A Simple Method for C-6 Modification of Guanine Nucleosides

Abstract

A facile method for the introduction of various substituents at the C-6 position of guanosine and 2′-deoxyguanosine is reported. In a simple, 1-step transformation, tert-butyldimethylsilyl protected guanosine and 2′-deoxyguanosine were converted to the O⁶-(benzotriazol-1-yl) derivatives via reaction with 1H-benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). The easily isolated, stable and storable, O⁶-(benzotriazol-1-yl) guanosine derivatives upon exposure to range of nucleophiles, under appropriate conditions, led to the C-6 modified 2-amino purine nucleoside analogues in good yields.

Introduction

The development of chemical methods to modify nucleosides continues to attract attention since unnatural and unusual nucleosides play a prominant role in biochemistry, biology and medicine.¹ Typically, modification at the C-6 position of the guanine nucleus requires the introduction of a suitable leaving group at that position, followed by S_NAr displacement with nucleophiles.

Although chlorination of guanosine can be readily accomplished from its triacetate derivative,^2,3 chlorination of the 2′-deoxy analogue has been reported to be low yielding.⁴ Therefore, improved methods have been reported for C-6 chlorination of 2′-deoxyguanosine 3′,5′-diacetate with careful control of reaction conditions.^5,6 In addition to C-6 chlorination of the guanine nucleosides, facile synthesis of the O6-arylsulfonates is also known.^6–12 Thus, the 6-chloro and O6-arylsulfonyl derivatives have served as the principal electrophilic nucleoside derivatives used in displacement reactions leading to C-6 modified 2-amino purine nucleosides.

Recently, we have developed a new class of O⁶-(benzotriazol-1-yl)inosine analogues that are easily prepared via a reaction of either protected or unprotected inosine and 2′-deoxyinosine with 1H-benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP).¹³ The basis for this chemistry resides in the postulated formation of C-6 phosphonium salts in the reaction of the hypoxanthine moiety with PPh₃/I₂¹⁴ or BOP.¹⁵ We reasoned that in the absence of other nucleophiles, the hydroxybenzotriazole from BOP should react as a nucleophile on the nucleoside phosphonium salts. This was indeed the case and not only were we able to synthesize O⁶-(benzotriazol-1-yl)inosine analogues, but we have been able to utilize the underlying chemistry to load inosine and 2′-deoxyinosine onto a polymer linker.^16,17 The intermediacy of nucleoside phosphonium salts has also proven important in our recently described N2-modification of 2′-deoxyguanosine,¹⁸ and in chemistry leading to N,N-modified adenosine analogues via reaction of protected inosine and 2′-deoxyinosine with hexaalkylphosphorus triamides produced in situ and I₂.¹⁹ In this paper, we report our findings on the synthesis of O⁶-(benzotriazol-1-yl)guanosine and 2′-deoxyguanosine, and their applications leading to C-6 modified 2-amino purine nucleoside analogues. While this work was in progress, preparation of O⁶-(benzotriazol-1-yl)-3′,5′-di-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine and its use for the synthesis of C-8 acetylarylamine adducts was reported.²⁰ However, no use of the O⁶-(benzotriazol-1-yl)guanine nucleosides for other types of functionalization is currently known.

Results and Discussion

In our initial work, we had shown that conversion of inosine and deoxyinosine to the O⁶-(benzotriazol-1-yl) derivatives could be accomplished via the use of BOP and i-Pr₂NEt as base. Although the reactions of the hypoxanthine nucleosides proceeded to completion,¹³ similar reactions of the guanine nucleosides proved to be quite slow. In fact, the literature reports 72 h even in a solvent such as DMF.²⁰ Therefore, we decided to reassess this reaction at the outset.

