Fluorescent 5-Pyrimidine and 8-Purine Nucleosides Modified with an N-Unsubstituted 1,2,3-Triazol-4-yl Moiety

Abstract

The Cu(I)- or Ag(I)-catalyzed cycloaddition between 8-ethynyladenine or guanine nucleosides and TMSN₃ gave 8-(1-H-1,2,3-triazol-4-yl) nucleosides in good yields. On the other hand, reactions of 5-ethynyluracil or cytosine nucleosides with TMSN₃ led to the chemoselective formation of triazoles via Cu(I)-catalyzed cycloaddition or vinyl azides via Ag(I)-catalyzed hydroazidation. These nucleosides with a minimalistic triazolyl modification showed excellent fluorescent properties with 8-(1-H-1,2,3-triazol-4-yl)-2′-deoxyadenosine (8-TrzdA), exhibiting a quantum yield of 44%. The 8-TrzdA 5′-triphosphate was incorporated into duplex DNA containing a one-nucleotide gap by DNA polymerase β.

Graphical Abstract

The natural nucleic acid has weak fluorescent properties.¹ However, modified nucleoside analogues with fluorescent properties serve as powerful tools to study nucleic acid interactions, activities, and structures.^2–9 The nucleoside derivatives with fluorescent nucleobases have been recently reviewed.¹⁰ The criteria⁴ for designing fluorescent nucleosides are (i) sensitiveness to the microenvironment, (ii) emission at long wavelengths, (iii) high quantum efficiency, and (iv) minimalistic modifications, which cause the least amount of distortion of natural hydrogen bonding.

Fluorescent properties of nucleosides bearing triazolyl modifications varied mostly by the (i) position of nucleobases, at which a triazolyl unit is attached, (ii) site of triazolyl attachment to the nucleobase (N1 vs C4), and (iii) additional substitutions either at nucleobase or triazolyl units. The syntheses of the N-substituted triazolyl analogues are mainly based on CuAAC and strain promoted click chemistry.^6,11–18 For example, the 8-(1H-1,2,3-triazol-4-yl)adenosine derivatives A and B (Figure 1), with triazolyl carbon attached to the C8 position of the purine ring, have quantum yields up to 64%,^14,19 while (1,2,3-triazol-1-yl)adenosines C and D, with triazolyl nitrogen attached to the C8 position of purine, display significantly lower quantum yields (0.21–1.7%).^6,15 The 2-(1,2,3-triazol-1-yl)adenosine analogues E and F with a triazole moiety at the C2 position have a moderate to relatively high quantum yield (2–20%).^6,11 The 5-azidouracil nucleosides click with cyclooctyne substrates, generating 5-(1,2,3-triazol-1-yl) nucleosides G for the living cell fluorescent imaging.⁶

Figure 1.

Structures and quantum yields of the selected triazoles

So far, only a few examples of nucleosides modified with the minimalistic (N-unsubstituted) (1H-1,2,3-triazol-4-yl) unit are reported. Thus, 5-(1H-1,2,3-triazol-4-yl)-2′-deoxyuridine (5-TrzdU) was synthesized by microwave-assisted CuAAC between the corresponding 5-ethynyluracil and in situ generated pivaloyloxymethyl (POM) azide followed by N-deprotection²⁰ or by the nucleobase-exchange reaction.²¹ N-POM-protected 5-TrzdU was incorporated into DNA via solid-phase synthesis.²⁰ The 5-TrzdU²⁰ and its analogues^17,22 stack in the major groove and increase the stability of the duplex DNA.

Strategies developed for the synthesis of N-unsubstituted triazoles include: (i) CuI-catalyzed [3+2] cycloaddition of terminal alkynes and trimethylsilyl azide (TMSN₃),²³ (ii) Pd-²⁴ or p-toluenesulfonic acid-catalyzed²⁵ cycloaddition between activated alkene and NaN₃, and (iii) deprotection of N-substituted triazoles.^12,26,27 Herein, we report the catalyst-dependent cycloaddition of acetylenic nucleosides with TMSN₃ for the synthesis of 5-pyrimidine and 8-purine nucleosides modified with a minimalistic N-unsubstituted 1,2,3-triazol-4-yl moiety and their fluorescent properties.

Synthesis.

Treatment of 8-ethynyl-2′-deoxyadenosine 1a²⁸ with TMSN₃ in the presence of CuI as a catalyst²³ gave 8-(1H-1,2,3-triazol-4-yl)-2′-deoxyadenosine (8-TrzdA, 2a; method A; Table 1, entry 1). Analogous treatment of TBDMS-protected 2′-deoxyadenosine 1b²⁸ with TMSN₃ yielded protected 8-TrzdA (2b, 47%; entry 2). 8-Ethynyl-2′-deoxyguanosine 1c²⁹ under analogous conditions provided 8-(1H-1,2,3-triazol-4-yl)-2′-deoxyguanosine (8-TrzdG, 2c; entry 3). Thus, cycloaddition of the 8-ethynylpurine nucleosides with TMSN₃ in the presence of CuI produced triazoles as sole products.

Table 1.

