheme 5). Compound 9a was successively treated with methylamine, ZnBr2 and cesium fluoride 146 (CsF) to remove the benzoyl-, the Boc-and the TBS groups respectively to give 12 in 5% yield over 147 three steps after HPLC purification. In comparison, a two-step strategy from 9b, followed by HPLC 148 purification, led to the formation of 12 in 4% yield. These results seem to indicate that in the pathway 149 2, the removal of the two Boc groups is less efficient than the two steps required for the deprotection 150 of the benzoyl and Boc groups in the pathway 1 (Scheme 5).

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We applied the strategy used for the deprotection of compound 9a to compound 11a.

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Unfortunately, efforts to remove the protecting group of the exocyclic amine were unsuccessful.

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Indeed, using the same successive steps, we observed the formation of the N-methylated compound 156 13 in 8% yield as a mixture of E and Z imines (Scheme 6). Other attempts were conducted with bases methodology, the propargyl group was introduced in one step at the N-6 position of adenosine Scheme 8. Access to SAM-adenosine conjugate 12.

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The unprotected azido partner 17 was prepared following a two-step procedure in 36% yield

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General procedure A for CuAAC reaction: To a solution of alkyne (1 eq) in THF (13 mL/mmol),

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were successively added azido compound 6 or 7 (1.2 eq), CuSO4 (0.3 eq, in water 3 mL/mmol) and 326 sodium ascorbate (0.6 eq, in water 3 mL/mmol). The heterogeneous mixture was stirred at room 327 temperature for 16 h. EtOAc was added and the organic layer was washed with brine, dried over

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Compound 9b: Following the general procedure A for CuAAC, starting from alkyne 2b (50 mg,

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Compound 11b: Following the general procedure A for CuAAC, starting from alkyne 3b (50 mg,

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The resulting precipitate was filtered and dissolved in water/MeOH (5:1) and aqueous ammonia 572 (25%, 2 mL) was added. The reaction mixture was stirred at room temperature for 30 min and the 573 solvent was removed under reduced pressure to provide 5'-chloroadenosine. 64 The resulting

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were successively added azido compound 17 (1.5 eq), CuSO4 (0.3 eq, in water 500 mL) and sodium 585 ascorbate (0.6 eq, in water 500 mL). The mixture was stirred at room temperature for 16 h and then 586 concentrated in vacuo. The crude product was purified by HPLC to afford the desired compounds.