Table 1 summarizes the results from these inital experiments. In order to avoid formation of the N⁶,N⁶-dimethyl-2,6-diaminopurine derivate from decomposition of DMF, we chose to conduct our first experiment in DMSO (Table 1, entry 1). In this solvent, elevated temperature and a prolonged reaction time were necessary. Upon switching to THF as solvent (entry 2), practically no reaction was observed, but when DBU was used as base a significantly fast reaction was observed (entry 3) that reached completion within 4 h. Finally, replacing THF with CH₃CN led to an improvement in reaction time and yield. In CH₃CN as solvent and with DBU as base, the reaction was complete within 2 h with a 65% isolated yield of 3 (entry 4). Under these conditions the 2′-deoxy analogue 4 could be prepared in 85% yield (the previously reported yield for 4 was 67%²⁰).

Table 1.

Initial studies at determining optimal conditions for synthesis of the O⁶-(benzotriazol-1-yl)guanosine analogues 3 and 4^a

Entry	Substrate	Solvent	Base	Temp	Time	Yieldb
1	1	DMSO	i-Pr2NEt	55 °C	24 h	54%
2	1	THF	i-Pr2NEt	rt	96 h	NRc
3	1	THF	DBU	rt	4 h	45%
4	1	CH3CN	DBU	rt	2 h	65%
5	2	CH3CN	DBU	rt	1 h	85%

^aReactions were performed using 2.0 molar equiv each of BOP and DBU at ~0.1 M nucleoside concentration.

^bWhere reported, yield is of isolated and purified products.

^cNo reaction was observed and only 1 was present.

Our observations on optimal solvent and base parallel those reported for the amination of 4-hydroxyquinazoline using BOP and n-BuNH₂.²¹ Besides solubility considerations, one major contributor to the differences in the reactions of inosine and guanosine derivatives should be the pK_a values of the acidic amide protons. Inosine is more acidic, with the amide pK_a of ~8; in guanosine it is ~9.^22,23 Nevertheless, both nucleosides can be conveniently converted to the O⁶-(benzotriazol-1-yl) derivatives under appropriate conditions.

With the synthesis of the O⁶-(benzotriazol-1-yl)guanine derivatives 3 and 4 optimized, the next step was an evaluation of their use for modification at the C-6 position. For this amine, alcohol, phenol and thiol nucleophiles were selected. In addition, our prior work¹³ provided a basis for this analysis and the results are shown in Table 2.

Table 2.

Reactions of 3 and 4 with various nucleophiles^a

Entry	Substrate	Nucleophile	Conditions	Yieldb
1	3		DME, 4 molar equiv morpholine, rt, 2 h	5: 87%
2	3		DME, 4 molar equiv Et2NH, rt, 19 h	6: 69%
3	3		DME, 4 molar equiv 1-methylpiperazine, 55 °C, 3.5 h	7: 82%
4	3		DME, 4 molar equiv S-(−)-α-methylbenzylamine, rt, 20.5 h	8: 92%
5	3	Methanol	CH3OH solvent, 2 molar equiv Cs2CO3, 55 °C, 2 h	9: 95%
6	3	Ethanol	CH3CH2OH solvent, 2 molar equiv Cs2CO3, 55 °C, 2.5 h	10: 71%
7	3	Allyl alcohol	CH2=CHCH2OH solvent, 2 molar equiv Cs2CO3, rt, 20 h	11: 88%
8	3		DME, 2 molar equiv benzyl mercaptan, 2 molar equiv Cs2CO3, rt, 24 h	12: 89%
9	4		1-pot reaction: CH3CN, 2 molar equiv BOP, 2 molar equiv DBU, 4 molar equiv morpholine, rt, 3 h	13: 91% (94%)d
10	4		DME, 4 molar equiv Me2NH,c rt, 22 h	14: 83% (86%)d
11	4		DME, 4 molar equiv benzyl amine, rt, 4 h	15: 81% (80%)d
12	4	CH3OH	CH3OH solvent, 2 molar equiv Cs2CO3, rt, 4 h	16: 79% (80%)d
13	4	Allyl alcohol	CH2=CHCH2OH solvent, 2 molar equiv Cs2CO3, rt, 7 h	17: 82% (71%)d
14	4		DME, 2 molar equiv phenol, 2 molar equiv, Cs2CO3, 60 °C, 2.5 h	18: 89% (86%)d
15	4		DME, 2 molar equiv 4-chlorophenol, 2 molar equiv, Cs2CO3, Cs2CO3 60 °C, 2.5 h	19: 88% (90%)d

^aReaction times have not been optimized for each entry.