Synthesis of 8-(1H-1,2,3-Triazol-4-yl)purine Nucleosides

entry	compound 1	method	compound 2	yielda [%]	compound 3	yielda [%]
1	1a	A	2a	48 (52b)	3a	0
2	1b	A	2b	47	3b	0
3	1c	A	2c	31 (36b)	3c	0
4	1a	B	2a	62	3a	0
5	1b	B	2b	71	3b	0
6	1c	B	2c	52	3c	0
7	1a	C	2a	58	3a	15
8	1b	C	2b	55	3b	0
9	1c	Cc	2c	60	3c	0

^aIsolated yields. Products were purified on a silica gel column.

^bProducts were purified on RP-HPLC.

^c4 h.

To avoid both loss of catalytic ability of Cu(I) and colorization of the reaction mixture, we found that treatment of 1a with TMSN₃ in the presence of in situ generated Cu(I) from CuSO₄/sodium ascorbate gave 2a with a 62% isolated yield (method B, entry 4). Similar treatment of 1b and 1c with TMSN₃ afforded triazoles 2b (entry 5) and 2c (entry 6) in higher yields than that by method A.

We also found that Ag₂CO₃-catalyzed hydroazidation of 1a with TMSN₃ (in DMF with 2 equiv of H₂O) produced 2a (58%) in addition to the vinyl azide 3a (15%; method C, entry 7). Analogous treatment of 1b provided 2b without a detected trace of vinyl azide 3b (entry 8). Subjection 1c to method C for 4 h also yielded 2c as the sole product (entry 9). It is noteworthy that Ag₂CO₃-catalyzed reactions between arylacetylenes and TMSN₃ provide vinyl azides as main products.^30,31

Transition-metal-catalyzed cycloaddition of TMSN₃ with alkyne has been extended into the 5-ethynyl pyrimidine nucleosides 4a–d (methods A–C, Table 2). Thus, CuI-catalyzed cycloaddition of 3′,5′-di-O-acetyl-5-ethynyl-2′-deoxycytidine 4a³² yielded 5-(1H-1,2,3-triazol-4-yl) product 5a (method A; 45%, entry 1), while unprotected 2′-deoxycytidine substrate 4b³² provided 5-(1H-1,2,3-triazol-4-yl)-2′-deoxycyti-dine 5b (5-TrzdC, entry 2). Protected 4c³³ and unprotected 5-ethynyl-2′-deoxyuridine 4d³³ gave 5-(1H-1,2,3-triazol-4-yl) products 5c (65%, entry 3) and 5d (5-TrzdU; 62%, entry 4). It appears that H₂O is required for the reaction to proceed²³ since the mixture of DMF/H₂O (9:1) as a solvent gave the best yields of products 5a–d. Hydroazidation of 4d in the presence of 2 equiv of H₂O in DMF gave 5d (50%, entry 5) as a sole product, showing that a stoichiometric amount of H₂O was sufficient for cycloaddition to occur.

Table 2.

Synthesis of 5-(1H-1,2,3-Triazol-4-yl) and 5-(1-Azidovinyl)pyrimidine Nucleosides

entry	compound 4	method	compound 5	yielda [%]	compound 6	yielda [%]
1	4a	A	5a	45	6a	0
2	4b	A	5b	48b	6b	0
3	4c	A	5c	65	6c	0
4	4d	A	5d	62	6d	0
5	4d	Ac	5d	50	6d	0
6	4a	B	5a	65	6a	0
7	4b	B	5b	60	6b	0
8	4c	B	5c	82	6c	0
9	4d	B	5d	72	6d	0
10	4a	C	5a	7	6a	51
11	4c	C	5c	0	6c	52

^aIsolated yields. Products were purified on a silica gel column.

^bYield was 53% when 5b was purified on RP-HPLC.

^cH₂O (2 equiv).

The CuSO₄/sodium ascorbate-catalyzed cycloaddition of TMSN₃ to alkynes 4a–d produced 5a–d in higher yields (60–82%; method B, entries 6–9) than by method A. However, treatment of alkyne 4a with TMSN₃ in the presence of Ag₂CO₃ provided triazole 5a in a low yield (7%), affording instead the 5-(1-azidovinyl)cytosine nucleoside 6a³⁴ as a major product (51%, entry 10). The analogous hydroazidation of 4c produced 5-(1-azidovinyl) 6c (52%) as the sole product (entry 11). It appears that paths for these reactions between nucleoside alkynes and TMSN₃ depends not only on the catalyst used but also on the nature of the nucleobases, to which the alkyne group is attached, leading selectively to triazoles or vinyl azides.

The CuSO₄/sodium ascorbate-catalyzed synthesis of triazoles from alkynes and TMSN₃ (method B) has a general character as illustrated with p-substituted phenylacetylenes 7a–c (Scheme 1). Thus, aryl alkyne 7a with EDG (CH₃O) gave triazole product 8a (63%), while aryl alkyne 7c with EWG (CF₃) yielded triazole 8c in a higher yield of 83%. Electron-withdrawing groups (EWGs) are known to promote the formation of triazoles in higher yields.^23,35

Scheme 1.

