Azide–Tetrazole Equilibrium of C-6 Azidopurine Nucleosides and Their Ligation Reactions with Alkynes

Abstract

Facile syntheses of C-6 azidopurine ribonucleosides and 2′-deoxyribonucleosides have been developed. For silyl and acetyl protected as well as unprotected nucleosides, access to the azido derivatives could be readily attained via displacement of BtO⁻ from the O⁶-(benzotriazol-1-yl)inosine nucleosides by azide anion. Use of diphenylphosphoryl azide/DBU as a simple route to the acetyl-protected azido nucleosides was also evaluated, but this proved to be inferior. Since these azido nucleosides can exist in an azide•tetrazole equilibrium, the effect of solvent polarity on this equilibrium was investigated. Subsequently, a detailed analysis of Cu-mediated azide-alkyne (“click”) ligation was undertaken. Biphasic CH₂Cl₂/H₂O medium proved to be best for the ligation reactions, suppressing the undesired azide reduction that was competing. Interestingly, although the tetrazolyl isomer predominates (ca 80%) in CD₂Cl₂ and in CD₂Cl₂/D₂O, the Cu-catalyzed click reactions proceed smoothly with the silyl-protected ribo and 2′-deoxyribonucleosides, leading to the C–6 triazolyl products in good to excellent yields. Thus, depletion of azido form from the reaction mixture shifts the azide•tetrazole equilibrium, eventually resulting in complete consumption of azide and tetrazole. In several cases, major and minor azide-alkyne ligation products were observed and characterization data are provided for both. In order to confirm the regiochemistry leading to the major isomer, one product was crystallized and evaluated by X-ray crystallography. The Cu-catalyzed azide-alkyne ligation is clearly efficient and significantly superior to thermal reactions, which were slow. Biological evaluation showed low cytotoxicities for the agents, suggesting their usefulness as biological probes.

Introduction

The Huisgen azide-alkyne cycloaddition reaction, leading to the formation of triazoles, is one of the most atom economical transformations.^1-7 In 2002, the use of Cu^I-mediated azide-alkyne ligation was reported to proceed in a facile and regioselective manner, producing the 1,4-disubstituted triazoles in preference to the 1,5-isomers.⁸ Soon after, a convenient method involving the use of Cu^II was discovered that was more reliable and superior than that utilizing Cu^I.⁹ This method has become highly popular and has impacted areas such as polymer, materials, carbohydrate and biological chemistry.

Nucleosides are central to a variety of biological processes, and the ability to modify nucleosides via facile approaches is of high importance for developing novel applications in biochemistry, biology and medicine. The nucleoside scaffold has proven to be highly versatile, modification of which has resulted in a wide range of pharmacologically important entities. For example, modification of the adenosine core has the potential to yield new compounds such as those possessing activity at the adenosine receptors.^10,11 Azide-alkyne click reactions have found a variety of applications in nucleoside chemistry recently,¹² and the importance of click reactions in the field of nucleosides has recently been reviewed.¹³ However, in the current literature azidopurine nucleoside derivatives have received very little attention as reactive partners in these reactions. To our knowledge, there are only two reports of azide-alkyne ligation reactions wherein C–2 azidopurine nucleoside analogues have been utilized.^14,15 Since there could be marked differences in reactivity at the various positions of purine nucleosides and because the azide-alkyne ligation reactions of 6-azidopurine nucleosides could be a powerful tool for biomolecular modification, we became interested in this class of reactions. The present paper describes a facile approach to 6-azidopurine nucleosides, their behavior in solution and their ability to partner in Cu-catalyzed ligation reactions with alkynes.

Results and Discussion

Synthesis of the 6-Azidopurine Nucleoside Derivatives

At the beginning of our studies, the first question was whether a reliable method to synthesize 6-azidopurine ribo and 2′-deoxyribonucleosides could be found. Three methods have been reported in the literature for the synthesis of 6-azido-9-β-d-ribofuranosylpurine. The oldest method involves conversion of 6-chloro-9-β-d-ribofuranosylpurine to an unstable 6-hydrazino derivative followed by treatment with nitrous acid.¹⁶ The acidic conditions are obviously not compatible with the labile 2′-deoxyribonucleosides. In a second method, 6-methylsulfonyl-9-β-d-ribofuranosylpurine was converted to the azidopurine nucleoside.¹⁷ It has been noted in these reports that displacement of chloride from 6-chloro-9-β-d-ribofuranosylpurine by azide ion results in decomposition.^16,17 Consistent with these observations, more recent syntheses of 6-azido-9-β-d-ribofuranosylpurine¹⁸ and 6-azido-9-β-d-arabinofuranosylpurine¹⁹ by replacement of chloride with azide gave low 21% and 38.4% yields of the respective azidopurine nucleoside derivatives. Given these relatively unsatisfactory methods, we opted to develop a new synthesis of 6-azido-9-(2-deoxy-β-d-ribofuranosyl)purine as well as 6-azido-9-β-d-ribofuranosylpurine.

We have recently reported facile, high-yielding syntheses of O⁶-(benzotriazol-1-yl)inosine and 2′-deoxyinosine derivatives.^20-22 We reasoned that these compounds could be convenient precursors to the 6-azidopurine nucleoside derivatives (Scheme 1).

SCHEME 1. Synthesis of C-6 Azidopurine Nucleoside Analogues from the O⁶-(Benzotriazol-1-yl)inosine Derivatives.

As shown in Scheme 1, silylated 2′-deoxyinosine 1a and inosine 1b as well as the unprotected nucleosides 1e and 1f were converted to the O⁶-(benzotriazol-1-yl) derivatives via known procedures.²⁰ Using similar methodology, the previously undescribed acetate-protected compounds 2c and 2d were prepared as well. Table 1 summarizes the conditions used for the conversion of 2a–f to azido nucleosides 3a–f.

TABLE 1. Conditions for the Conversion of O⁶-(Benzotriazol-1-yl)inosine Derivatives 2a–f to the Azido Nucleosides 3a–f.

entry	substrate	conditions	product: yielda
1	2a	NaN3, DMSO, 40 °C, 2 h	3a: 99%
2	2b	NaN3, DMSO, 40 °C, 3.5 h	3b: 96%
3	2c	NaN3, DMSO, room temperature, 1 h	3c: 95%
4	2d	NaN3, DMSO, room temperature, 1 h	3d: 75%
5	2e	Polymer-supported N3−, H2O, 50 °C, 3.5 h	3e: 59%
6	2f	Polymer-supported N3−, DMF, 50 °C, 5 h	3f: 70%

^aYields of isolated and purified products.

Although reactions of 2a and 2b could be conducted in DMF as solvent, competing formation of small amounts of the N⁶,N⁶-dimethylamino nucleoside derivative prompted the use of DMSO. These reactions proceeded smoothly and in high yields. Reactions of the unprotected derivatives 2e and 2f were somewhat problematic considering solubility as well as ease of product isolation. In both cases, polymer-supported azide²³ proved to be useful for the conversion. With the deoxy derivative 2e, reasonable reaction was attained in water. We have demonstrated that O⁶-(benzotriazoly-1-yl) nucleoside analogues are relatively stable towards hydroxylic solvents such as water and alcohol under neutral conditions.^20,24 Thus, the modest yield in this case may not be due to hydrolysis. With the ribose derivative 2f, DMF proved to be optimal for workup and isolation reasons. In principle, cleavage of protecting groups in 3a–d should also readily yield the unprotected compounds 3e, f.

Next, we considered a more direct access to protected 6-azidopurine nucleosides by conversion of the amide carbonyl to a leaving group in situ. For this, we decided to use diphenylphosphoryl azide [(PhO)₂P(O)N₃, DPPA] in conjunction with a base. Mechanistically, deprotonation of the amide and reaction with DPPA would produce a diphenyl phosphate intermediate that could undergo subsequent S_NAr reaction with azide anion (Scheme 2).

SCHEME 2. Synthesis of 3c and 3d via Reaction of 1c and 1d with (PhO)₂P(O)N₃/DBU.

On the basis of this rationale, 1c and 1d were exposed to DPPA and DBU in THF at 0 °C followed by warming to 60 °C. Azido nucleosides 3c and 3d were produced in yields of 67% and 41%, respectively, after purification. Interestingly, a similar reaction of silylated derivative 1a led to only a 36% isolated yield of 3a, perhaps indicating some influence of the saccharide protecting group on the efficiency of the S_NAr reaction. Thus, displacement reactions on O⁶-(benzotriazol-1-yl) derivatives 2a–f by azide offer generally good access to C–6 azidopurine nucleosides.

The Azide-Tetrazole Equilibium of the 6-Azidopurine Nucleoside Derivatives

It is known that both 6-azidopurine as well as the corresponding ribonucleoside can exist in the alternate tetrazolyl forms.^16,25,26 In fact, in DMSO-d₆, 6-azidopurine as well as its N7- and N9-benzyl derivatives exist exclusively as the tetrazolyl isomers, and the azido forms are observable only upon protonation with trifluoroacetic acid.^25,26 Similarly, in the solid state 6-azidopurine ribonucleoside exists as the tetrazolyl isomer lacking an IR absorption at 2000-2200 cm^-1 corresponding to the azido tautomer.^16,18 Since the subsequent stage of our work entailed the development of azide-alkyne ligation chemistry, we wanted to evaluate the influence solvent polarity elicits on the equilibrium involving the azido nucleosides and the corresponding tetrazolo[5,1-i]purinyl isomers. For this analysis, ¹H NMR spectroscopy appeared to be optimal since the H–2 and H–8 resonances of the azido and tetrazolyl isomers are well resolved and diagnostic. In the tetrazolyl forms of 6-azidopurine, its N9- and N7-benzyl derivatives, H–2 appears at ∼9.7-10 ppm, whereas H–8 appears at ∼8.6-9.4 ppm.²⁶ On the other hand, in the azido isomers of the same purine derivatives, the line separation between the H–2 and H–8 resonances is much smaller, with H–2 appearing at ∼9.1-9.2 ppm and the H–8 at ∼8.8-9.1 ppm (see Table S1 in the Supporting Information).²⁶ Thus, H–2 of the tetrazolyl isomer appears farther downfield compared to its H–8 resonance as well as the H–2 and H–8 resonances of the azido tautomer.²⁶ On the basis of these as well as NOESY data from 3b (in acetone-d₆) and 3d (in CDCl₃) showing correlation between H–1′ and H–8, we determined the chemical shifts of purinyl H–2, H–8 and the saccharide H–1′ in the azido (A) and tetrazolyl (T) forms of compounds 3a–f. These results are shown in Table 2.

TABLE 2. Chemical Shifts (δ ppm) of Purinyl H–2, H–8 and the Sugar H–1′ in the Azido and Tetrazolyl Isomers of 3a–f.

		azido form (A)			tetrazolyl form (T)
compound	solvent	H–2	H–8	H–1′	H–2	H–8	H–1′
3a	CDCl3	8.63	8.33	6.47	9.51	8.57	6.61
	THF-d8	8.58	8.36	6.45	9.78	8.54	6.60
	CD2Cl2	8.63	8.33	6.47	9.51	8.57	6.61
	acetone-d6	8.65	8.51	6.54	9.91	8.69	6.70
	DMSO-d6	--	--	--	10.14	8.82	6.57
3b	CDCl3	8.65	8.41	6.10	9.49	8.66	6.25
	THF-d8	8.60	8.46	6.07	9.82	8.66	6.24
	CD2Cl2	8.64	8.40	6.09	9.52	8.67	6.25
	acetone-d6	8.68	8.59	6.17	9.97	8.78	6.33
	DMSO-d6	--	--	--	10.19	8.88	6.17
3c	CDCl3	8.67	8.18	6.47	9.53	8.40	6.59
	THF-d8	8.60	8.35	6.48	9.81	8.52	6.62
	CD2Cl2	8.65	8.19	6.48	9.54	8.42	6.60
	acetone-d6	8.68	8.53	6.57	9.95	8.71	6.73
	DMSO-d6	--	--	--	10.16	8.87	6.61
3d	CDCl3	8.68	8.15	6.21	9.54	8.38	6.33
	THF-d8	8.62	8.35	6.27	9.83	8.52	6.41
	CD2Cl2	8.67	8.16	6.22	9.56	8.41	6.36
	acetone-d6	8.70	8.53	6.36	9.97	8.71	6.52
	DMSO-d6	--	--	--	10.19	8.88	6.46
3ea	THF-d8	8.57	8.46	6.48	9.78	8.68	6.64
	DMSO-d6	--	--	--	10.13	8.88	6.57
3fa	DMSO-d6	--	--	--	10.15	8.93	6.15

^aSolubility constraints precluded assessment in other solvents.

For determining the proportion of the azido and tetrazolyl forms in 3a–f, the H–2 and H–8 resonances were related to the H–1′ resonances of the two isomers. The integrals of the H–1′ resonances were then used to determine the ratios of the two isomeric forms. Several solvents were selected with varying dielectric constants (ε) and the results are shown in Table 3.

TABLE 3. Percentage Populations of the Azido (A) and Tetrazolyl (T) Isomers of 3a–f in Various Solvents^a.

compound	CDCl3 ε = 4.8		THF-d8 ε = 7.6		CD2Cl2 ε = 8.9		acetone-d6 ε = 20.6		DMSO-d6 ε = 46.5
	%A	%T	%A	%T	%A	%T	%A	%T	%A	%T
3a	46.0	54.0	14.8	85.2	20.8	79.2	7.5	92.5	0.1	99.9
3b	47.6	52.4	16.5	83.5	19.7	80.3	8.2	91.8	0.1	99.9
3c	56.9	43.1	14.1	85.9	27.5	72.5	6.2	93.8	0.9	99.1
3d	61.9	38.1	16.9	83.1	31.4	68.6	7.6	92.4	1.9	98.1
3e	ndb	ndb	15.1	84.9	ndb	ndb	ndb	ndb	0.1	99.9
3f	ndb	ndb	ndb	ndb	ndb	ndb	ndb	ndb	0.9	99.1

^aThe dielectric constants (ε) of the corresponding protio solvents are reported (reference 27).

