6-azido and 6-azidomethyl uracil nucleosides

Abstract Azido nucleosides have been utilized for click reactions, metabolic incorporation into cellular DNA, and fluorescent imaging of live cells. Two classes of 6-azido modified uracil nucleosides; one with azido group directly attached to uracil ring and second with azido group attached via methylene linker are described. The 6-azido-2’-deoxyuridine (6-AdU) was prepared in 55% overall yield by lithiation-based regioselective C6-iodination of silyl protected 2’-deoxyuridine followed by treatment with sodium azide and deprotection with TBAF. Lithiation-based C6-alkylation of the protected uridine with methyl iodide followed by the oxidation of the 6-methyl product with selenium dioxide and the subsequent mesylation and azidation of the resulting 6-hydroxymethyl group gave after deprotection 6-azidomethyluridine (6-AmU) in 61% overall yield. Direct lithiation-based C6-hydroxymethylation followed by mesylation/azidation sequence and deprotection provided 6-AmU or 6-azidomethyl-2’-deoxyuridine (6-AmdU). Yields for the lithiation-based regioselective C6-iodination and alkylation were higher for uridine than 2’-deoxyuridine derivatives and they appear to be less dependent on the sugar protection group used. Strain promoted click reactions of 6-AdU and 6-AmdU with symmetrically fused cyclopropyl cyclooctyne (OCT) provided fluorescent triazoles. DFT-calculated dihedral angles and energy differences for the favored anti and syn conformation of 6-AdU and 6-AmdU versus their C5 azido counterparts are discussed. Graphical abstract

[34][35] As part of our continuing investigations on azido modified nucleosides as precursors to NCRs, herein we report 6-azido modified uracil nucleosides with azido group attached to uracil ring directly (6-AdU) or via methylene linker (6-AmU or 6-AmdU), their strain promoted click reactions with cyclooctyne, and DFT-calculated energy differences for the favored anti and syn conformation versus their C5 azido counterparts.
The 6-azidomethyl-2′-deoxyuridine (6-AmdU, 15) was synthesized beginning with C6-hydroxylmethylation [27] of 3′,5′-O-TIPDS-2′-deoxyuridine 1a with ethyl formate followed by reduction with NaBH 4 to yield 11 (37%; Scheme 5).Mesylation of the hydroxyl group in 11 followed by displacement reaction with azide gave 13 in 39% yield.Desilylation of 13 with NH 4 F in MeOH [37] (50 °C) provided 6-AmdU 15 (62%).It is noteworthy that deprotection with excess of NH 4 F in MeOH must be carried out cautiously and monitored by TLC to avoid degradation and formation of byproducts.In attempt to increase yields and to examine stability of the C6 substituted products, 3′,5′-O-TBDMS-2′-deoxyuridine 1b was subjected to the same sequence of reactions.Thus, 6-azidomethyl 14 was obtained in 45% yield from 12 but deprotection with  NH 4 F/MeOH led to glycosylic bond cleavage and isolation of methyl 2-deoxyribofuranose as major product.Attempted desilylation of 13 or 14 with TBAF failed to give 15.6-AmdU appears to be less stable than 5-AmdU, [12,21] as degradation of 6-AmdU was observed during deprotection with TBAF or NH 4 F/MeOH and purification on silica gel.Still, using silica gel deacidified with a diluted solution of methanolic ammonia (MeOH/NH 3 ) [38] allowed for purification of 15 with minimal loss of product.Less acidic Florosil® has been used [27] to purify C6-substituted deoxyuridine analogs to minimize degradation due to instability.Analysis of C6-alkylation of 2′-deoxyuridine 1a-b vs uridine 5 derivatives revealed that yields for C6-hydroxylmethylation decreased from 67% for 5 to 37% and 33% for 1a, and 1b.This effect is likely attributed to the reported instability differences between C6-substituted ribose and 2-deoxyribose nucleosides. [27]Same trend was also observed for the C6-iodination of uridine [26,39] vs 2′-deoxyuridine 2 as yields decreased from 75% to 40%.Overall, yields for the lithiation-based regioselective C6-iodination and alkylation are much higher for uridine than 2′-deoxyuridine derivatives and they appear to be less dependent on the sugar protection group used.
To examine the potential of 6-AdU and 6-AmdU for metabolic labeling and fluorescence imaging, [40] strain promoted click reactions with symmetrically fused cyclopropyl cyclooctyne (OCT) 16 were tested.The reaction between 6-AdU (4) and 16 (1.3equiv.) in MeOH at ambient temperature proceeded slowly but heating at 50 °C for 18 h produced  2. The 6-TrzdU 17 emits a band at 330-580 nm with a maximum emission at 410 nm.The 6-mTrzdU 18 emits a band at 275-380 nm with a maximum emission at 305 nm.The 6-TrzdU 17 showed larger Stokes shift of 103 nm compared to 26 nm for compound 18.42] The preferred syn, C3'-endo (North) puckering of C6-substituted nucleosides like 6-methyluridine is attributed to the minimization of repulsive interactions between the sugar substituents and the C2 oxygen of the uracil base. [26,32,43,44]From the glycosidic torsional angle χ or (O4'-C1'-N1-C2) dihedral angle, the syn-or anti-conformation of C6 vs. C5-substituted nucleosides can be interpreted (Figure 2).Comparison of Scheme 6. strain-promoted click reaction of 6Adu 4 and 6Amdu 15 with cyclooctyne 16. the 1 H NMR spectra of 5-AmdU vs. 6-AmdU in D 2 O also provides insight into the conformation of 6-AmdU.Schweizer et al., studied 5-and 6-methyl substituted pyrimidine nucleosides and reported a significant deshielding at H2' and H3' along with the concomitant upfield shield for H1' in the 6-methyl derivatives, therefore concluding that 6-substituted derivatives are not in the normal anti conformation. [43]They attributed that deshielding and shielding effects of the sugar protons to C2-keto anisotropic effects.
A DFT computational study of 6-AmdU and 5-AmdU was conducted to further evaluate their syn and anti conformational preferences with respect to rotation around the N1-C1' bond defined by the (O4'-C1'-N1-C2) dihedral angle (Figure 2).In these calculations, we have employed the ωB97XD functional [45] with the −311 G** basis set for geometry optimizations of various conformations and computation of their vibrational frequencies utilizing the Gaussian 16 software package. [46]The calculated energies and frequencies were used to assess relative Gibbs free energies at 298 K.
Relative energy of conformers may also be affected by the presence of solvents.