Synthesis of p-Substituted Phenyl Triazoles by Method B

In summary, N-unsubstituted triazolyl analogues of the four natural bases of DNA were prepared by treatment of 5-ethynylpyrimidine or 8-ethynylpurine 2′-deoxynucleosides with TMSN₃ in the presence of CuI or CuSO₄/sodium ascorbate as a catalyst. Interestingly, Ag-catalyzed reactions of TMSN₃ with 8-alkyne purines gave mainly triazole products 2a–c (Table 1), while with 5-alkyne pyrimidines provided mainly Markovnikov addition products 6a or 6c (Table 2), probably due to electronic differences between pyrimidine and purine imidazole rings. NMR studies showed that triazolyl nucleosides 2 and 5 are stable in D₂O/DMSO-d₆ solution (500 μL/50 μL; 2 mg/mL) at 37 °C for 72 h.

A reaction mechanism for the formation of triazoles might involve [3+2] cycloaddition or an initial formation of vinyl azide followed by 1,5-electrocyclization and tautomerization. However, attempts to validate the latter mechanism by treatment with 8-(1-azidovinyl)-2′-deoxyadenosine 3a using method C failed to produce the desired triazole 2a (Scheme 2). Also treatment of 5-(1-azidovinyl)-2′-deoxyuridine 6d using method B did not produce triazole 5d. Thus, the formation of the triazolyl unit involving [3+2] cycloaddition²³ of alkyne with in situ generated HN₃ seems to be plausible (Figure S1).

Scheme 2.

Attempted Conversion of Vinyl Azide to Triazole

Enzymatic Incorporation of 8-TrzdATP into DNA.

Treatment of 8-TrzdA 2a with POCl₃ in the presence of a proton sponge³⁶ followed by the addition of tributylammonium pyrophosphate (TBAPP) and tributylamine (TBA) yielded 8-TrzdATP 9 (Scheme 3).

Scheme 3.

Synthesis of 8-TrzdATP and Its Incorporation into DNA by Human DNA Polymerase β (Pol β)

We also examined the incorporation of the triazolyl nucleotides into an oligonucleotide DNA substrate containing a nucleotide gap by pol β. The pol β efficiently incorporated 8-TrzdATP 9 into the one-nucleotide gap substrate containing a phosphate group on the downstream strand (Figure 2, Table S3). Incorporation of 8-TrzdATP increased with increasing concentrations of pol β (10–200 nM; lanes 2–6). Ten nM pol β was sufficient to insert 8-TrzdATP into the DNA to generate ~10% of the DNA synthesis product (lane 2). The 200 nM concentration of pol β resulted in 40% of incorporation of 8-TrzdATP (lane 6). Incorporation of 9 was extended by pol β in duplex DNA in the presence of dGTP (Figure S4). Polymerase-catalyzed incorporation of the 8-substituted purine nucleotides is known to depend on the size of the substituent and preference for syn/anti conformation of the base.^37,38

Figure 2.

(A) Incorporation of 8-TrzdATP 9 into duplex DNA containing a one-nucleotide gap by pol β. Substrates were ³²P-labeled at the 5′-end of the upstream strand of the substrate. Lane 1 indicates substrate only. Lanes 2–6 indicate the DNA synthesis product at increasing concentrations of pol β (10–200 nM). Lane 7 indicates the reaction with pol β without 8-TrzdATP. (B) Bar chart illustrating the quantification of the pol β DNA synthesis product.

Fluorescent Properties.

The normalized fluorescence emission, absorption, and excitation spectra for 1H-1,2,3-triazol-4-yl nucleosides in methanol are shown in Figure 3. Their photophysical data are summarized in Table 3. The 8-TrzdA 2a with the C4 of triazolyl attaching to the C8 of adenine exhibits the highest quantum yield (Φ_F) of 44% and emits at 300–480 nm with the maximum emission at 355 nm. The silyl-protected 8-TrzdA analogue 2b has a Φ_F of 48% (Table S1). The emission of 8-TrzdG 2c starts at 300 nm but extends to 540 nm, and the maximum emission is at 364 nm with a Φ_F of 9%. Emission λ_max of 2a is pH- and solvent-independent (Figure S2, Table S2).

Figure 3.

Normalized fluorescence emission, absorption, and excitation spectra for (A) 8-TrzdA, (B) 8-TrzdG, (C) 5-TrzdC, and (D) 5-TrzdU in MeOH. Absorption and excitation spectra were normalized to one at the absorption band of the lowest energy.

Table 3.

Photophysical Data for Compounds 2a, 2c, 5b, and 5d^a

	2a	2c	5b	5d
εmax (M−1 cm−1)	17950	14800	18750	12400
λmax (abs) (nm)	287	283	295	291
λmax (exc) (nm)	295	299	311	238
λmax (exc) (nm)	235		254	302
λmax (emi) (nm)	355	364	407	408
Stokes shift (nm)	68	65	112	117
ΦF	0.44	0.09	0.02	0.004
τ1 (ns)	0.69	0.61	0.10	0.14
τ2 (ns)	2.07	3.22	4.35	1.06
τaverage (ns)	1.55	2.47	3.72	0.805
f1 (%)	37	28	15	27
f2 (%)	63	71	85	73

^aIn MeOH

The 5-pyrimidine analogues 5-TrzdU 5d and 5-TrzdC 5b showed a larger Stokes shift of ~110 nm with a maximum emission approximately at 408 nm and lower quantum yields. The 5-TrzdC emits at 320–550 nm (Φ_F = 2%), while 5-TrzdU emits at 320–500 nm (Φ_F = 0.4%). Acetyl-protected 5a and 5c have similar fluorescent properties (Table S1). The emission spectra of 5b show a strong pH dependence in aqueous phosphate buffer with a bathochromic shift of the emission band maximum from 378 nm (pH 4.0) to 435 nm (pH 7.0) and to 433 nm (pH 12.0). Emission maxima in DMSO and ACN are similar to those observed in phosphate buffer at a neutral and basic pH (Figure S3, Table S2).