^bSolubility constraints precluded assessment.

With this analysis, it became clear that generally the proportion of the tetrazolyl isomer increases with increasing solvent dielectric constant. This is consistent with prior results on 6-azidopurine where lower solvent dielectric constant has been implicated in tetrazole destabilization.²⁵ In fact, in chloroform, about 40–50% of the tetrazolyl form is present. In THF as well as in dichloromethane, which are close in dielectric constant, the tetrazolyl form is major (the triacetates 3c and 3d show a slightly higher proportion of the azido form in chloroform). Finally, in acetone and DMSO the tetrazolyl form predominates. In the case of unprotected 3e, where analysis was possible in THF, a greater proportion of the azido isomer is observed in THF than in DMSO. The ratio of these isomers in 3e is also consistent with data for the other azidopurine nucleoside derivatives shown in Table 3. It stands to reason therefore, that only the tetrazolyl form of azidopurine nucleoside 3f could be detected in the solid state and as a solution in DMSO.^16,18,28 These observations then led to the obvious question: Do the azide-tetrazole mixtures undergo efficient ligation (“click”) reactions with alkynes? The answer to this is not obvious in the context of a recent report showing 2-azido-3-phenylpyridine to exist as the 8-phenyltetrazolo[1,5-a]pyridine, and being recalcitrant towards the Staudinger reaction with PPh₃.²⁹ Also, it has been speculated that some of the lower-yielding azide-alkyne ligations involving a C–2 azidopurine nucleoside could be due to the presence of the tetrazolyl tautomer.¹³

Thus, we began screening conditions for the Cu-catalyzed azide-alkyne ligation^8,9 reaction between 3a and phenylacetylene. At the outset, the CuSO₄/Na ascorbate system in tert-BuOH/H₂O were chosen.⁹ However, under these conditions, an incomplete reaction was observed after 3.5 h at room temperature, with only 26% product formation. Importantly, 11% reduction of the azide to the amine was observed (entry 1 in Table 4), and the resulting disilyl 2′-deoxyadenosine was isolated and identified. This clearly points to the facile reducibility of the azide functionality in 3a, which contrasts with the reactivity of simpler azides where such reduction is generally not observed under the click reaction conditions.

TABLE 4. Evaluation of Conditions for the Reaction of 3a with Phenylacetylene.

entry	catalyst	solvent	temp, time	resulta,b
1	CuSO4/Na ascorbate	1:1 tert-BuOH/H2O	rt, 3.5 h	Incomplete reaction 3a: 63%, 4a: 26%, reduction: 11%
2	CuSO4/Na ascorbate	1:1 THF/H2O	rt, 6 h	Incomplete reaction 3a: 46%, 4a: 30%, reduction: 24%
3	CuSO4/Na ascorbate	1:1 Toluene/H2O	rt, 6 h	Incomplete reaction 3a: 67%, 4a: 22%, reduction: 11%
4	CuSO4/Na ascorbate	1:1 CHCl3/H2O	rt, 2 h	4a: 78% yieldc
5	CuSO4/Na ascorbate	1:1 CH2Cl2/H2O	rt, 3.5 h	4a: 81% yield
6	Cu(Phen)(PPh3)Br	1:1 CH2Cl2/H2O	rt, 24 h	4a: 46% yield
7	Cu(PPh3)3Br	1:1 CH2Cl2/H2O	rt, 15 h	4a: 73% yield, an uncharacterized byproduct was also formed
8	CuIMes	1:1 CH2Cl2/H2O	rt, 20 h	Incomplete reaction 3a: 60%, 4a: 29%, reduction: 11%
9	CuIPr	1:1 CH2Cl2/H2O	rt, 24 h	No reaction, only 3a present
10	CuCl	1:1 CH2Cl2/H2O	rt, 5 h	4a: 76% yield
11	CuTC	1:1 CH2Cl2/H2O	rt, 6 h	4a: 63% yield
12	Cu/C and 2 molar equiv Et3N	1,4-Dioxane	rt, 15 h then 60 °C, 1.5 h	4a: 51%, reduction: 38%
13	Cu/C	1,4-Dioxane	rt, 23 h	No reaction, only 3a present
14	Cu/C	1:1 CH2Cl2/H2O	rt, 2.5 h	No reaction, only 3a present

^aIn reactions that proceeded to completion, yield reported is that of isolated and purified 4a.

^bIn some cases the reduction product, 3′,5′-bis-O-(tert-butyldimethylsilyl)-2′-deoxyadenosine, was isolated and characterized. In other cases this product was detected by TLC (UV visualization) and the amount formed was estimated by ¹H NMR analyses of the crude reaction mixtures.

^cBy TLC, reaction was less clean compared to entry 5.

On the basis of the results obtained above, we screened azide-alkyne ligation conditions extensively (Table 4), and in subsequent reactions TLC analysis was conducted to include detection (UV visualization) of the azide reduction. Replacing tert-BuOH with THF or toluene did not provide any significant improvement (entries 2 and 3). Several other Cu catalysts shown in Figure 1 were also evaluated, and Table 4 lists some representative results.

FIGURE 1. Other Copper Catalysts Tested for the Azide-Alkyne Ligation.

From these experiments two important features emerged. Use of a biphasic CH₂Cl₂/H₂O system³⁰ was important for successful reaction. Secondly and more significantly, despite a major proportion of the tetrazolyl tautomer in this solvent system, reactions with phenylacetylene proceeded smoothly. To illustrate this point, ¹H NMR data for 3a and 3b were obtained in CD₂Cl₂/10% D₂O. In this solvent the amounts of the azido and tetrazolyl forms of 3a were 18.4% and 81.6%, respectively, whereas for 3b they were 21% and 79%, respectively. These ratios are very similar to the data obtained in CD₂Cl₂ alone (Table 3), indicating that the azide/tetrazole ratio is not substantially altered in the presence of water.

Once conditions leading to successful click reactions were realized, attention was focused on the issue of regiochemistry. In reactions of simpler systems, only 1,4-disubstituted triazoles are formed in the Cu-catalyzed azide-alkyne ligation reactions.⁹ We wanted to assess whether this was also the case with these nucleoside substrates, which can demonstrate complex reactivities. Thus, a reaction of 3a and 4-ethynyltoluene was conducted under the optimized conditions. In addition to a major product, a minor, more polar material was observed by TLC (spectral data could not be obtained on the minor product as it was formed to a very low extent). It proved difficult, however, to ascertain the exact regioisomeric structure of the major product. Therefore, we attempted structure evaluation by X-ray analysis. Gratifyingly, the major product from the reaction of 3a and 4-ethynyltoluene could be crystallized as long needles from hexanes. The X-ray structure (Figure 2) clearly indicated this to be the anticipated 1,4-disubstituted triazole derivative (a pdb file as well as ORTEP are included in the Supporting Information). The crystal structure shows that the purine and the triazole rings are nearly coplanar, with a syn-conformation of the purine around the glycosidic bond, and a 3′-endo sugar ring pucker.

FIGURE 2. X-Ray Structure of the Click Reaction Product of 3a and 4-Ethynyltoluene.

In order to compare the catalyzed reaction with the uncatalyzed process, two different reactions were conducted. In one case, a mixture of 3a and 4-ethynyltoluene was heated at 85 °C in 1:3 MeOH–H₂O. This reaction, which was incomplete at 36 h, gave 48% of the 1,4-disubstituted triazole, 5% of the 1,5-isomer and 18% of recovered 3a. A second reaction between 3a and 4-ethynyltoluene was conducted in toluene at 65 °C. This reaction was incomplete at 120 h and showed ∼10% 3a, ∼80% of the 1,4-disubstituted triazole, as well as ∼10% of the 1,5-isomer (estimates based upon ¹H NMR analysis of the crude reaction mixture). These results clearly indicate that the rate of the ligation reaction benefits significantly by Cu catalysis.

The next stage involved an analysis of the generality of the azide-alkyne ligation reaction using the optimized conditions (CuSO₄/Na ascorbate in CH₂Cl₂/H₂O). A range of alkynes underwent successful ligation with 3a and 3b in good yields and within reasonably short reaction times. In many cases, particularly with the ribosides, additional minor products could be isolated. From the NMR as well as the HRMS data, these minor compounds are quite possibly the 1,5-disubstituted azide-alkyne ligation products. Where possible, characterization data for the minor products are presented in the Experimental Section as well.

These results also indicate a point of departure in the reactivity of nucleoside azides in comparison to those of simpler azides. From a mechanistic consideration³¹ (Scheme 3), it appears that a major and a minor process are in operation, leading to the two regioisomeric products. Since no product formation was observed by simply stirring 4-ethynyltoluene and 3b in CH₂Cl₂/H₂O at room temperature for 48 h, it is likely that the minor 1,5-disubstituted products also arise via a catalyzed process (after 48 h at 100 °C, 32% of 3b, 16% of the major and 2.5% of the minor cycloaddition products were isolated from this reaction). Although it is difficult to rationalize why the reaction partitions through the minor pathway, it is possible that presence of the subsitutent (R) and the nucleoside (Nuc) on vicinal carbons is not completely sterically disfavored.

SCHEME 3. Plausible Mechanistic Pathways Leading to the Regioisomeric Products.

We were curious to evaluate the azide-alkyne ligation reactions of 4-azidoquinazoline, which is indicated in the literature to exist as the corresponding tetrazolo[1,5-c]quinazoline.³² Several attempts were directed at the reaction of this compound with phenylacetylene (Scheme 4), and these included the reaction conditions used herein, CuSO₄/Na ascorbate in tert-BuOH/H₂O at room temperature and at 50 °C, as well as a reaction in the presence of excess alkyne in refluxing toluene. No product formation was observed under any of these conditions. These results are also indicative of the markedly different reactivity 6-azidopurine nucleoside derivatives possess in comparison to this simpler model.

SCHEME 4. Attempted Ligation of Tetrazolo[1,5-c]quinazoline with Phenylacetylene.

Finally, it was of interest to determine the biological activities of these new triazolyl nucleoside analogues. Ribo and 2′-deoxyribonucleosides are typically of low toxicity to human cells. Compounds 4a–g and 5a–g were desilylated to yield the free nucleosides. The desilylated compounds were tested for antiproliferative activity (range of concentrations tested was 1–100 μM, 72 hour continuous exposure) against a panel of human cancer cell lines: wild type p53^+/+ and p53^−/− HCT116 colon carcinoma cells; and paclitaxel sensitive (1A9) and resistant (1A9/PTX10 and 1A9/PTX22) ovarian carcinoma cells. Only weak antiproliferative or cytotoxic actions were noted for compounds 6e, 6f, 6g, 7d and 7f (Table S2 in the Supporting Information). All these new agents are presently being tested for inhibitory activities against a panel of kinases, phosphatases, polymerases and reverse transcriptases, and the results will be reported in due course.

Conclusions

We have demonstrated that O⁶-(benzotriazol-1-yl)inosine and 2′-deoxyinosine derivatives are excellent substrates for conversion to the C–6 azidopurine nucleoside derivatives in a simple operation using azide anion. The azido nucleosides exist in equilibrium with the tetrazolyl forms and increasing solvent dielectric constant generally increases the proportion of the tetrazolyl tautomer. Despite the azide•tetrazole equilibrium, successful azide-alkyne click reactions can be accomplished using CuSO₄/Na ascorbate. However, the biphasic CH₂Cl₂/H₂O solvent system is required for successful reaction, where the competing reduction of the azide to the amine is suppressed. In contrast to click reactions of simpler organic azides, those of the C-6 azido nucleoside analogues are less regioselective and minor amounts of the 1,5-disubstituted triazoles were isolated and characterized in some cases. By X-ray crystallographic analysis, the structure of the major product from a reaction between 3a and 4-ethynyltoluene was shown to be the 1,4-disubstituted triazole. To our knowledge, this is the first report describing a simple, unifying synthesis of C-6 azidopurine 2′-deoxyribo- and ribonucleosides, their behavior in solution and use in click reactions with alkynes. In contrast to the successful reactions of the 6-azidopurine nucleosides, azide-alkyne ligation reactions of tetrazolo[1,5-c]quinazoline were unsuccessful. This demonstrates markedly different behavior of the 6-azidopurine nucleosides in comparison to the simpler heterocycle.

Experimental

Please see the Supporting Information for General Experimental Methods.

3′,5′-Di-O-acetyl-O⁶-(benzotriazol-1-yl)-2′-deoxyinosine (2c)

In a clean, dry 100 mL round-bottomed flask, equipped with a stirring bar were placed 2′-deoxyinosine 3′,5′-diacetate 1c (300 mg, 0.893 mmol) and BOP (790 mg, 1.79 mmol) in anhydrous CH₂Cl₂ (8.0 mL). To this stirred mixture was added (iso-Pr)₂NEt (0.31 mL, 1.79 mmol) and the stirring was continued at room temperature for 22 h. The mixture was evaporated to dryness and the residue was redissolved in EtOAc. The mixture was washed with water (3 ×) and then with brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel using 30% EtOAc in hexanes to provide 2c as a white foamy solid (382 mg, 94% yield). R_f (silica gel, 10% EtOAc in hexanes) = 0.70. ¹H NMR (500 MHz, CDCl₃): δ 8.44 (s, 1H, Ar–H), 8.33 (s, 1H, Ar–H), 8.14 (d, 1H, Ar–H, J = 8.3 Hz), 7.58-7.41 (m, 3H, Ar–H), 6.52 (t, 1H, H–1′, J = 6.5 Hz), 5.46 (br s, 1H, H–3′), 4.44-4.36 (m, 3H, 1H–4′, 2H–5′), 3.00 (app quint, 1H, H–2′, J_app ∼ 7.1 Hz), 2.71 (ddd, 1H, H–2′, J = 2.6, 6.0, 14.1 Hz), 2.15 and 2.10 (2s, 6H, OCOCH₃). ¹³C NMR (125 MHz, CDCl₃): δ 170.5, 170.4, 159.4, 153.7, 151.9, 143.7, 143.3, 129.0, 125.1, 120.8, 108.8, 85.5, 83.1, 74.4, 63.8, 37.9, 21.1, 21.0. HRMS (ESI) calculated for C₂₀H₁₉N₇O₆Na [M + Na]⁺: 476.1295, found: 476.1292.