Conclusion
In summary, 6-azido-and 6-azidomethyluridine and 2′-deoxyuridine derivatives (4, 10, or 15) were synthesized via regioselective lithiation-based C6-iodination or alkylation of the protected uridine or 2′-deoxyuridine substrates (1a or 5).Subsequent displacement of 6-iodo or mesyloxy group in 6-(mesyloxy)methyl substituents with NaN 3 followed by deprotections provided C6-azido modified uracil nucleosides.Yields for the C6-iodination and alkylation were higher for uridine than 2′-deoxyuridine derivatives, and they appear to be less depended on the sugar protection group used.Strain promoted click reactions of 6-AdU and 6-AmdU with symmetrically fused cyclopropyl cyclooctyne (OCT) provided fluorescent triazoles.DFT-calculated dihedral angles and energy differences for the anti and syn conformation of 6-AdU and 6-AmdU versus their C5 azido counterparts showed flexibility of these species with respect to rotation around the C1'-N1 bond.Studies on metabolic incorporation of these 6-azido uracil nucleosides into DNA for fluorescent imaging and their antiviral and anticancer evaluation will be published elsewhere.Lithium diisopropylamide (LDA) was generated from freshly distilled diisopropylamine (DIPA) and n-BuLi (2.5 M/hexanes) at 0 °C.For lithiation reactions, all starting materials were dried in vacuum pistol over (P 2 O 5 ) at refluxing MeOH for 5-8 h.Purification of 15 was performed on deacidified silica gel saturated for 1 h with methanolic ammonia [38] and then thoroughly flushed with DCM.

Figure 3 .
Figure 3. dFt-calculated dihedral angles and energy differences for the favored anti and syn conformations of 5-Amdu and 6-Amdu.

Table 1 .
optimization of the oxidation of 6-methyluridine 6 to 6-hydroxymethyl derivative 8 via route A.

Table 2 .
Photophysical data for 17 and 18 in MeoH.
13NMR spectra at 400 MHz and13C NMR at 100.6 MHz were recorded using a Bruker 400 MHz instrument with solutions in CDCl 3 , D 2 O, or MeOH-d 4 .All chemical shift values are reported in parts per million (ppm) and referenced to the residual solvent peaks of CDCl 3 (7.26pp),D 2 O (4.79 ppm), or MeOH-d 4 (3.31 ppm) for 1 H NMR and CDCl 3 (77.2ppm) oreOH-d 4 (49.15ppm)peaksfor13C NMR spectra, with coupling constant (J) values reported in Hz.HRMS were obtained in TOF (ESI) mode.TLC was performed on Merck Kieselgel 60-F 254 , and products were detected with 254 nm light.Merck Kieselgel 60 (230-400 mesh) was used for column chromatography.All reagents and solvents were purchased from commercial suppliers and dried using standard procedures.Cyclooctyne 16 was purchased from TCI and was used without further purification.Ethyl formate was stirred for 48 h over CaH 2 at rt and distilled prior to use.DCM and Et 3 N were stirred for 24 h at rt over CaH 2 and distilled prior to use. 1 C NMR δ −5.12, 18.62, 25.51, 26.07, 27.40, 51.22, 64.07, 81.87, 84.23, 89.60, 91.87, 103.95, 114.12, 149.67, 150.08, 161.50.