All triazoles showed biphasic fluorescence decay. The triazoles present a fast lifetime of 0.1–4.4 ns (Table 3). Compound 5b shows the longest lifetime of 4.35 ns and longest average lifetime of 3.7 ns. Compounds 2a (63%), 2c (71%), 5b (85%), and 5d (73%) showed a larger contribution of the long lifetime (τ₂).

Fluorescent N-unsubstituted 1,2,3-triazol-4-yl deoxynucleo-sides have been synthesized by catalyst-dependent reactions between 5-ethynylpyrimidine or 8-ethynylpurine nucleosides with TMSN₃. CuI or CuSO₄/sodium ascorbate-catalyzed cycloaddition gave triazoles products for both purine and pyrimidine nucleosides. In contrast, Ag₂CO₃-catalyzed reactions with 8-ethynylpurine nucleosides produced 8-triazolyls, whereas 5-ethynylpyrimidine nucleosides followed the hydroazidation pathway to give 5-(1-azidovinyl) as a major product. The triazoles showed good fluorescent properties with 8-TrzdA, exhibiting the highest quantum yield of 44%. 8-TrzdATP was incorporated into duplex DNA containing a one-nucleotide gap by human DNA polymerase β. Metabolic incorporation of 8-purine and 5-pyrimidine 1,2,3-triazol-4-yl nucleosides into DNA for fluorescent imaging and their antiviral and anticancer evaluation will be published elsewhere.³⁹

EXPERIMENTAL SECTION

General Information.

¹H NMR spectra at 400 MHz and ¹³C NMR at 100.6 MHz were recorded in DMSO-d₆ unless otherwise noted. All chemical shift values are reported in parts per million (ppm) and referenced to the residual solvent peaks of DMSO-d₆ (2.50 ppm) for ¹H NMR and DMSO-d₆ (39.52 ppm) peaks for ¹³C NMR spectra, with coupling constant (J) values reported in hertz (Hz). HRMS were obtained in TOF (ESI) mode. TLC was performed on Merck silica gel 60-F₂₅₄, and products were detected with 254 nm light. Merck silica gel 60 (230–400 mesh) was used for column chromatography. All reagents and solvents were purchased from commercial suppliers and used without further purification. Experimental procedures for the fluorescent characterization of the triazolyl nucleosides and protocols for the incorporation of 8-TrzdATP 9 by human DNA polymerase are detailed in the Supporting Information.

8-(1H-1,2,3-Triazol-4-yl)-2′-deoxyadenosine (2a).

Procedure A: CuI as a Catalyst.

The stirred solution of 1a²⁸ (27.5 mg, 0.1 mmol) in DMF/H₂O (1 mL, 9:1, v/v) was degassed with Ar for 15 min. TMSN₃ (26.3 μL, 23 mg, 0.2 mmol) and CuI (1 mg, 0.005 mmol) were then added, and the resulting mixture was further degassed for another 5 min and was stirred at 90 °C for 5 h. After the mixture cooled to ambient temperature, the volatiles were evaporated and the residue was column chromatographed (CHCl₃/MeOH, 100:0 → 85:15) to give 2a (15.0 mg, 48%): UV (MeOH) λ_max 203, 228, 287 nm (ε 15 900, 16 050, 17 950), λ_min 213, 250 nm (ε 13 800, 5100); ¹H NMR δ 2.20 (ddd, J = 12.9, 5.9, 1.7 Hz, 1H), 3.12–3.19 (m, 1H), 3.49–3.55 (m, 1H), 3.67–3.72 (m, 1H), 3.90 (q, J = 3.7 Hz, 1H), 4.46–4.51 (m, 1H), 5.27 (d, J = 3.7 Hz, 1H), 5.77–5.80 (m, 1H), 7.07 (t, J = 7.2 Hz, 1H), 7.51 (s, 2H), 8.13 (s, 1H), 8.44 (s, 1H), 15.72 (s, 1H); ¹³C{¹H} NMR δ 38.1, 62.4, 71.6, 85.9, 88.4, 119.4, 130.6, 137.8, 141.6, 149.8, 152.1, 156.1; HRMS (ESI) m/z calcd for C₁₂H₁₄N₈O₃Na [M + Na]⁺ 341.1081, found 341.1062.

Note that purification of the crude reaction mixture on RP-HPLC (solvent A, 100% ACN; solvent B, 5% ACN/H₂O; gradient 0% A → 15% A in 30 min, flow rate = 2 mL/min) gave 2a (52%).

Procedure B: CuSO₄/Sodium Ascorbate as a Catalyst.