2′,3′,5′-Tri-O-acetyl-O⁶-(benzotriazol-1-yl)inosine (2d)

This compound was prepared by the procedure described for 2c using 1d (300 g, 0.761 mmol), BOP (673 mg, 1.52 mmol), anhydrous CH₂Cl₂ (8.0 mL) and (iso-Pr)₂NEt (0.26 mL, 1.52 mmol). The crude product was purified by column chromatography on silica gel using 40% EtOAc in hexanes to provide 2d as a white foamy solid (353 mg, 91% yield). R_f (silica gel, 20% EtOAc in hexanes) = 0.32. ¹H NMR (500 MHz, CDCl₃): δ 8.45 (s, 1H, Ar–H), 8.30 (s, 1H, Ar–H), 8.14 (d, 1H, Ar–H, J = 8.3 Hz), 7.57-7.46 (m, 3H, Ar–H), 6.27 (d, 1H, H–1′, J = 5.1 Hz), 5.96 (t, 1H, H–2′, J = 5.4 Hz), 5.60 (t, 1H, H–3′, J = 5.1 Hz), 4.51-4.38 (m, 3H, H–4′, 2H–5′), 2.16, 2.14 and 2.10 (3s, 9H, OCOCH₃). ¹³C NMR (125 MHz, CDCl₃): δ 170.4, 169.7, 169.5, 159.5, 153.8, 152.1, 143.6, 143.5, 129.0, 125.1, 120.7, 120.3, 108.8, 87.1, 80.7, 73.3, 70.6, 63.0, 53.6, 20.9, 20.7, 20.5. HRMS (ESI) calculated for C₂₂H₂₁N₇O₈Na [M + Na]⁺: 534.1349, found: 534.1351.

6-Azido-9-[2-deoxy-3,5-di-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (3a)

In a clean, dry 50 mL round-bottomed flask, equipped with a stirring bar were placed O⁶-(benzotriazol-1-yl)-2′,3′-bis-O-(tert-butyldimethylsilyl)-2′-deoxyinosine (2a) (1.036 g, 1.73 mmol) and NaN₃ (326 mg, 5.0 mmol) in anhydrous DMSO (8.6 mL). The reaction mixture was flushed with nitrogen gas and stirred under a nitrogen balloon at 40 °C for 2 h. The reaction mixture was transferred to a separatory funnel and partitioned between EtOAc and a 1:1 mixture of water–brine. The organic layer was washed with 1:1 water –brine mixture (4 ×), then with water (3 ×) and finally with brine. The organic layer was dried over Na₂SO₄ and evaporated under reduced pressure. Filtration of the crude material through a silica gel plug using 20% EtOAc in hexanes solution afforded 3a as a clear gum (871 mg, 99%). R_f (silica gel, 1% MeOH in CH₂Cl₂) = 0.12. ¹H NMR (500 MHz, DMSO-d₆): δ 10.14 (s, 1H, Ar–H), 8.82 (s, 1H, Ar–H), 6.57 (t, 1H, H–1′, J = 6.5 Hz), 4.64 (m, 1H, H–3′), 3.91 (m, 1H, H–4′), 3.82 (dd, 1H, H–5′, J = 5.2, 11.0 Hz), 3.70 (dd, 1H, H–5′, J = 4.0, 11.0 Hz), 2.88 (app quint, 1H, H–2′, J_app ∼ 6.0 Hz), 2.46 (m, 1H, H–2′), 0.89 and 0.82 (2s, 18H, tert-Bu), 0.11, 0.02 and −0.001 (3s, 12H, Si–CH₃). ¹³C NMR (125 MHz, DMSO-d₆): δ 145.3, 142.5, 141.5, 135.9, 120.4, 87.3, 84.2, 71.5, 62.3, 39.9, 25.7, 25.6, 17.9, 17.6, −4.8, −5.0, −5.6. HRMS (ESI) calculated for C₂₂H₃₉N₇O₃Si₂Na [M + Na]⁺: 528.2545, found: 528.2534.

6-Azido-9-[2,3,5-tri-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (3b)

In a clean, dry round-bottomed flask equipped with a stirring bar were placed O⁶-(benzotriazol-1-yl)-2′,3′,5′-tri-O-(tert-butyldimethylsilyl)inosine (2b) (2.55 g, 3.51 mmol) and NaN₃ (684 mg, 10.53 mmol) in anhydrous DMSO (17.5 mL). The reaction mixture was flushed with nitrogen gas and stirred under a nitrogen balloon at 40 °C for 3.5 h. The reaction mixture was transferred to a separatory funnel and partitioned between EtOAc and a 1:1 mixture of water–brine. The organic layer was washed with 1:1 water–brine mixture (4 ×), then with water (3 ×) and finally with brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. Filtration of the crude material through a silica gel plug using 20% EtOAc in hexanes afforded 3b as a white foam (2.14 g, 96% yield). R_f (silica gel, 20% EtOAc in hexanes) = 0.46. ¹H NMR (500 MHz, DMSO-d₆): δ 10.19 (s, 1H, Ar–H), 8.88 (s, 1H, Ar–H), 6.17 (d, 1H, H–1′, J = 5.5 Hz), 4.79 (t, 1H, H–2′, J = 4.7 Hz), 4.37 (m, 1H, H–3′) 4.10-4.06 (m, 1H, H–4′), 4.02 (dd, 1H, H–5′, J = 5.6, 11.0 Hz), 3.80 (dd, 1H, H–5′, J = 3.0, 11.0 Hz), 0.93, 0.91 and 0.74 (3s, 27H, tert-Bu), 0.14, 0.11, −0.05 and −0.28 (4s, 18H, Si–CH₃). ¹³C NMR (125 MHz, DMSO-d₆): δ 145.3, 142.5, 141.8, 136.2, 120.3, 87.8, 85.3, 75.2, 71.6, 62.1, 25.8, 25.7, 25.4, 18.0, 17.7, 17.4, −4.6, −4.9, −5.4, −5.5, −5.6. HRMS (ESI) calculated for C₂₈H₅₃N₇O₄Si₃Na [M + Na]⁺: 658.3359, found: 658.3340.

6-Azido-9-(2-deoxy-3,5-di-O-acetyl-β-d-ribofuranosyl)purine (3c)

From the O⁶-(benzotriazol-1-yl)-2′-deoxyinosine diacetate

In a clean, dry vial, equipped with a stirring bar were placed O⁶-(benzotriazol-1-yl)-2′-deoxyinosine diacetate 2c (249 mg, 0.550 mmol) and NaN₃ (107 mg, 1.65 mmol) in DMSO (2.5 mL). The mixture was flushed with nitrogen gas and stirred at room temperature for 1 h at which time TLC indicated the reaction to be complete. The mixture was diluted with EtOAc and transferred to a separatory funnel. The mixture was extracted with 1:1 water-brine (3 ×), water (3 ×) and finally once with brine. The organic layer was dried over Na₂SO₄ and evaporated under reduced pressure. Chromatography of the crude material on a silica gel column using 40% EtOAc in hexanes afforded 3c as a white foamy material (195 mg, 95% yield).

From 2′-deoxyinosine diacetate

In a clean, dry reaction vial, equipped with a stirring bar were placed 2′-deoxyinosine-3′,5′-diacetate (1c) (500 mg, 1.487 mmol) and THF (3.0 mL). The mixture was cooled with stirring to 0 °C in an ice bath. DPPA (0.48 mL, 2.23 mmol) and DBU (0.34 mL, 2.23 mmol) were added. The nitrogen gas-flushed mixture was allowed to stir at the ice bath temperature for 5 min, then at room temperature for 10 min. Finally, the mixture was stirred in a 60 °C sandbath for 1 hr. Another aliquot of DPPA (0.48 mL, 2.23 mmol) and DBU (0.34 mL, 2.23 mmol) were added and the reaction was continued for an additional hour. The mixture was evaporated to dryness, the residue was dissolved in CH₂Cl₂ and washed with water followed by brine. Chromatographic purification on a silica gel column using 2% MeOH/50% EtOAc/48% hexanes afforded 3c as a yellow foam (362 mg, 67%). R_f (silica gel, 80% EtOAc in hexanes) = 0.26. ¹H NMR (500 MHz, DMSO-d₆): δ 10.16 (s, 1H, Ar–H), 8.87 (s, 1H, Ar–H), 6.61 (t, 1H, H–1′, J = 6.8 Hz), 5.50-5.41 (m, 1H, H–3′), 4.37-4.29 (m, 2H, H–4′, H–5′), 4.24 (dd, 1H, H–5′, J = 5.5, 11.4 Hz), 3.15 (app quint, 1H, H–2′, J_app ∼ 7.1 Hz), 2.74-2.66 (m, 1H, H–2′), 2.11 and 2.01 (2s, 6H, OCOCH₃). ¹³C NMR (125 MHz, DMSO-d₆): δ 170.1, 170.0, 145.4, 142.8, 141.6, 136.1, 120.6, 84.4, 82.1, 74.0, 63.4, 36.1, 20.8, 20.5. HRMS (ESI) calculated for C₁₄H₁₅N₇O₅Na [M + Na]⁺: 384.1027, found: 384.1027.

6-Azido-9-(2,3,5-tri-O-acetyl-β-d-ribofuranosyl)purine (3d)

From the O⁶-(benzotriazol-1-yl)inosine triacetate

In a clean, dry vial, equipped with a stirring bar were placed O⁶-(benzotriazol-1-yl)inosine triacetate 2d (281 mg, 0.550 mmol) and NaN₃ (107 mg, 1.65 mmol) in DMSO (2.5 mL). The mixture was flushed with nitrogen gas and stirred at room temperature for 1 h at which time TLC indicated the reaction to be complete. The mixture was diluted with EtOAc and transferred to a separatory funnel. The mixture was extracted with 1:1 water-brine (3 ×), water (3 ×) and finally once with brine. The organic layer was dried over Na₂SO₄ and evaporated under reduced pressure. Chromatography of the crude material on a silica gel column using 40% EtOAc in hexanes afforded 3d as a white foamy material (173 mg, 75% yield).

From inosine triacetate

In a clean, dry reaction vial, equipped with a stirring bar were placed inosine-2′,3′,5′-triacetate (1d) (600 mg, 1.521 mmol) and THF (3.6 mL). The mixture was cooled with stirring to 0 °C in an ice bath. DPPA (0.49 mL, 2.28 mmol) and DBU (0.35 mL, 2.28 mmol) were added. The nitrogen gas-flushed mixture was and allowed to stir at the ice bath temperature for 5 min, then at room temperature for 10 min. Finally, the mixture was stirred in a 60 °C sandbath for 1 hr. Another aliquot of DPPA (0.49 mL, 2.28 mmol) and DBU (0.35 mL, 2.28 mmol) were added and the reaction was continued for an additional hour. The mixture was evaporated to dryness, the residue was dissolved in CH₂Cl₂ and washed with water followed by brine. Chromatographic purification on a silica gel column using 60% EtOAc in hexanes afforded 3d as a yellow foam (266 mg, 41% yield). R_f (silica gel, 80% EtOAc in hexanes) = 0.35. ¹H NMR (500 MHz, DMSO-d₆): δ 10.19 (s, 1H, Ar–H), 8.88 (s, 1H, Ar–H), 6.46 (d, 1H, H–1′, J = 5.0 Hz), 5.98 (t, 1H, H–2′, J = 5.4 Hz), 5.63 (t, 1H, H–3′, J = 5.3 Hz), 4.47 (br s, 1H, H–4′), 4.43 (dd, 1H, H–5′, J = 2.9, 12.0 Hz), 4.30 (dd, 1H, H–5′, J = 5.3, 12.0 Hz), 2.14, 2.05 and 2.04 (3s, 3H, OCOCH₃). ¹³C NMR (125 MHz, DMSO-d₆): δ 170.0, 169.4, 169.2, 145.4, 143.3, 141.3, 136.3, 120.8, 86.4, 79.8, 72.6, 69.8, 62.7, 20.5, 20.3, 20.2. HRMS (ESI) calculated for C₁₆H₁₇N₇O₇Na [M + Na]⁺: 442.1082, found: 442.1077.