The stirred solution of 1a (27.5 mg, 0.1 mmol) and CuSO₄·5H₂O (2.5 mg, 0.01 mmol) in DMF/H₂O (1 mL, 9:1, v/v) was degassed with Ar for 15 min. TMSN₃ (26.3 μL, 23 mg, 0.2 mmol) and sodium ascorbate (4 mg, 0.02 mmol) were then added, and the resulting mixture was further degassed for another 5 min and was stirred at 90 °C for 5 h. After the mixture was cooled to ambient temperature, the volatiles were evaporated and the residue was column chromatographed (CHCl₃/MeOH, 100:0 → 85:15) to give 2a (19.7 mg, 62%) with the spectroscopic data as described above.

Procedure C: Ag₂CO₃ as a Catalyst.

Ag₂CO₃ (2.8 mg, 0.01 mmol) was added to a solution of 1a (27.5 mg, 0.1 mmol), TMSN₃ (26.3 μL, 23 mg, 0.2 mmol), and H₂O (3.6 μL, 3.6 mg, 0.2 mmol) in DMF (1 mL). The resulting mixture was stirred at 80 °C for 1 h. After the mixture cooled to ambient temperature, the volatiles were evaporated under the reduced pressure and the residue was column chromatographed (CHCl₃/MeOH, 100:0 → 85:15) to give 3a (4.8 mg, 15%, see below for spectroscopic characterization) followed by 2a (18.4 mg, 58%).

3′,5′-Di-O-tert-butyldimethylsilyl-8-(1H-1,2,3-triazol-4-yl)-2′-deoxyadenosine (2b).

Treatment of 1b²⁸ (201.6 mg, 0.4 mmol) with CuI by Procedure A (column chromatography; hexane/EtOAc 50:50 → 0:100) gave 2b (103.2 mg, 47%): UV (MeOH) λ_max 225, 285 nm (ε 17 500, 14 100), λ_min 247 nm (ε 3900); ¹H NMR δ −0.13 (s, 3H), −0.07 (s, 3H), 0.12 (s, 6H), 0.77 (s, 9H), 0.90 (s, 9H), 2.20–2.27 (m, 1H), 3.57–3.66 (m, 2H), 3.77 (dd, J = 9.0, 4.7 Hz, 1H), 3.88 (dd, J = 10.8, 6.0 Hz, 1H), 4.88 (“q”, J = 4.2 Hz, 1H), 7.01 (t, J = 6.5 Hz, 1H), 7.36 (s, 2H), 8.13 (s, 1H), 8.41 (s, 1H); ¹³C{¹H} NMR δ −5.6, −5.5, −4.9, −4.7, 17.8, 17.9, 25.6, 25.7, 36.4, 62.3, 72.2, 84.5, 86.6, 119.3, 130.6, 138.0, 142.0, 150.2, 152.3, 156.0; HRMS (ESI) m/z calcd for C₂₄H₄₃N₈O₃Si₂ [M + H]⁺ 547.2991, found 547.3004.

Treatment of 1b (201.6 mg, 0.4 mmol) with CuSO₄/sodium ascorbate by Procedure B (column chromatography; hexane/EtOAc 50:50 → 0:100) gave 2b (155.6 mg, 71%).

Treatment of 1b (201.6 mg, 0.4 mmol) with Ag₂CO₃ by Procedure C (column chromatography; hexane/EtOAc 50:50 → 0:100) gave 2b (120.4 mg, 55%).

8-(1H-1,2,3-Triazol-4-yl)-2′-deoxyguanosine (2c).

Treatment of 1c²⁹ (29.1 mg, 0.1 mmol) with CuI by Procedure A (column chromatography; CHCl₃/MeOH, 95:5 → 80:20) gave 2c (10.2 mg, 31%): UV (MeOH) λ_max 205, 283 nm (ε 14 450, 14 800); λ_min 238 (ε 2600); ¹H NMR δ 2.07–2.13 (m, 1H), 3.12–3.19 (m, 1H), 3.50 (dd, J = 11.7, 5.3 Hz, 1H), 3.65 (dd, J = 11.8, 4.8 Hz, 1H), 3.78–3.81 (m, 1H), 4.38–4.44 (m, 1H), 5.05 (“s”, 1H), 5.18 (d, J = 3.7 Hz, 1H), 6.43 (s, 2H), 8.34 (t, J = 7.8 Hz, 1H), 10.83 (s, 1H), 15.42 (s, 1H); ¹³C{¹H} NMR δ 37.4, 62.3, 71.4, 84.8, 88.0, 117.6, 128.8, 138.8, 151.9, 153.1, 154.0, 156.5; HRMS (ESI) m/z calcd for C₁₂H₁₅N₈O₄ [M + H]⁺ 335.1211, found 335.1214.

Note that purification of the crude reaction mixture on RP-HPLC (C18, A, 100% ACN; B, 5% ACN/H₂O; 0% A → 15% A in 30 min, flow rate = 2 mL/min) gave 2c (36%).

Treatment of 1c (29.1 mg, 0.1 mmol) with CuSO₄/sodium ascorbate by Procedure B (column chromatography; CHCl₃/MeOH, 95:5 → 80:20) gave 2c (26.0 mg, 52%).