6-Azido-9-(2-deoxy-β-d-ribofuranosyl)purine (3e)

In a clean, dry reaction vial, equipped with a stirring bar were placed O⁶-(benzotriazol-1-yl)-2′-deoxyinosine 2e (150 mg, 0.406 mmol) and polymer-supported azide²³ (530 mg, 0.406 mmol). Water (3.75 mL) was added, the reaction mixture was flushed with nitrogen gas and allowed to stir in a 50 °C sandbath for 3.5 hr. The reaction mixture was filtered and evaporated to dryness under reduced pressure. The residue was washed with Et₂O to afford 3e as a white powder (66 mg, 59% yield). R_f (silica gel, 10% MeOH in CH₂Cl₂) = 0.2. ¹H NMR (500 MHz, DMSO-d₆): δ 10.13 (s, 1H, Ar–H), 8.88 (s, 1H, Ar–H), 6.57 (t, 1H, H–1′, J = 6.5 Hz), 5.40 (d, 1H, OH, J = 4.3 Hz), 4.98 (t, 1H, OH J = 5.4 Hz), 4.46 (m, 1H, H–3′), 3.93 (app q, 1H, H–4′, J_app ∼ 4.1 Hz), 3.68-3.61 (m, 1H, H–5′), 3.59-3.53 (m, 1H, H–5′), 2.75 (app quint, 1H, H–2′, J_app ∼ 6.5 Hz), 2.44 (ddd, 1H, H–2′, J = 3.9, 6.3, 13.3 Hz). ¹³C NMR (125 MHz, DMSO-d₆): δ 145.4, 142.5, 141.6, 135.9, 120.5, 88.2, 84.4, 70.3, 61.3, 39.8. HRMS (ESI) calculated for C₁₀H₁₁N₇O₃Na [M + Na]⁺: 300.0816, found: 300.0818.

6-Azido-9-(β-d-ribofuranosyl)purine (3f)

In a clean, dry reaction vial, equipped with a stirring bar were placed O⁶-(benzotriazol-1-yl)inosine 2f (150 mg, 0.389 mmol) and polymer-supported azide²³ (530 mg, 0.406 mmol). DMF (3.9 mL) was added, the reaction mixture was flushed with nitrogen gas and allowed to stir in a 50 °C sandbath for 5 hr. The reaction mixture was filtered and the DMF was coevaporated with toluene. The residue was washed with Et₂O to afford 3f as a white powder (80 mg, 70% yield). R_f (silica gel, 10% MeOH in CH₂Cl₂) = 0.19. ¹H NMR (500 MHz, DMSO-d₆): δ 10.15 (s, 1H, Ar–H), 8.93 (s, 1H, Ar–H), 6.15 (d, 1H, H–1′, J = 5.1 Hz), 5.63 (d, 1H, OH, J = 5.6 Hz), 5.30 (d, 1H, OH, J = 5.2 Hz), 5.10 (t, 1H, OH, J = 5.3 Hz), 4.57 (q, 1H, H–2′, J = 5.2 Hz), 4.20 (q, 1H, H–3′, J = 4.6 Hz), 4.02 (m, 1H, H–4′), 3.76-3.68 (m, 1H, H–5′), 3.65-3.56 (m, 1H, H–5′). ¹³C NMR (125 MHz, DMSO-d₆): δ 145.4, 142.6, 141.9, 136.1, 120.2, 88.3, 85.7, 74.5, 70.0, 60.9. HRMS (ESI) calculated for C₁₀H₁₁N₇O₄Na [M + Na]⁺: 316.0765, found: 316.0765.

Typical Procedure for Azide-Alkyne Ligation: Synthesis of 6-[(4-phenyl)-1,2,3-triazol-1-yl]-9-[2-deoxy-3,5-di-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (4a)

In a clean, dry vial equipped with a stirring bar were placed the azido nucleoside 3a (240 mg, 0.474 mmol), CH₂Cl₂ (1.6 mL) and phenylacetylene (104 μL, 0.949 mmol). An aqueous solution of sodium ascorbate (0.047 mmol, 0.95 mL of freshly prepared 0.05 M solution) was added followed by an aqueous solution of CuSO₄ (24 μmol, 0.60 mL of freshly prepared 0.04 M solution). The mixture was stirred at room temperature for 4 h, at which time TLC indicated the reaction to be complete. The reaction mixture was diluted with EtOAc and washed with water. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. Chromatographic purification on a silica gel column using 20% EtOAc in hexanes afforded 4a as an off-white foam (222 mg, 77% yield). R_f (silica gel, 20% EtOAc in hexanes) = 0.18. ¹H NMR (500 MHz, CDCl₃): δ 9.35 (s, 1H, Ar–H), 8.96 (s, 1H, Ar–H), 8.59 (s, 1H, Ar–H), 8.02 (d, 2H, Ar–H, J = 7.2 Hz), 7.49 (t, 2H, Ar–H, J = 7.6 Hz), 7.40 (t, 1H, Ar–H, J = 7.3 Hz), 6.61 (t, 1H, H–1′, J = 6.4 Hz), 4.65 (m, 1H, H–3′), 4.09 (q, 1H, H–4′, J = 3.2 Hz), 3.91 (dd, 1H, H–5′, J = 3.8, 11.3 Hz), 3.81 (dd, 1H, H–5′, J = 2.9, 11.3 Hz), 2.68 (app quint, 1H, H–2′, J_app ∼ 6.4 Hz), 2.54 (ddd, 1H, H–2′, J = 3.8, 6.1, 13.1 Hz), 0.93 and 0.92 (2s, 18H, tert-Bu), 0.13 and 0.11 (2s, 12H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 154.0, 152.3, 148.5, 144.9, 144.7, 130.1, 129.1, 128.9, 126.4, 123.4, 120.1, 88.5, 85.2, 72.1, 62.9, 41.8, 26.2, 25.9, 18.6, 18.2, −4.4, −4.6, −5.1, −5.2. HRMS (ESI) calculated for C₃₀H₄₅N₇O₃Si₂Na [M + Na]⁺: 630.3015, found: 630.2999.

6-[4-(4-Methylphenyl)-1,2,3-triazol-1-yl]-9-[2-deoxy-3,5-di-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (4b)

Synthesized from 3a (195 mg, 0.385 mmol) and 4-ethynyltoluene (98 μL, 0.773 mmol). Chromatography of the crude reaction mixture on a silica gel column using 10% EtOAc in hexanes yielded 4b as a white foamy solid (209 mg, 87% yield). R_f (silica gel, 20% EtOAc in hexanes) = 0.22. ¹H NMR (500 MHz, CDCl₃): δ 9.30 (s, 1H, Ar–H), 8.95 (s, 1H, Ar–H), 8.58 (s, 1H, Ar–H), 7.90 (d, 1H, Ar–H, J = 8.0 Hz), 7.29 (d, 2H, Ar–H, J = 8.0 Hz), 6.61 (t, 1H, H–1′, J = 6.3 Hz), 4.65 (m, 1H, H–3′), 4.08 (q, 1H, H–4′, J = 3.2 Hz), 3.91 (dd, 1H, H–5′, J = 3.9, 11.2 Hz), 3.81 (dd, 1H, H–5′, J = 2.9, 11.2 Hz), 2.68 (app quint, 1H, H–2′, J_app ∼ 6.4 Hz), 2.54 (ddd, 1H, H–2′, J = 3.8, 6.1, 13.1 Hz), 2.41 (s, 3H, CH₃), 0.93 and 0.92 (2s, 18H, tert-Bu), 0.12 and 0.11 (2s, 12H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 154.0, 152.4, 148.5, 145.0, 144.7, 138.8, 129.8, 127.2, 126.3, 123.4, 119.7, 88.5, 85.2, 72.1, 63.0, 41.8, 26.2, 26.0, 21.6, 18.6, 18.2, −4.4, −4.6, −5.1, −5.2. HRMS (ESI) calculated for C₃₁H₄₇N₇O₃Si₂Na [M + Na]⁺: 644.3171, found: 644.3154.

6-[4-(4-Methoxyphenyl)-1,2,3-triazol-1-yl]-9-[2-deoxy-3,5-di-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (4c)

Synthesized from 3a (204 mg, 0.400 mmol) and 4-ethynylanisole (102.5 μL, 0.807 mmol). Chromatography of the crude reaction mixture a silica gel column using 10% EtOAc in hexanes yielded 4c as a white foamy solid (231 mg, 90% yield). R_f (silica gel, 20% EtOAc in hexanes) = 0.43. ¹H NMR (500 MHz, CDCl₃): δ 9.25 (s, 1H, Ar–H), 8.95 (s, 1H, Ar–H), 8.58 (s, 1H, Ar–H), 7.94 (d, 2H, Ar–H, J = 8.8 Hz), 7.01 (d, 2H, Ar–H, J = 8.8 Hz), 6.61 (t, 1H, H–1′, J = 6.4 Hz), 4.65 (m, 1H, H–3′), 4.08 (q, 1H, H–4′, J = 3.2 Hz), 3.91 (dd, 1H, H–5′, J = 3.8, 11.3 Hz), 3.87 (s, 3H, OCH₃), 3.81 (dd, 1H, H–5′, J = 2.9, 11.3 Hz), 2.67 (app quint, 1H, H–2′, J_app ∼ 6.4 Hz), 2.54 (ddd, 1H, H–2′, J = 3.8, 6.1, 13.1 Hz), 0.93 and 0.92 (2s, 18H, tert-Bu), 0.12 and 0.11 (2s, 12H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 160.2, 154.0, 152.3, 148.3, 145.0, 144.6, 127.8, 123.3, 122.7, 119.1, 114.5, 88.5, 85.1, 72.1, 62.9, 55.6, 41.8, 26.2, 25.9, 18.6, 18.2, −4.4, −4.6, −5.1, −5.2. HRMS (ESI) calculated for C₃₁H₄₇N₇O₄Si₂Na [M + Na]⁺: 660.3120, found: 660.3105.

6-[4-(4-Fluorophenyl)-1,2,3-triazol-1-yl]-9-[2-deoxy-3,5-di-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (4d)

Synthesized from 3a (210 mg, 0.415 mmol) and 1-ethynyl-4-fluorobenzene (95 μL, 0.830 mmol). Chromatography of the crude reaction mixture on a silica gel column using 20% EtOAc in hexanes yielded 4d as a yellow, crystalline solid (240 mg, 92% yield). R_f (silica gel, 40% EtOAc in hexanes) = 0.36. ¹H NMR (500 MHz, CDCl₃): δ 9.30 (s, 1H, Ar–H), 8.96 (s, 1H, Ar–H), 8.60 (s, 1H, Ar–H), 7.99 (dd, 2H, Ar–H, J_H,H = 8.6 Hz, J_F,H = 5.4 Hz), 7.17 (t, 2H, Ar–H, J_H,H = J_F,H = 8.6 Hz), 6.61 (t, 1H, H–1′, J = 6.3 Hz), 4.65 (m, 1H, H–3′), 4.08 (q, 1H, H–4′, J = 3.2 Hz), 3.91 (dd, 1H, H–5′, J = 3.7, 11.2 Hz), 3.81 (dd, 1H, H–5′, J = 2.9, 11.2 Hz), 2.68 (app quint, 1H, H–2′, J_app ∼ 6.4 Hz), 2.54 (ddd, 1H, H–2′, J = 3.8, 6.0, 13.0 Hz), 0.93 and 0.92 (2s, 18H, tert-Bu), 0.12 and 0.11 (2s, 12H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 163.0 (d, ¹J_C,F = 248.1 Hz), 153.8, 152.1, 147.4, 144.6, 144.5, 128.0 (d, ³J_C,F = 8.2 Hz), 126.0 (d, ⁴J_C,F = 3.2 Hz), 123.1, 119.6, 115.9 (d, ²J_C,F = 21.8 Hz), 88.3, 84.9, 71.9, 62.7, 41.6, 25.9, 25.6, 18.4, 18.0, −4.6, −4.8, −5.3, −5.5. HRMS (ESI) calculated for C₃₀H₄₄FN₇O₃Si₂Na [M + Na]⁺: 648.2920, found: 648.2911.

6-[4-(N-Phthalimidomethyl)-1,2,3-triazol-1-yl]-9-[2-deoxy-3,5-di-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (4e)

Synthesized from 3a (190 mg, 0.376 mmol) and N-propargylphthalimide (139 mg, 0.751 mmol). Chromatography of the crude reaction mixture on a silica gel column using 10% EtOAc in hexanes yielded 4e as an off-white, foamy solid (246 mg, 95% yield). R_f (silica gel, 20% EtOAc in hexanes) = 0.15. ¹H NMR (500 MHz, CDCl₃): δ 9.10 (s, 1H, Ar–H), 8.90 (s, 1H, Ar–H), 8.55 (s, 1H, Ar–H), 7.88 (dd, 2H, Ar–H, J = 3.1, 5.4 Hz), 7.73 (dd, 2H, Ar–H, J = 3.0, 5.4 Hz), 6.57 (t, 1H, H–1′, J = 6.4 Hz), 5.16 (s, 2H, NCH₂), 4.64 (m, 1H, H–3′), 4.06 (q, 1H, H–4′, J = 3.3 Hz), 3.89 (dd, 1H, H–5′, J = 3.9, 11.2 Hz), 3.79 (dd, 1H, H–5′, J = 3.1, 11.2 Hz), 2.64 (app quint, 1H, H–2′, J_app ∼ 6.4 Hz), 2.52 (ddd, 1H, H–2′, J = 3.8, 6.0, 13.0 Hz), 0.92 and 0.90 (2s, 18H, tert-Bu), 0.11, 0.092 and 0.088 (3s, 12H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 167.8, 154.1, 152.2, 144.8, 143.6, 134.3, 132.3, 123.7, 123.6, 123.5, 88.5, 85.1, 72.1, 62.9, 41.8, 33.3, 26.2, 25.9, 18.6, 18.2, −4.5, −4.6, −5.2, −5.3. HRMS (ESI) calculated for C₃₃H₄₆N₈O₅Si₂Na [M + Na]⁺: 713.3022, found: 713.2999.

6-[4-(Ferrocenyl)-1,2,3-triazol-1-yl]-9-[2-deoxy-3,5-di-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (4f)

Synthesized from 3a (209 mg, 0.413 mmol) and ethynylferrocene (173 mg, 0.820 mmol). Chromatography of the crude reaction mixture on a silica gel column using 15% acetone in hexanes yielded 4f as a brown, foamy solid (247 mg, 83% yield). R_f (silica gel, 15% acetone in hexanes) = 0.22. ¹H NMR (500 MHz, CDCl₃): δ 8.99 (s, 1H, Ar–H), 8.94 (s, 1H, Ar–H), 8.58 (s, 1H, Ar–H), 6.60 (t, 1H, H–1′, J = 6.3 Hz), 4.88 (br t, 2H, ferrocenyl–H), 4.65 (m, 1H, H–3′), 4.36 (t, 2H, ferrocenyl–H, J = 1.8 Hz), 4.12 (s, 5H, ferrocenyl–H), 4.08 (q, 1H, H–4′, J = 3.2 Hz), 3.91 (dd, 1H, H–5′, J = 3.9, 11.2 Hz), 3.81 (dd, 1H, H–5′, J = 2.9, 11.2 Hz), 2.67 (app quint, 1H, H–2′, J_app ∼ 6.4 Hz), 2.54 (ddd, 1H, H–2′, J = 3.9, 6.0, 13.0 Hz), 0.93 and 0.92 (2s, 18H, tert-Bu), 0.12 and 0.11 (2s, 12H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 154.0, 152.3, 147.9, 144.9, 144.5, 123.3, 118.8, 88.5, 85.1, 74.6, 72.1, 69.9, 69.2, 67.3, 62.9, 41.8, 26.2, 25.9, 18.7, 18.2, −4.4, −4.6, −5.1, −5.2. HRMS (ESI) calculated for C₃₄H₄₉FeN₇O₃Si₂Na [M + Na]⁺: 738.2677, found: 738.2677.

Minor isomer: 6-[5-(Ferrocenyl)-1,2,3-triazol-1-yl]-9-[2-deoxy-3,5-di-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (4f′)

Obtained 18.4 mg (6% yield) of a reddish-brown solid. R_f (silica gel, 15% acetone in hexanes) = 0.14. ¹H NMR (500 MHz, CDCl₃): δ 8.97 (s, 1H, Ar–H), 8.55 (s, 1H, Ar–H), 7.89 (s, 1H, Ar–H), 6.61 (t, 1H, H–1′, J = 6.5 Hz), 4.64 (m, 1H, H–3′), 4.58 (m, 2H, ferrocenyl–H), 4.27 (m, 2H, ferrocenyl–H), 4.07 (q, 1H, H–4′, J = 3.1 Hz), 3.99 (s, 5H, ferrocenyl–H), 3.88 (dd, 1H, H–5′, J = 3.9, 11.2 Hz), 3.80 (dd, 1H, H–5′, J = 2.9, 11.2 Hz), 2.64 (app quint, 1H, H–2′, J_app ∼ 6.4 Hz), 2.52 (ddd, 1H, H–2′, J = 3.7, 6.0, 13.0 Hz), 0.93 and 0.90 (2s, 18H, tert-Bu), 0.12, 0.09 and 0.08 (3s, 12H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 154.1, 151.9, 147.1, 145.5, 138.9, 133.3, 128.6, 88.5, 85.1, 72.1, 70.6, 70.0, 69.6, 69.5, 63.0, 41.9, 26.2, 26.0, 18.7, 18.2, −4.4, −4.6, −5.1, −5.2. HRMS (ESI) calculated for C₃₄H₄₉FeN₇O₃Si₂Na [M + Na]⁺: 738.2677, found: 738.2655.

6-[4-(1-Butyl)-1,2,3-triazol-1-yl]-9-[2-deoxy-3,5-di-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (4g)

Synthesized from 3a (190 mg, 0.375 mmol) and 1-hexyne (82.5 μL, 0.751 mmol). Chromatography of the crude reaction mixture on a silica gel column using 20% EtOAc in hexanes yielded 4g as white solid (166 mg, 75% yield). R_f (silica gel, 20% EtOAc in hexanes) = 0.16. ¹H NMR (500 MHz, CDCl₃): δ 8.91 (s, 1H, Ar–H), 8.81 (s, 1H, Ar–H), 8.53 (s, 1H, Ar–H), 6.58 (t, 1H, H–1′, J = 6.3 Hz), 4.64 (m, 1H, H–3′), 4.07 (m, 1H, H–4′), 3.89 (dd, 1H, H–5′, J = 3.9, 11.2 Hz), 3.79 (dd, 1H, H–5′, J = 2.9, 11.2 Hz), 2.87 (t, 2H, butyl–CH₂, J = 7.8 Hz), 2.66 (app quint, 1H, H–2′, J_app ∼ 6.4 Hz), 2.50 (ddd, 1H, H–2′, J = 3.8, 5.9, 13.0 Hz), 1.76 (quint, 2H, butyl–CH₂, J = 7.6 Hz), 1.44 (sextet, 2H, butyl–CH₂, J = 7.4 Hz), 0.95 (t, 3H, butyl–CH₃, J = 7.3 Hz), 0.92 and 0.90 (2s, 18H, tert-Bu), 0.11 and 0.09 (2s, 12H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 153.9, 152.3, 149.4, 145.1, 144.5, 123.3, 121.4, 88.5, 85.1, 72.1, 62.9, 41.7, 31.5, 26.2, 25.9, 25.5, 22.5, 18.6, 18.2, 14.0, −4.5, −4.6, −5.2, −5.3. HRMS (ESI) calculated for C₂₈H₄₉N₇O₃Si₂Na [M + Na]⁺: 610.3328, found 610.3328.

6-[4-(Phenyl)-1,2,3-triazol-1-yl]-9-[2,3,5-tri-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (5a)

Synthesized from 3b (200 mg, 0.314 mmol) and phenylacetylene (69.0 μL, 0.628 mmol). Chromatography of the crude reaction mixture on a silica gel column using 20% EtOAc in hexanes yielded 5a as brown crystalline solid (210 mg, 90% yield). R_f (silica gel, 20% acetone in hexanes) = 0.49. ¹H NMR (500 MHz, CDCl₃): δ 9.34 (s, 1H, Ar–H), 8.95 (s, 1H, Ar–H), 8.64 (s, 1H, Ar–H), 8.0 (d, 2H, Ar–H, J = 7.5 Hz), 7.45 (t, 2H, Ar–H, J = 7.6 Hz), 7.36 (t, 1H, Ar–H, J = 7.4 Hz), 6.2 (d, 1H, H–1′, J = 5.1 Hz), 4.65 (t, 1H, H–2′, J = 4.6 Hz), 4.33 (t, 1H, H–3′, J = 3.9 Hz), 4.18 (q, 1H, H–4′, J = 3.0 Hz), 4.04 (dd, 1H, H–5′, J = 3.5, 11.3 Hz), 3.82 (dd, 1H, H–5′, J = 2.3, 11.3 Hz), 0.97, 0.94 and 0.79 (3s, 27H, tert-Bu), 0.17, 0.16, 0.11, 0.10, −0.02 and −0.23 (6s, 18H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 154.3, 152.3, 148.4, 144.9, 130.0, 129.0, 128.7, 126.7, 123.2, 120.1, 88.6, 86.0, 76.5, 72.1, 62.6, 26.2, 25.9, 25.7, 18.7, 18.2, 18.0, −4.2, −4.4, −4.5, −4.8, −5.2. HRMS (ESI) calculated C₃₆H₅₉N₇O₄Si₃Na [M + Na]⁺: 760.3829, found: 760.3826.

Minor isomer: 6-[5-(Phenyl)-1,2,3-triazol-1-yl]-9-[2,3,5-tri-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (5a′)

Obtained 4.4 mg (2% yield) of a brown solid. R_f (silica gel, 20% acetone in hexanes) = 0.19. ¹H NMR (500 MHz, CDCl₃): δ 8.82 (s, 1H, Ar–H), 8.55 (s, 1H, Ar–H), 7.93 (s, 1H, Ar–H), 7.36-7.28 (m, 5H, Ar–H), 6.19 (d, 1H, H–1′, J = 5.1 Hz), 4.60 (t, 1H, H–2′, J = 4.7 Hz), 4.31 (t, 1H, H–3′, J = 3.8 Hz), 4.17 (m, 1H, H–4′), 4.02 (dd, 1H, H–5′, J = 3.7, 11.5 Hz), 3.81 (dd, 1H, H–5′, J = 2.4, 11.5 Hz), 0.95, 0.94 and 0.79 (3s, 27H, tert-Bu), 0.13, 0.11, 0.10, −0.02 and −0.25 (5s, 18H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 154.5, 152.1, 146.9, 145.7, 139.5, 133.9, 129.4, 128.8, 127.7, 127.1, 88.7, 86.1, 76.5, 72.1, 62.7, 26.3, 26.0, 25.9, 18.8, 18.3, 18.0, −4.2, −4.4, −4.5, −4.8, −5.1. HRMS (ESI) calculated C₃₆H₅₉N₇O₄Si₃Na [M + Na]⁺: 760.3829, found: 760.3829.

6-[4-(4-Methylphenyl)-1,2,3-triazol-1-yl]-9-[2,3,5-tri-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (5b)

Synthesized from 3b (200 mg, 0.314 mmol) and 4-ethynyltoluene (79.5 μL, 0.629 mmol). Chromatography of the crude reaction mixture on a silica gel column using 10% acetone in hexanes yielded 5b as a light-yellow solid (214 mg, 90% yield). R_f (silica gel, 10% acetone in hexanes) = 0.29. ¹H NMR (500 MHz, CDCl₃): δ 9.31 (s, 1H, Ar–H), 8.96 (s, 1H, Ar–H), 8.67 (s, 1H, Ar–H), 7.90 (d, 2H, Ar–H, J = 8.0 Hz), 7.29 (d, 2H, Ar–H, J = 8.0 Hz), 6.22 (d, 1H, H–1′, J = 5.2 Hz), 4.66 (t, 1H, H–2′, J = 4.6 Hz), 4.33 (t, 1H, H–3′, J = 3.9 Hz), 4.19 (q, 1H, H–4′, J = 3.1 Hz), 4.05 (dd, 1H, H–5′, J = 3.5, 11.4 Hz), 3.83 (dd, 1H, H–5′, J = 2.5, 11.4 Hz), 2.41 (s, 3H, CH₃), 0.98, 0.95, and 0.80 (3s, 27H, tert-Bu), 0.18, 0.17, 0.12, 0.11, −0.01 and −0.22 (6s, 18H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 154.4, 152.4, 148.6, 145.1, 145.0, 138.8, 129.8, 127.3, 126.4, 123.3, 119.7, 88.7, 86.1, 76.5, 72.2, 62.7, 26.3, 26.1, 25.8, 21.5, 18.8, 18.3, 18.0, −4.2, −4.4, −4.5, −4.8, −5.1. HRMS (ESI) calculated for C₃₇H₆₁N₇O₄Si₃Na [M + Na]⁺: 774.3985, found: 774.3986.

6-[4-(4-Methoxyphenyl)-1,2,3-triazol-1-yl]-9-[2,3,5-tri-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (5c)

Synthesized from 3b (200 mg, 0.314 mmol) and 4-ethynylanisole (81.3 μL, 0.628 mmol). Chromatography of the crude reaction mixture on a silica gel column using 20% EtOAc in hexanes yielded 5c as a white, foamy solid (228 mg, 94% yield). R_f (silica gel, 20% EtOAc in hexanes) = 0.29. ¹H NMR (500 MHz, CDCl₃): δ 9.27 (s, 1H, Ar–H), 8.95 (s, 1H, Ar–H), 8.63 (s, 1H, Ar–H), 7.94 (d, 2H, Ar–H, J = 8.8 Hz), 7.01 (d, 2H, Ar–H, J = 8.8 Hz), 6.22 (d, 1H, H–1′, J = 5.8 Hz), 4.66 (t, 1H, H–2′, J = 4.7 Hz), 4.33 (t, 1H, H–3′, J = 3.9 Hz), 4.19 (q, 1H, H–4′, J = 3.0 Hz), 4.05 (dd, 1H, H–5′, J = 3.6, 11.5 Hz), 3.87 (s, 3H, OCH₃), 3.83 (dd, 1H, H–5′, J = 2.4, 11.5 Hz), 0.98, 0.95 and 0.80, (3s, 27H, tert-Bu), 0.18, 0.17, 0.12, 0.11, −0.01 and −0.23 (6s, 18H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 160.2, 154.4, 152.4, 148.4, 145.0, 144.9, 127.8, 123.3, 122.8, 119.2, 114.5, 88.7, 86.0, 76.5, 72.1, 62.7, 55.6, 26.3, 26.0, 25.8, 18.8, 18.3, 18.0, −4.2, −4.4, −4.5, −4.8, −5.1. HRMS (ESI) calculated for C₃₇H₆₁N₇O₅Si₃Na [M + Na]⁺: 790.3934, found: 790.3933.

Minor isomer: 6-[5-(4-Methoxyphenyl)-1,2,3-triazol-1-yl]-9-[2,3,5-tri-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (5c′)

Obtained 9.1 mg (3% yield) of a clear, gummy material. R_f (silica gel, 20% EtOAc in hexanes) = 0.10. ¹H NMR (500 MHz, CDCl₃): δ 8.84 (s, 1H, Ar–H), 8.55 (s, 1H, Ar–H), 7.88 (s, 1H, Ar–H), 7.22 (d, 2H, Ar–H, J = 8.8 Hz), 6.83 (d, 2H, Ar–H, J = 8.8 Hz), 6.19 (d, 1H, H–1′, J = 5.1 Hz), 4.60 (t, 1H, H–2′, J = 4.7 Hz), 4.32 (t, 1H, H–3′, J = 3.9 Hz), 4.17 (m, 1H, H–4′), 4.02 (dd, 1H, H–5′, J = 3.7, 11.5 Hz), 3.81 (merged with OCH₃ resonance, 1H, H–5′), 3.80 (s, 3H, OCH₃), 0.95, 0.94 and 0.79 (3s, 27H, tert-Bu), 0.13, 0.11, 0.10, −0.02 and −0.25 (5s, 18H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 160.5, 154.5, 152.1, 146.9, 145.7, 139.4, 133.4, 130.2, 127.9, 119.3, 114.3, 88.7, 86.0, 76.5, 72.1, 62.7, 55.5, 26.3, 26.0, 25.8, 18.8, 18.3, 18.0, −4.2, −4.4, −4.5, −4.8, −5.1. HRMS (ESI) calculated for C₃₇H₆₁N₇O₅Si₃Na [M + Na]⁺: 790.3934, found: 790.3944.