Treatment of 1c (29.1 mg, 0.1 mmol) with Ag₂CO₃ by Procedure C (column chromatography; CHCl₃/MeOH, 95:5 → 80:20) gave 2c (30.1 mg, 60%).

8-(1-Azidovinyl)-2′-deoxyadenosine (3a):

4.8 mg, 15%; Procedure C, see above; ¹H NMR δ 2.19 (ddd, J = 13.1, 6.3, 2.3 Hz, 1H), 3.20–3.27 (m, 1H), 3.47–3.54 (m, 1H), 3.67 (dt, J = 11.8, 4.0 Hz, 1H), 3.89 (q, J = 4.1 Hz, 1H), 4.46–4.51 (m, 1H), 5.31 (d, J = 4.1 Hz, 1H), 5.40 (d, J = 2.0 Hz, 1H), 5.46 (dd, J = 8.1, 4.0 Hz, 1H), 5.56 (d, J = 2.0 Hz, 1H), 6.36 (dd, J = 8.1, 6.4 Hz, 1H), 7.56 (s, 2H), 8.15 (s, 1H); ¹³C{¹H} NMR δ 37.4, 62.1, 71.3, 85.6, 88.4, 108.7, 118.6, 134.2, 143.9, 149.5, 152.8, 156.4; HRMS (ESI) m/z calcd for C₁₂H₁₄N₈O₃Na⁺ [M + Na]⁺ 341.1081, found 341.1088.

3′,5′-Di-O-Acetyl-5-(1H-1,2,3-triazol-4-yl)-2′-deoxycyti-dine (5a).

Treatment of 4a⁴⁰ (134.0 mg, 0.4 mmol) with CuI by Procedure A (column chromatography; CHCl₃/MeOH, 100:0 → 90:10) to give 5a (67.6 mg, 45%): UV (MeOH) λ_max 208, 238, 293 nm (ε 13 050, 9400, 4150), λ_min 225, 271 nm (ε 8350, 3150); ¹H NMR δ 1.99 (s, 3H), 2.08 (s, 3H), 2.36 (ddd, J = 14.1, 5.8, 2.0 Hz, 1H), 2.44–2.47 (m, 1H), 4.20–4.23 (m, 1H), 4.26–4.35 (m, 2H), 5.20–5.22 (m, 1H), 6.21 (dd, J =7.6, 6.2 Hz, 1H), 7.67 (s, 1H), 7.90 (s, 1H), 8.07 (s, 1H), 8.24 (s, 1H), 15.28 (s, 1H); ¹³C{¹H} NMR δ 20.5, 20.7, 36.6, 63.7, 74.3, 81.6, 86.0, 97.2, 126.9 (HMQC showed a cross peak to proton at 8.24 ppm), 139.8, 153.6, 162.4, 170.0, 170.2; HRMS (ESI) m/z calcd for C₁₅H₁₉N₆O₆⁺ [M + H]⁺ 379.1361, found 379.1372.

Treatment of 4a (134.0 mg, 0.4 mmol) with CuSO₄/sodium ascorbate by using Procedure B (column chromatography; CHCl₃/MeOH, 100:0 → 90:10) gave 5a (97.6 mg, 65%).

5-(1H-1,2,3-Triazol-4-yl)-2′-deoxycytidine (5b).

Treatment of 4b⁴⁰ (25.1 mg, 0.1 mmol) with CuI by using Procedure A (column chromatography; CHCl₃/MeOH, 100:0 → 80:20) gave 5b (13.9 mg, 48%): UV (MeOH) λ_max 207, 238, 296 nm (ε 18 750, 13 900, 5500), λ_min 224, 273 nm (ε 11 900, 3500); ¹H NMR δ 2.08–2.14 (m, 1H), 2.21 (ddd, J = 13.2, 6.1, 4.7 Hz, 1H), 3.59–3.66 (m, 1H), 3.68–3.75 (m, 1H), 3.82 (q, J = 3.3 Hz, 1H), 4.24–4.30 (m, 1H), 5.24 (s, 1H), 5.29 (s, 1H), 6.18 (t, J = 6.1 Hz, 1H), 7.68 (s, 1H), 7.80 (s, 1H), 8.07 (s, 1H), 8.60 (s, 1H), 15.18 (s, 1H); ¹³C{¹H} NMR δ 41.4, 61.2, 70.0, 85.9, 88.0, 96.9, 126.8, 140.6, 142.0, 154.2, 162.7; HRMS (ESI) m/z calcd for C₁₁H₁₅N₆O₄⁺ [M + H]⁺ 295.1149, found 295.1160.

Note that the purification of the crude reaction mixture on RP-HPLC (C18, A, 100% ACN; B, 5% ACN/H₂O; 0% A → 15% A in 30 min, flow rate = 2 mL/min) gave 5b (53%).

Treatment of 4b (25.1 mg, 0.1 mmol) with CuSO₄/sodium ascorbate by using Procedure B (column chromatography; CHCl₃/MeOH, 100:0 → 80:20) gave 5b (17.4 mg, 60%).

3′,5′-Di-O-Acetyl-5-(1H-1,2,3-triazol-4-yl)-2′-deoxyuridine (5c).

Treatment of 4c³³ (33.6 mg, 0.1 mmol) with CuI by using Procedure A (column chromatography; CHCl₃/MeOH, 100:0 → 92:8) gave 5c (24.6 mg, 65%): UV (MeOH) λ_max 231, 293 nm (ε 10 700, 9600), λ min 258 nm (ε 2900); ¹H NMR δ 2.08 (s, 3H), 2.13 (s, 3H), 2.36–2.46 (m, 2H), 4.22–4.26 (m, 2H), 4.27–4.31 (m, 1H), 5.21–5.26 (m, 1H), 6.25 (t, J = 6.3 Hz, 1H), 8.18 (s, 1H), 8.31 (s, 1H), 11.82 (s, 1H), 15.19 (s, 1H); ¹³C{¹H} NMR δ 20.7, 20.8, 36.7, 63.8, 74.2, 81.7, 84.9, 105.5, 128.2 (HMQC showed a cross peak to proton at 8.18 ppm), 135.8, 149.6, 161.2, 170.1, 170.4; HRMS (ESI) m/z calcd for C₁₅H₁₈N₅O₇⁺ [M + H]⁺ 380.1201, found 380.1208.

Treatment of 4c (33.6 mg, 0.1 mmol) with CuSO₄/sodium ascorbate by using Procedure B (column chromatography; CHCl₃/MeOH, 100:0 → 92:8) gave 5c (31.0 mg, 82%).

5-(1H-1,2,3-Triazol-4-yl)-2′-deoxyuridine (5d).

Treatment of 4d³³ (25.1 mg, 0.1 mmol) with CuI by using Procedure A (column chromatography; CHCl₃/MeOH, 100:0 → 85:15) gave 5d^20,21 (18.4 mg, 62%): UV (MeOH) λ_max 231, 292 nm (ε 12 400, 11 450), λ_min 259 nm (ε 3700); ¹H NMR δ 2.18 (dd, J = 6.3, 4.7 Hz, 2H), 3.55–3.63 (m, 2H), 3.84 (q, J = 3.4 Hz, 1H), 4.25–4.31 (m, 1H), 5.04 (“s”, 1H), 5.29 (d, J = 4.1 Hz, 1H), 6.22 (t, J = 6.6 Hz, 1H), 8.14 (s, 1H), 8.49 (s, 1H), 11.68 (s, 1H), 15.10 (s, 1H); ¹³C{¹H} NMR δ 39.9, 61.3, 70.6, 84.7, 87.6, 105.1, 131.8, 135.8, 137.0, 149.7, 161.2; HRMS (ESI) m/z calcd for C₁₁H₁₄N₅O₅⁺, [M + H]⁺ 296.0989, found 296.0983.

Treatment of 4d (25.1 mg, 0.1 mmol) with CuI by using modified Procedure A with 2 equiv of H₂O and DMF as a solvent (column chromatography; CHCl₃/MeOH, 100:0 → 85:15) gave 5d (14.9 mg, 50%).

Treatment of 4d (25.1 mg, 0.1 mmol) with CuSO₄/sodium ascorbate by using Procedure B (column chromatography; CHCl₃/MeOH, 100:0 → 85:15) gave 5d (21.2 mg, 72%).

3′,5′-Di-O-Acetyl-5-(1-azidovinyl)-2′-deoxycytidine (6a).

Treatment of 4a (134.0 mg, 0.4 mmol) with Ag₂CO₃ by using Procedure C (column chromatography; CHCl₃/MeOH, 100:0 → 90:10) gave 6a (75.6 mg, 51%) followed by 5a (10.4 mg, 7%). Compound 6a: ¹H NMR (CDCl₃) δ 2.02–2.14 (m, 1H), 2.08 (s, 3H), 2.10 (s, 3H), 2.75 (ddd, J = 14.3, 5.5, 2.0 Hz, 1H), 4.31–4.41 (m, 3H), 5.03 (d, J = 1.5 Hz, 1H), 5.11 (d, J = 1.4 Hz, 1H), 5.21 (dt, J = 6.3, 1.8 Hz, 1H), 5.83 (s, 2H) 6.28 (dd, J = 8.0, 5.6 Hz, 1H), 7.80 (s, 1H); ¹³C[¹H] NMR (CDCl₃) δ 20.8, 21.0, 39.0, 64.0, 74.4, 82.9, 86.8, 101.8, 103.0, 139.9, 140.2, 154.3, 162.7, 170.4, 170.6; HRMS (ESI) m/z calcd for C₁₅H₁₉N₆O₆⁺ [M + H]⁺ 379.1361, found 379.1355.

3′,5′-Di-O-Acetyl-5-(1-azidovinyl)-2′-deoxyuridine (6c).

Treatment of 4c (67.2 mg, 0.2 mmol) with Ag₂CO₃ by using Procedure C (hexane/EtOAc 50:50) gave 6c⁴¹ (41.2 mg, 52%): ¹H NMR δ 2.07 (s, 6H), 2.33–2.46 (m, 2H), 4.24–4.29 (m, 3H), 5.06 (“s”, 1H), 5.19–5.21 (m, 1H), 6.00 (“s”, 1H), 6.14 (t, J = 6.4 Hz, 1H), 7.83 (s, 1H), 11.73 (s, 1H); ¹³C[¹H] NMR δ 20.4, 20.8, 36.7, 63.8, 74.2, 81.8, 85.4, 101.6, 107.3, 136.9, 138.3, 149.3, 160.9, 170.1, 170.2; HRMS (ESI) m/z calcd for C₁₅ H₁₈N₅O₇⁺ [M + H]⁺ 380.1201, found 380.1195.