6-[4-(4-Fluorophenyl)-1,2,3-triazol-1-yl]-9-[2,3,5-tri-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (5d)

Synthesized from 3b (200 mg, 0.314 mmol) and 1-ethynyl-4-fluorobenzene (72.0 μL, 0.629 mmol). Chromatography of the crude reaction mixture on a silica gel column using 10% EtOAc in hexanes yielded 5d as a light-yellow, foamy solid (194 mg, 82% yield). R_f (silica gel, 20% EtOAc in hexanes) = 0.41. ¹H NMR (500 MHz, CDCl₃): δ 9.32 (s, 1H, Ar–H), 8.96 (s, 1H, Ar–H), 8.64 (s, 1H, Ar–H), 7.99 (dd, 2H, Ar–H, J_H,H = 8.6 Hz, J_F,H = 5.3 Hz), 7.17 (t, 2H, Ar–H, J_H,H = J_F,H = 8.6 Hz), 6.23 (d, 1H, H–1′, J = 5.1 Hz), 4.66 (t, 1H, H–2′, J = 4.6 Hz), 4.33 (t, 1H, H–3′, J = 3.9 Hz), 4.19 (m, 1H, H–4′), 4.05 (dd, 1H, H–5′, J = 3.3, 11.3 Hz), 3.84 (dd, 1H, H–5′, J = 2.2, 11.3 Hz), 0.98, 0.95 and 0.80 (3s, 27H, tert-Bu), 0.18, 0.17, 0.11, −0.013 and −0.23 (5s, 18H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 163.2 (d, ¹J_C,F = 248.1 Hz), 154.5, 152.4, 147.6, 145.1, 144.9, 128.2 (d, ³J_C,F = 8.2 Hz), 126.3 (d, ⁴J_C,F = 5.1 Hz), 123.3, 119.9, 116.1 (d, ²J_C,F = 21.8 Hz), 88.7, 86.1, 76.5, 72.1, 62.7, 26.3, 26.1, 25.8, 18.8, 18.3, 18.1, −4.2, −4.4, −4.5, −4.8, −5.1. HRMS (ESI) calculated for C₃₆H₅₈FN₇O₄Si₃Na [M + Na]⁺: 778.3734, found: 778.3721.

Minor isomer: 6-[5-(4-Fluorophenyl)-1,2,3-triazol-1-yl]-9-[2,3,5-tri-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (5d′)

Obtained 2.9 mg (1% yield) of a clear, gummy material. R_f (silica gel, 20% EtOAc in hexanes) = 0.08. ¹H NMR (500 MHz, CDCl₃): δ 8.83 (s, 1H, Ar–H), 8.56 (s, 1H, Ar–H), 7.91 (s, 1H, Ar–H), 7.29 (dd, 2H, Ar–H, J_H,H = 8.7 Hz, J_F,H = 5.2 Hz), 7.01 (t, 2H, Ar–H, J_H,H = J_F,H = 8.7 Hz), 6.19 (d, 1H, H–1′, J = 5.1 Hz), 4.58 (t, 1H, H–2′, J = 4.6 Hz), 4.31 (t, 1H, H–3′, J = 3.9 Hz), 4.19 (m, 1H, H–4′), 4.02 (dd, 1H, H–5′, J = 3.4, 11.5 Hz), 3.81 (dd, 1H, H–5′, J = 2.2, 11.5 Hz), 0.95, 0.94 and 0.79 (3s, 27H, tert-Bu), 0.13, 0.11, 0.10, −0.02 and −0.25 (5s, 18H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 163.4 (d, ¹J_C,F = 250.3 Hz), 154.6, 152.1, 146.5, 145.7, 138.6, 133.9, 130.8 (d, ³J_C,F = 8.6 Hz), 127.6, 123.3, 116.0 (²J_C,F = 22.0 Hz), 88.7, 86.1, 76.6, 72.1, 62.7, 26.3, 26.0, 25.8, 18.8, 18.3, 18.0, −4.2, −4.4, −4.5, −4.8, −5.1. HRMS (ESI) calculated for C₃₆H₅₈FN₇O₄Si₃Na [M + Na]⁺: 778.3734, found: 778.3739.

6-[4-(N-Phthalimidomethyl)-1,2,3-triazol-1-yl]-9-[2,3,5-tri-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (5e)

Synthesized from 3b (200 mg, 0.314 mmol) and N-propargylphthalimide (0.132 g, 0.628 mmol). Chromatography of the crude reaction mixture on a silica gel column using 15% EtOAc in hexanes yielded 5e as an off-white, foamy solid (196 mg, 76% yield). R_f (silica gel, 25% EtOAc in hexanes) = 0.22. ¹H NMR (500 MHz, CDCl₃): δ 9.11 (s, 1H, Ar–H), 8.90 (s, 1H, Ar–H), 8.61 (s, 1H, Ar–H), 7.88 (dd, 2H, Ar–H, J = 2.9, 5.4 Hz), 7.72 (dd, 2H, Ar–H, J = 2.9, 5.4 Hz), 6.19 (d, 1H, H–1′, J = 4.9 Hz), 5.16 (s, 2H, NCH₂), 4.63 (t, 1H, H–2′, J = 4.3 Hz), 4.31 (m, 1H, H–3′), 4.17 (m, 1H, H–4′), 4.03 (dd, 1H, H–5′, J = 3,4, 11.7 Hz), 3.81 (dd, 1H, H–5′, J = 2.4, 11.7 Hz), 0.96, 0.94 and 0.78 (3s, 27H, tert-Bu), 0.16, 0.15, 0.10, −0.03 and −0.25 (5s, 18H, Si-CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 167.6, 154.2, 152.1, 145.0, 144.6, 143.4, 134.1, 132.1, 123.5, 123.4, 123.2, 88.5, 85.8, 76.2, 71.9, 62.4, 33.1, 26.1, 25.8, 25.6, 18.5, 18.1, 17.8, −4.4, −4.6, −4.7, −5.0, −5.3. HRMS (ESI) calculated for C₃₉H₆₀N₈O₆Si₃Na [M + Na]⁺: 843.3836, found: 843.3833.

Minor isomer: 6-[5-(N-Phthalimidomethyl)-1,2,3-triazol-1-yl]-9-[2,3,5-tri-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (5e′)

Obtained 8.5 mg (3% yield) of an off-white, gummy material. R_f (silica gel, 25% EtOAc in hexanes) = 0.10. ¹H NMR (500 MHz, CDCl₃): δ 8.95 (s, 1H, Ar–H), 8.66 (s, 1H, Ar–H), 7.86 (dd, 2H, Ar–H, J = 3.1, 5.4 Hz), 7.75 (dd, 2H, Ar–H, J = 3.4, 5.4 Hz), 7.69 (s, 1H, Ar–H), 6.26 (d, 1H, H–1′, J = 5.1 Hz), 5.55 (s, 2H, NCH₂), 4.64 (t, 1H, H–2′, J = 4.6 Hz), 4.34 (t, 1H, H–3′, J = 3.9 Hz), 4.21-4.17 (m, 1H, H–4′), 4.04 (dd, 1H, H–5′, J = 3.4, 11.5 Hz), 3.83 (dd, 1H, H–5′, J = 2.2, 11.5 Hz), 0.96, 0.95 and 0.82 (3s, 27H, tert-Bu), 0.16, 0.15, 0.12, 0.001 and −0.19 (5s, 18H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 167.5, 154.7, 151.7, 146.6, 145.5, 135.2, 134.6, 134.5, 132.0, 125.7, 123.9, 88.6, 86.0, 76.6, 72.1, 62.7, 33.3, 26.4, 26.1, 25.9, 18.8, 18.3, 18.1, −4.1, −4.4, −4.5, −4.7, −5.1. HRMS (ESI) calculated for C₃₉H₆₀N₈O₆Si₃Na [M + Na]⁺: 843.3836, found: 843.3830.

6-[4-(Ferrocenyl)-1,2,3-triazol-1-yl]-9-[2,3,5-bis-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (5f)

Synthesized from 3b (200 mg, 0.314 mmol) and ethynylferrocene (0.132 g, 0.628 mmol). Chromatography of the crude reaction mixture on a silica gel column using 5% acetone in hexanes yielded 5f as a reddish-brown, foamy solid (189 mg, 71% yield). R_f (silica gel, 10% acetone in hexanes) = 0.18. ¹H NMR (500 MHz, CDCl₃): δ 8.99 (s, 1H, Ar–H), 8.94 (s, 1H, Ar–H), 8.64 (s, 1H, Ar–H), 6.21 (d, 1H, H–1′, J = 5.1 Hz), 4.88 (br s, 2H, ferrocenyl–H), 4.65 (t, 1H, H–2′, J = 4.5 Hz), 4.36 (br s, 2H, ferrocenyl–H), 4.33 (t, 1H, H–3′, J = 4.0 Hz), 4.19 (m, 1H, H–4′), 4.12 (s, 5H, ferrocenyl–H), 4.05 (dd, 1H, H–5′, J = 3.3, 11.5 Hz), 3.83 (dd, 1H, H–5′, J = 2.0, 11.5 Hz), 0.98, 0.94 and 0.81 (3s, 27H, tert-Bu), 0.18, 0.16, 0.11, 0.107, −0.01 and −0.20 (6s, 18H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 154.4, 152.4, 147.9, 145.0, 144.9, 123.3, 118.8, 88.7, 85.9, 76.4, 74.7, 72.1, 69.9, 69.2, 67.3, 62.7, 26.3, 26.0, 25.9, 18.8, 18.3, 18.0, −4.2, −4.4, −4.5, −4.7, −5.1. HRMS (ESI) calculated for C₄₀H₆₃FeN₇O₄Si₃Na [M + Na]⁺: 868.3491, found: 868.3487.

Minor isomer: 6-[5-(Ferrocenyl)-1,2,3-triazol-1-yl]-9-[2,3,5-bis-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (5f′)

Obtained 28.3 mg (10% yield) of a brown solid. R_f (silica gel, 40% EtOAc in hexanes) = 0.41. ¹H NMR (500 MHz, CDCl₃): δ 8.98 (s, 1H, Ar–H), 8.60 (s, 1H, Ar–H), 7.89 (s, 1H, Ar–H), 6.22 (d, 1H, H–1′, J = 5.1 Hz), 4.60 (t, 1H, H–2′, J = 4.6 Hz), 4.41 (m, 2H, ferrocenyl–H), 4.32 (t, 1H, H–3′, J = 4.0 Hz), 4.25 (m, 2H, ferrocenyl–H), 4.18 (q, 1H, H–4′, J = 3.1 Hz), 4.02 (dd, 1H, H–5′, J = 3.4, 11.5 Hz), 3.99 (s, 5H, ferrocenyl–H), 3.81 (dd, 1H, H–5′, J = 2.4, 11.5 Hz), 0.943, 0.939 and 0.81 (3s, 27 H, tert-Bu), 0.14, 0.13, 0.10, −0.005 and −0.20 (5s, 18H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 154.4, 152.0, 147.1, 145.7, 138.9, 133.3, 128.5, 88.7, 85.9, 76.6, 72.0, 70.0, 69.5, 69.4, 62.6, 53.6, 26.3, 26.0, 25.9, 18.8, 18.3, 18.0, −4.2, −4.4, −4.5, −4.7, −5.1. HRMS (ESI) calculated for C₄₀H₆₃FeN₇O₄Si₃Na [M + Na]⁺: 868.3491, found: 868.3489.

6-[4-(1-Butyl)-1,2,3-triazol-1-yl]-9-[2,3,5-bis-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]purine (5g)

Synthesized from 3b (200 mg, 0.314 mmol) and 1-hexyne (72.2 μL, 0.628 mmol). Chromatography of the crude reaction mixture on a silica gel column using 5% acetone in hexanes yielded 5g as a yellow, foamy solid (184 mg, 81% yield). R_f (silica gel, 10% acetone in hexanes) = 0.22. ¹H NMR (500 MHz, CDCl₃): δ 8.92 (s, 1H, Ar–H), 8.85 (s, 1H, Ar–H), 8.59 (s, 1H, Ar–H), 6.20 (d, 1H, H–1′, J = 5.1 Hz), 4.65 (t, 1H, H–2′, J = 4.6 Hz), 4.32 (t, 1H, H–3′, J = 3.9 Hz), 4.18 (q, 1H, H–4′, J = 3.0 Hz), 4.03 (dd, 1H, H–5′, J = 3.7, 11.3 Hz), 3.82 (dd, 1H, H–5′, J = 2.5, 11.3 Hz), 2.88 (t, 2H, butyl–CH₂, J = 7.7 Hz), 1.77 (quint, 2H, butyl–CH₂, J = 7.6 Hz), 1.45 (sextet, 2H, butyl–CH₂, J = 7.4 Hz), 0.97, 0.94 and 0.79 (overlapping 3s and t, 30H, tert-Bu and butyl–CH₃), 0.17, 0.16, 0.11, 0.10, −0.02 and −0.24 (6s, 18H, Si–CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 154.4, 152.4, 149.4, 145.2, 144.8, 123.3, 121.4, 88.7, 86.1, 76.4, 72.2, 62.7, 31.6, 26.3, 26.1, 25.8, 25.5, 22.5, 18.8, 18.3, 18.1, 14.0, −4.2, −4.4, −4.5, −4.8, −5.1. HRMS (ESI) calculated for C₃₄H₆₃N₇O₄Si₃Na [M + Na]⁺: 740.4142, found 740.4139.

General method for desilylation of the click products. Synthesis of 6-(4-phenyl-1,2,3-triazol-1-yl)-9-(2-deoxy-β-d-ribofuranosyl)purine (6a)

In a clean, dry plastic vial equipped with a stirring bar was placed the nucleoside derivative 4a (91.2 mg, 0.150 mmol) in anhydrous THF (2.0 mL). Et₃N•3HF (85 μL, 0.525 mmol) was added to the stirring mixture at room temperature. After 34 h at room temperature TLC indicated complete consumption of the starting material at which time the mixture was evaporated to dryness under a stream of nitrogen gas. Chromatographic purification on a silica gel column using 3-5% MeOH in hexanes afforded 6a as white powder (54.6 mg, 96% yield). R_f (silica gel, 10% MeOH in CH₂Cl₂) = 0.30. ¹H NMR (500 MHz, DMSO-d₆): δ 9.62 (s, 1H, Ar–H), 9.04 (s, 1H, Ar–H), 9.03 (s, 1H, Ar–H), 8.07 (d, 2H, Ar–H, J = 7.3 Hz), 7.53 (t, 2H, Ar–H, J = 7.6 Hz), 7.43 (t, 1H, Ar–H, J = 7.4 Hz), 6.57 (t, 1H, H–1′, J = 6.5 Hz), 5.40 (d, 1H, OH, J = 4.2 Hz), 5.01 (t, 1H, OH, J = 5.5 Hz), 4.49 (m, 1H, H–3′), 3.94 (app q, 1H, H–4′, J_app ∼ 3.6 Hz), 3.66 (m, 1H, H–5′), 3.07 (m, 1H, H–5′), 2.83 (m, 1H, H–2′), 2.43 (m, 1H, H–2′, superimposed with solvent). ¹³C NMR (125 MHz, acetone-d₆ + 4 drops DMSO-d₆): δ 155.2, 152.7, 148.5, 147.2, 145.6, 131.2, 130.0, 129.6, 126.9, 124.5, 121.6, 89.8, 86.0, 72.1, 62.9, 41.7. HRMS (ESI) calculated for C₁₈H₁₈N₇O₃ [M + H]⁺: 380.1466, found: 380.1465.

6-[4-(4-Methylphenyl)-1,2,3-triazol-1-yl]-9-(2-deoxy-β-d-ribofuranosyl)purine (6b)

Desilylation of 4b (93.3 mg, 0.150 mmol) with Et₃N•3HF (0.15 mL, 0.901 mmol) and chromatographic purification as for 6a afforded 6b as white powder (43.9 mg, 74% yield). R_f (silica gel, 10% MeOH in CH₂Cl₂) = 0.32. ¹H NMR (500 MHz, DMSO-d₆): δ 9.55 (s, 1H, Ar–H), 9.03 (s, 1H, Ar–H), 9.02 (s, 1H, Ar–H), 7.95 (d, 2H, Ar–H, J = 7.8 Hz), 7.33 (d, 2H, Ar–H, J = 7.8 Hz), 6.57 (t, 1H, H–1′, J = 6.6 Hz), 5.40 (d, 1H, OH, J = 4.1 Hz), 5.01 (t, 1H, OH, J = 5.2 Hz), 4.49 (br m, 1H, H–3′), 3.93 (app q, 1H, H–4′, J_app = 3.8 Hz), 3.66 (m, 1H, H–5′), 3.57 (m, 1H, H–5′), 2.83 (app quint, 1H, H–2′, J_app ∼ 6.5 Hz), 2.43 (ddd, 1H, H-2′, J = 4.5, 6.5, 13.1 Hz). ¹³C NMR (125 MHz, acetone-d₆ + 15 drops DMSO-d₆): δ 154.8, 152.3, 148.0, 146.9, 145.1, 138.9, 130.2, 127.9, 126.4, 124.0, 120.8, 89.3, 85.3, 71.5, 62.3, 40.7, 21.3. HRMS (ESI) calculated for C₁₉H₂₀N₇O₃ [M + H]⁺: 394.1622, found: 394.1619.

6-[4-(4-Methoxyphenyl)-1,2,3-triazol-1-yl]-9-(2-deoxy-β-d-ribofuranosyl)purine (6c)

Desilylation of 4c (95.7 mg, 0.150 mmol) with Et₃N•3HF (85 μL, 0.525 mmol) and chromatographic purification as for 6a afforded 6c as white powder (50.8 mg, 83% yield). R_f (silica gel, 10% MeOH in CH₂Cl₂) = 0.32. ¹H NMR (500 MHz, DMSO-d₆): δ 9.50 (s, 1H, Ar–H), 9.02 (s, 1H, Ar–H), 9.01 (s, 1H, Ar–H), 7.99 (d, 2H, Ar–H, J = 8.8 Hz), 7.08 (d, 2H, Ar–H, J = 8.8 Hz), 6.57 (t, 1H, H–1′, J = 6.6 Hz), 4.48 (m, 1H, H–3′), 3.93 (app q, 1H, H–4′, J_app ∼ 4.0 Hz), 3.66 (dd, 1H, H–5′, J = 4.6, 11.7 Hz), 3.57 (dd, 1H, H-5′, J = 4.5, 11.7 Hz), 2.82 (app quint, 1H, H–2′, J_app ∼ 6.5 Hz), 2.43 (ddd, 1H, H-2′, J = 3.9, 6.3, 13.5 Hz). ¹³C NMR (125 MHz, acetone-d₆ + 15 drops DMSO-d₆): δ 160.9, 155.0, 152.6, 148.2, 147.0, 145.4, 128.1, 124.3, 123.5, 120.4, 115.3, 89.6, 85.7, 71.8, 62.6, 55.9, 41.3. HRMS (ESI) calculated for C₁₉H₂₀N₇O₄ [M + H]⁺: 410.1571, found: 410.1570.

6-[4-(4-Fluorophenyl)-1,2,3-triazol-1-yl]-9-(2-deoxy-β-d-ribofuranosyl)purine (6d)

Desilylation of 4d (93.9 mg, 0.150 mmol) with Et₃N•3HF (85 μL, 0.525 mmol) and chromatographic purification as for 6a afforded 6d as white solid (50.8 mg, 83% yield). R_f (silica gel, 10% MeOH in CH₂Cl₂) = 0.37. ¹H NMR (500 MHz, DMSO-d₆): δ 9.62 (s, 1H, Ar–H), 9.04 (s, 1H, Ar–H), 9.03 (s, 1H, Ar–H), 8.13 (dd, 2H, Ar–H, J_H,H = 8.6 Hz, J_F,H = 5.5 Hz), 7.37 (t, 2H, Ar–H, J_H,H = J_F,H = 8.6 Hz), 6.57 (t, 1H, H–1′, J = 6.3 Hz), 5.39 (br s, 1H, OH), 5.01 (br s, 1H, OH), 4.49 (m, 1H, H–3′), 3.94 (app q, 1H, H–4′, J_app ∼ 4.1 Hz), 3.66 (dd, 1H, H–5′, J = 4.5, 11.4 Hz), 3.57 (dd, 1H, H–5′, J = 4.2, 11.4 Hz), 2.83 (app quint, 1H, H-2′, J_app ∼ 6.5 Hz), 2.43 (ddd, 1H, H–2′, J = 3.7, 6.3, 13.3 Hz). ¹³C NMR (125 MHz, acetone-d₆ + 10 drops DMSO-d₆): δ 163.4 (d, ¹J_C,F = 245.7 Hz), 155.2, 152.6, 147.3, 147.2, 145.5, 129.0 (d, ³J_C,F = 8.3 Hz), 127.6 (d, ⁴J_C,F = 3.2 Hz), 124.5, 121.4, 116.8 (²J_C,F = 21.9 Hz), 89.7, 85.9, 72.0, 62.7, 41.5. HRMS (ESI) calculated for C₁₈H₁₇FN₇O₃ [M + H]⁺: 398.1371, found: 398.1386.

6-[4-(N-Phthalimidomethyl)-1,2,3-triazol-1-yl]-9-(2-deoxy-β-d-ribofuranosyl)purine (6e)

Desilylation of 4e (112.9 mg, 0.163 mmol) with Et₃N•3HF (85 μL, 0.525 mmol) and chromatographic purification as for 6a afforded 6e as white solid (64.5 mg, 85% yield). R_f (silica gel, 10% MeOH in CH₂Cl₂) = 0.27. ¹H NMR (500 MHz, DMSO-d₆): δ 9.22 (s, 1H, Ar–H), 9.00 (s, 1H, Ar–H), 8.98 (s, 1H, Ar–H), 7.92 (m, 2H, Ar–H), 7.87 (m, 2H, Ar–H), 6.54 (t, 1H, H–1′, J = 6.6 Hz), 5.03 (s, 2H, NCH₂), 4.46 (m, 1H, H–3′), 3.92 (app q, 1H, H–4′, J_app ∼ 4.0 Hz), 3.64 (dd, 1H, H–5′, J = 4.5, 11.8 Hz), 3.55 (dd, 1H, H–5′, J = 4.5, 11.8 Hz), 2.79 (app quint, 1H, H–2′, J_app ∼ 6.5 Hz), 2.41 (ddd, 1H, H–2′, J = 3.9, 6.3, 13.3 Hz). ¹³C NMR (125 MHz, acetone-d₆ + 15 drops DMSO-d₆): δ 168.2, 154.8, 152.4, 147.0, 144.9, 144.2, 135.2, 132.7, 124.4, 123.9, 89.3, 85.4, 71.5, 62.4, 41.0, 33.5. HRMS (ESI) calculated for C₂₁H₁₉N₈O₅ [M + H]⁺: 463.1473, found: 463.1476.

6-[4-(Ferrocenyl)-1,2,3-triazol-1-yl]-9-(2-deoxy-β-d-ribofuranosyl)purine (6f)

Desilylation of 4f (107.3 mg, 0.150 mmol) with Et₃N•3HF (85 μL, 0.525 mmol) and chromatographic purification as for 6a afforded 6f as an orange solid (51.8 mg, 71% yield). R_f (silica gel, 10% MeOH in CH₂Cl₂) = 0.26. ¹H NMR (500 MHz, DMSO-d₆): δ 9.23 (s, 1H, Ar–H), 9.02 (s, 1H, Ar–H), 9.01 (s, 1H, Ar–H), 6.57 (t, 1H, H–1′, J = 6.6 Hz), 4.95 (br t, 2H, ferrocenyl–H, J = 1.5 Hz), 4.48 (m, 1H, H–3′), 4.40 (br t, 2H, ferrocenyl–H, J = 1.5 Hz), 4.10 (s, 5H, ferrocenyl–H), 3.94 (app q, 1H, H–4′, J_app ∼ 4.3 Hz), 3.66 (dd, 1H, H–5′, J = 4.6, 11.7 Hz), 3.57 (dd, 1H, H–5′, J = 4.4, 11.7 Hz), 2.81 (app quint, 1H, H–2′, J_app ∼ 6.5 Hz), 2.43 (ddd, 1H, H-2′, J = 4.0, 6.3 13.3 Hz). ¹³C NMR (125 MHz, acetone-d₆ + 10 drops DMSO-d₆): δ 155.3, 152.8, 148.2, 147.1, 145.8, 124.5, 120.4, 90.0, 86.2, 76.1, 72.3, 70.6, 70.0, 68.0, 63.0, 41.9. HRMS (ESI) calculated for C₂₂H₂₁FeN₇O₃Na [M + Na]⁺: 510.0948, found: 510.0948.

6-[4-(1-Butyl)-1,2,3-triazol-1-yl]-9-(2-deoxy-β-d-ribofuranosyl)purine (6g)

Desilylation of 4g (88.2 mg, 0.150 mmol) with Et₃N•3HF (85 μL, 0.525 mmol) and chromatographic purification as for 6a afforded 6g as a clear, gummy material (38.0 mg, 70% yield). R_f (silica gel, 10% MeOH in CH₂Cl₂) = 0.35. ¹H NMR (500 MHz, DMSO-d₆): δ 8.98 (s, 1H, Ar–H), 8.97 (s, 1H, Ar–H), 8.96 (s, 1H, Ar–H), 6.55 (t, 1H, H–1′, J = 6.6 Hz), 5.39 (d, 1H, OH, J = 4.4 Hz), 4.99 (t, 1H, OH, J = 5.5 Hz), 4.47 (m, 1H, H–3′), 3.93 (app q, 1H, H–4′, J_app ∼ 4.0 Hz), 3.65 (m, 1H, H–5′), 3.56 (m, 1H, H–5′), 2.84-2.76 (m, 3H, H–2′ and butyl–CH₂), 2.42 (ddd, 1H, H–2′, J = 3.9, 6.3, 13.3 Hz), 1.64 (quint, 2H, butyl–CH₂, J = 7.5 Hz), 1.38 (sextet, 2H, butyl–CH₂, J = 7.4 Hz), 0.93 (t, 3H, butyl–CH₃, J = 7.3 Hz). ¹³C NMR (125 MHz, acetone-d₆ + 15 drops DMSO-d₆): δ 154.7, 152.4, 149.0, 146.7, 145.3, 123.8, 122.5, 89.3, 85.4, 71.5, 62.4, 41.0, 31.8, 25.4, 22.6, 14.1. HRMS (ESI) calculated for C₁₆H₂₂N₇O₃ [M + H]⁺: 360.1779, found: 360.1791.