4-(4-Methoxyphenyl)-1H-1,2,3-triazole (8a).

Treatment of 7a (13.2 mg, 0.1 mmol) with CuSO₄/sodium ascorbate by using Procedure B (column chromatography; hexane/EtOAc, 100:0 → 70:30) gave 8a^23,42 (11 mg, 63%): ¹H NMR δ 3.80 (s, 3H), 7.02 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 8.8 Hz, 2H), 8.22 (s, 1H); ¹³C{¹H} NMR δ 55.6, 114.5, 114.8, 123.3, 127.4, 145.8, 159.7; HRMS (ESI) m/z calcd for C₉H₁₀N₃O⁺ [M +H]⁺ 176.0818, found 176.0822.

4-Phenyl-1H-1,2,3-triazole (8b).

Treatment of 7b (20 mg, 0.2 mmol) with CuSO₄/sodium ascorbate by using Procedure B (column chromatography; hexane/EtOAc, 100:0 → 70:30) gave 8b^23,42 (21 mg, 73%): ¹H NMR δ 7.39 (t, J = 7.2 Hz, 1H), 7.50 (t, J = 7.5 Hz, 2H), 7.86 (d, J = 7.3 Hz, 2H), 8.34 (s, 1H); ¹³C{¹H} NMR δ 126.0, 127.7, 128.5, 129.4, 130.8, 145.6; HRMS (ESI) m/z calcd for C₈H₈N₃⁺ [M + H]⁺ 146.0713, found 146.0708.

4-(4-Trifluoromethylphenyl)-1H-1,2,3-triazole (8c).

Treatment of 7c (17 mg, 0.1 mmol) with CuSO₄/sodium ascorbate by using Procedure B (column chromatography; hexane/EtOAc, 100:0 → 70:30) gave 8c⁴² (17.6 mg, 83%): ¹H NMR δ 7.82 (d, J = 8.4 Hz, 2H), 8.10 (d, J = 8.1 Hz, 2H), 8.54 (s, 1H); ¹³C{¹H} NMR δ 124.7 (q, J = 271.8 Hz), 126.4 (q, J = 3.8 Hz), 126.5, 127.9, 128.8 (q, J = 31.9 Hz), 135.0, 144.7; ¹⁹F NMR δ −1.0; HRMS (ESI) m/z calcd for C₉H₇F₃N₃⁺ [M +H]⁺ 214.0587, found 214.0593.

8-(1H-1,2,3-Triazol-4-yl)-2′-deoxyadenosine 5′-triphosphate (8-TrzdATP, 9).

POCl₃ (28 μL, 46 mg, 0.3 mmol) was added to a stirred solution of 8-TrzdA 2a (48 mg, 0.15 mmol) and proton sponge (80 mg, 0.375 mmol) in (MeO)₃PO (2 mL) at 0 °C. The resulting mixture was stirred at 0 °C for 30 min; then tributylammomium pyrophosphate solution in DMF (0.5 M; 1.5 mL, 0.75 mmol) followed by Bu₃N (106.8 μL, 83.4 mg, 0.45 mmol) were added, and stirring was continued at 0 °C for 2 min. The reaction was quenched by adjusting the pH to 7.5 with 2 M TEAB buffer. The residue was dissolved in H₂O (5 mL) and was extracted with EtOAc (3 × 5 mL). The water layer was evaporated and coevaporated with a mixture of EtOH/H₂O (1:1, 5 mL). The residue was column chromatographed (DEAE–Sephadex, TEAB 0.1 M → 0.6 M) and the appropriate fractions were evaporated in a vacuum and coevaporated 5 times with a mixture of EtOH/H₂O (1:1, 10 mL) to give 8-TrzdATP triethylammonium salt 9 (33.4 mg, 30%): ¹H NMR (D₂O) δ 2.33 (ddd, J = 13.4, 6.5, 3.9, 1H), 3.22–3.26 (m, 1H), 4.04–4.10 (m, 1H), 4.14 (“q”, J = 5.2 Hz, 1H), 4.18–4.24 (m, 1H), 4.66 (“quint”, J = 3.9 Hz, 1H), 6.74 (t, J = 7.8 Hz, 1H), 8.20 (s, 1H), 8.41 (s, 1H); ³¹P NMR (D₂O) δ –23.22 (t, J = 21.0 Hz, 1P_β), −11.34 (d, J = 21.0 Hz, 1P_α), −10.22 (d, J = 21.0 Hz, 1P_γ); ¹³C{¹H} NMR (D₂O) δ 36.2, 65.2, 70.6, 84.3, 84.8, 118.6, 128.8, 136.1, 143.0, 149.9, 152.6, 155.0; HRMS m/z calcd for C₁₂H₁₆N₈O₁₂P₃ [M – H]^– 557.0106, found 557.0091.