6-[4-(Phenyl)-1,2,3-triazol-1-yl]-9-(β-d-ribofuranosyl)purine (7a)

Desilylation of 5a (110.7 mg, 0.150 mmol) with Et₃N•3HF (0.12 mL, 0.75 mmol) and chromatographic purification on a silica gel column using 3-7% MeOH in hexanes afforded 7a as a white, fluffy solid (57.3 mg, 96% yield). R_f (silica gel, 10% MeOH in CH₂Cl₂) = 0.28. ¹H NMR (500 MHz, DMSO-d₆): δ 9.62 (s, 1H, Ar–H), 9.09 (s, 1H, Ar–H), 9.05 (s, 1H, Ar–H), 8.05 (d, 2H, Ar–H, J = 7.3 Hz), 7.53 (t, 2H, Ar–H, J = 7.5 Hz), 7.43 (t, 1H, Ar–H, J = 7.6 Hz) 6.15 (d, 1H, H–1′, J = 4.9 Hz), 5.62 (d, 1H, OH, J = 5.6 Hz), 5.28 (d, 1H, OH, J = 5.1 Hz), 5.14 (t, 1H, OH, J = 5.1 Hz), 4.65 (m, 1H, H–2′), 4.24 (m, 1H, H–3′), 4.03 (m, 1H, H-4′), 3.74 (m, 1H, H-5′), 3.62 (m, 1H, H-5′). ¹³C NMR (125 MHz, THF-d₈ + 4 drops DMSO-d₆): δ 155.5, 152.7, 148.5, 147.2, 146.0, 131.6, 129.8, 129.3, 126.9, 124.7, 121.2, 90.4, 87.7, 76.5, 71.9, 62.6. HRMS (ESI) calculated for C₁₈H₁₈N₇O₄ [M + H]⁺: 396.1415, found: 396.1416.

6-[4-(4-Methylphenyl)-1,2,3-triazol-1-yl]-9-(β-d-ribofuranosyl)purine (7b)

Desilylation of 5b (112.8 mg, 0.15 mmol) with Et₃N•3HF (0.12 mL, 0.75 mmol) and chromatographic purification as for 7a afforded 7b as a white solid (59.2 mg, 96% yield). R_f (silica gel, 10% MeOH in CH₂Cl₂) = 0.28. ¹H NMR (500 MHz, DMSO-d₆): δ 9.56 (s, 1H, Ar–H), 9.08 (s, 1H, Ar–H), 9.04 (s, 1H, Ar–H), 7.96 (d, 2H, Ar–H, J = 7.8 Hz), 7.33 (d, 2H, Ar–H, J = 7.8 Hz), 6.15 (d, 1H, H–1′, J = 4.9 Hz), 5.62 (d, 1H, OH, J = 5.8 Hz), 5.28 (d, 1H, OH, J = 5.1 Hz), 5.14 (t, 1H, OH, J = 5.3 Hz), 4.65 (m, 1H, H–2′), 4.24 (m, 1H, H–3′), 4.02 (m, 1H, H–4′), 3.74 (m, 1H, H–5′), 3.62 (m, 1H, H–5′), 2.37 (s, 3H, CH₃). ¹³C NMR (125 MHz, THF-d₈ + 4 drops DMSO-d₆): δ 155.5, 152.7, 148.6, 147.1, 145.9, 139.0, 130.4, 128.8, 126.8, 124.7, 120.7, 90.5, 87.7, 76.4, 71.9, 62.6, 21.5. HRMS (ESI) calculated for C₁₉H₂₀N₇O₄ [M + H]⁺: 410.1571, found: 410.1572.

6-[4-(4-Methoxyphenyl)-1,2,3-triazol-1-yl]-9-(β-d-ribofuranosyl)purine (7c)

Desilylation of 5c (115.2 mg, 0.150 mmol) with Et₃N•3HF (0.12 mL, 0.75 mmol) and chromatographic purification as for 7a afforded 7c as a white solid (51.3 mg, 81% yield). R_f (silica gel, 10% MeOH in CH₂Cl₂) = 0.30. ¹H NMR (500 MHz, DMSO-d₆): δ 9.51 (s, 1H, Ar–H), 9.07 (s, 1H, Ar–H), 9.04 (s, 1H, Ar–H), 8.01 (d, 2H, Ar–H, J = 8.8 Hz), 7.08 (d, 2H, Ar–H, J = 8.8 Hz), 6.15 (d, 1H, H-1′, J = 5.4 Hz), 4.65 (t, 1H, H–2′, J = 5.0 Hz), 4.23 (t, 1H, H–3′, J = 4.5 Hz), 4.03 (app q, 1H, H–4′, J_app ∼ 4.9 Hz), 3.83 (s, 3H, OCH₃), 3.74 (dd, 1H, H-5′, J = 3.9, 11.7 Hz), 3.62 (dd, 1H, H-5′, J = 3.9, 11.7 Hz). ¹³C NMR (125 MHz, THF-d₈ + 4 drops DMSO-d₆): δ 161.2, 155.5, 152.7, 148.5, 147.2, 145.9, 128.2, 124.6, 124.0, 120.1, 115.3, 90.3, 87.6, 76.5, 71.8, 62.5, 55.8. HRMS (ESI) calculated for C₁₉H₂₀N₇O₅ [M + H]⁺: 426.1520, found: 426.1521.

6-[4-(Fluorophenyl)-1,2,3-triazol-1-yl]-9-(β-d-ribofuranosyl)purine (7d)

Desilylation of 5d (113.4 mg, 0.15 mmol) with Et₃N•3HF (0.12 mL, 0.75 mmol) and chromatographic purification as for 7a afforded 7d as a white solid (44.0 mg, 71% yield). R_f (silica gel, 10% MeOH in CH₂Cl₂) = 0.26. ¹H NMR (500 MHz, DMSO-d₆): δ 9.63 (s, 1H, Ar–H), 9.08 (s, 1H, Ar–H), 9.05 (s, 1H, Ar–H), 8.13 (dd, 2H, Ar–H, J_H,H = 8.9 Hz, J_F,H = 5.5 Hz), 7.37 (t, 2H, Ar–H, J_H,H = J_F,H = 8.9 Hz), 6.15 (d, 1H, H–1′, J = 5.1 Hz), 5.62 (d, 1H, OH, J = 5.8 Hz), 5.28 (d, 1H, OH, J = 5.3 Hz), 5.14 (t, 1H, OH, J = 5.3 Hz), 4.65 (m, 1H, H–2′), 4.24 (m, 1H, H–3′), 4.03 (m, 1H, H–4′), 3.74 (m, 1H, H–5′), 3.62 (m, 1H, H–5′). ¹³C NMR (125 MHz, THF-d₈ + 4 drops DMSO-d₆): δ 163.7 (d, ¹J_C,F = 246.3 Hz), 155.6, 152.7, 147.6, 147.3, 145.8, 128.9 (d, ³J_C,F = 8.2 Hz), 128.0 (d, ⁴J_C,F = 3.2 Hz), 124.7, 121.2, 116.7 (d, ²J_C,F = 21.8 Hz), 90.4, 87.7, 76.5, 71.8, 62.5. HRMS (ESI) calculated for C₁₈H₁₇FN₇O₄ [M + H]⁺: 414.1321, found: 414.1319.

6-[4-(N-Phthalimidomethyl)-1,2,3-triazol-1-yl]-9-(β-d-ribofuranosyl)purine (7e)

Desilylation of 5e (115.0 mg, 0.15 mmol) with Et₃N•3HF (0.12 mL, 0.75 mmol) and chromatographic purification as for 7a afforded 7e as a white solid (61.7 mg, 85% yield). R_f (silica gel, 10% MeOH in CH₂Cl₂) = 0.26. ¹H NMR (500 MHz, DMSO-d₆): δ 9.22 (s, 1H, Ar–H), 9.05 (s, 1H, Ar–H), 8.99 (s, 1H, Ar–H), 7.94-7.91 (m, 2H, Ar–H), 7.89–7.86 (m, 2H, Ar–H), 6.12 (d, 1H, H–1′, J = 5.1 Hz), 5.03 (s, 2H, NCH₂), 4.60 (t, 1H, H–2′, J = 5.2 Hz), 4.21 (t, 1H, H–3′, J = 4.3 Hz), 4.01 (app q, 1H, H–4′, J_app ∼ 3.8 Hz), 3.72 (dd, 1H, H–5′, J = 3.6, 12.0 Hz), 3.60 (dd, 1H, H–5′, J = 3.8, 12.0 Hz). ¹³C NMR (125 MHz, THF-d₈ + 4 drops DMSO-d₆): δ 168.3, 155.5, 152.7, 147.3, 145.7, 144.5, 135.2, 133.5, 124.6, 124.5, 124.1, 90.2, 87.6, 76.5, 71.8, 62.5, 33.8. HRMS (ESI) calculated for C₂₁H₁₉N₈O₆ [M + H]⁺: 479.1422, found: 479.1432.

6-[4-(Ferrocenyl)-1,2,3-triazol-1-yl]-9-(β-d-ribofuranosyl)purine (7f)

Desilylation of 5f (126.9 mg, 0.15 mmol) with Et₃N•3HF (0.12 mL, 0.75 mmol) and chromatographic purification as for 7a afforded 7f as an orange solid (73.4 mg, 97% yield). R_f (silica gel, 10% MeOH in CH₂Cl₂) = 0.28. ¹H NMR (500 MHz, DMSO-d₆): δ 9.23 (s, 1H, Ar–H), 9.07 (s, 1H, Ar–H), 9.02 (s, 1H, Ar–H), 6.14 (d, 1H, H–1′, J = 5.1 Hz), 4.95 (br t, 2H, ferrocenyl–H), 4.64 (t, 1H, H-2′, J = 4.9 Hz), 4.41 (br t, 2H, ferrocenyl–H), 4.23 (t, 1H, H–3′, J = 4.4 Hz), 4.10 (s, 5H, ferrocenyl–H), 4.03 (app q, 1H, H–4′, J_app ∼ 3.9 Hz), 3.74 (dd, 1H, H–5′, J = 3.9, 11.8 Hz), 3.62 (dd, 1H, H–5′, J = 3.9, 11.8 Hz). ¹³C NMR (125 MHz, THF-d₈ + 4 drops DMSO-d₆): δ 155.5, 152.7, 148.0, 147.1, 145.8, 124.5, 120.0, 90.4, 87.6, 76.5, 76.3, 71.8, 70.5, 69.7, 62.5. HRMS (ESI) calculated for C₂₂H₂₂FeN₇O₄ [M + H]⁺: 504.1077, found: 504.1060.

6-[4-(1-Butyl)-1,2,3-triazol-1-yl]-9-(β-d-ribofuranosyl)purine (7g)

Desilylation of 5g (107.7 mg, 0.15 mmol) with Et₃N•3HF (0.12 mL, 0.75 mmol) and chromatographic purification as for 7a afforded 7g as a white solid (53.5 mg, 95% yield). R_f (silica gel, 10% MeOH in CH₂Cl₂) = 0.28. ¹H NMR (500 MHz, DMSO-d₆): δ 9.03 (s, 1H, Ar–H), 8.99 (s, 1H, Ar–H), 8.97 (s, 1H, Ar–H), 6.12 (d, 1H, H–1′, J = 5.1 Hz), 5.60 (br s, 1H, OH), 5.27 (br s, 1H, OH), 5.12 (br s, 1H, OH), 4.63 (t, 1H, H–2′, J = 4.7 Hz), 4.22 (t, 1H, H–3′, J = 4.2 Hz), 4.01 (app q, 1H, H–4′, J_app ∼ 3.8 Hz), 3.72 (dd, 1H, H–5′, J = 3.4, 11.7 Hz), 3.61(dd, 1H, H–5′, J = 3.2, 11.7 Hz), 2.80 (t, 2H, butyl–CH₂, J = 7.6 Hz), 1.70 (quint, 2H, butyl–CH₂, J = 7.5 Hz), 1.39 (sextet, 2H, butyl–CH₂, J = 7.4 Hz), 0.94 (t, 3H, butyl–CH₃, J = 7.3 Hz). ¹³C NMR (125 MHz, THF-d₈ + 4 drops DMSO-d₆): δ 155.3, 152.7, 149.2, 146.9, 146.0, 124.4, 122.4, 90.4, 87.7, 76.4, 71.9, 62.6, 32.4, 26.1, 23.3, 14.4. HRMS (ESI) calculated for C₁₆H₂₂N₇O₄ [M + H]⁺: 376.1728, found: 376.1731.

SCHEME 5. Desilylation of the Click Products.

TABLE 5. Azide-Alkyne Click Reactions of Nucleosides 3a and 3b.

entry	substrate	alkyne	reaction time and temp	product: yielda
1	3a		4.0 h, room temp	4a: 77%
2	3b		4.0 h, room temp	5a: 90%
				5a′: 2%b
3	3a		4.0 h, room temp	4b: 87%
4	3b		4.0 h, room temp	5b: 90%
5	3a		4.0 h, room temp	4c: 90%
6	3b		3.5 h, room temp	5c: 94%
				5c′: 3%b
7	3a		4.5 h, room temp	4d: 92%
8	3b		4.0 h, room temp	5d: 82%
				5d′: 1%b
9	3a		7.0 h, 40 °C	4e: 95%
10	3b		4.0 h, 40 °C	5e: 76%
				5e′: 3%b
11	3a		4.0 h, room temp	4f: 83%
				4f′: 6%b
12	3b		4.0 h, room temp	5f: 71%
				5f′: 10%b
13	3a		4.5 h, room temp	4g: 75%
14	3b		4.5 h, room temp	5g: 81%

^aYields of isolated and purified products.

^bThe isomeric 1,5-disubstituted triazole was also isolated as a minor byproduct.