^bYields of isolated and purified products.

^cA 40 wt% solution of Me₂NH in water was used.

^dYields in parentheses are those obtained from 3′,5′-di-O-(tert-butyldimethylsilyl)-O⁶-[(2,4,6-trimethylphenyl)sulfonyl]-2′-deoxyguanosine (reported in reference ²⁴).

Our first examples on the S_NAr displacement involved reactions of the ribose derivative 3 and secondary amines (morpholine, diethylamine and 1-methylpiperazine) in 1,2-dimethoxyethane (DME) as solvent, without additional base. These reactions were complete at room temperature or at 55 °C, with good to high product yields (Table 2, entries 1–3). Reacton with S-(−)-α-methylbenzyl amine also proceeded smoothly (entry 4), indicating that steric bulk proximal to the nitrogen atom does not interfere with the reaction. Displacements with alcohols (CH₃OH, CH₃CH₂OH and CH₂=CHCH₂OH) were conducted using the alcohol as solvent with Cs₂CO₃ as base, and each proceeded smoothly (entries 5–7). Reaction with benzyl mercaptan was conducted in DME with Cs₂CO₃ as base (entry 8). These results clearly demonstrated the utility of the O⁶-(benzotriazol-1-yl)guanosine derivative 3 for the synthesis of C-6 modified 2-amino purine nucleosides.

With the disilyl 2′-deoxyguanosine 2, we initially explored the possibility of a 2-step, 1-pot transformation where the O⁶-(benzotriazol-1-yl) derivative was generated in situ. Thus, 2 was exposed to BOP and DBU in CH₃CN. After disapparance of 2 (1 h), 4 molar equiv of morpholine was added. This reaction was complete within an additional 2 h and yielded the 6-morpholinyl product 13 in 91% yield. This is consistent with the reported 1-pot reaction of inosine with BOP and i-Pr₂NEt, followed by displacement with morpholine.¹⁵ Similarly, we attempted a 2-step, 1-pot synthesis of the methyl ether 16 under identical conditions. However, this reaction only showed a trace of product after 52 h at room temperature. Thus it appears that the 1-pot protocol is currently only feasible with amines, although at this time we have not performed any extensive experiments on the etherification. In this context, reaction of 4 with MeNH₂ was performed using a 40 wt% solution in water (entry 10). The good product recovery indicates that any competing reaction with water was minimal in the presence of the amine.

Reactions with phenols (entries 14 and 15) also proceed smoothly. On the basis of these results, it becomes clear that isolation of the nucleoside O⁶-(benzotriazol-1-yl) derivatives is important for fully exploiting their reactivity with a wide range of nucleophiles. The isolated compounds 3 and 4 possess excellent reactivity towards the range of nucleophiles as exemplified in Table 2 (entries 14 and 15).

We then became interested in evaluating the mechanistic pathway involved in the formation of 3 and 4 for comparison to the reactions of inosine and 2′-deoxyinosine.¹³ For this we utilized ³¹P{¹H} NMR (Figure 1). The resonances for BOP appear at 45.1 ppm and −143.1 ppm (PF₆⁻) in CD₃CN (panel A in Figure 1). Upon addition of 1, no change was observed (panel B). However, two new signals at 35.7 ppm and 26.1 ppm were observed immediately upon addition of DBU (panel C). The resonance at 26.1 ppm corresponding to HMPA continued to grow (pure HMPA in CH₃CN appears at 25.6 ppm) as the one at 35.7 ppm was depleted (panel D), indicating the likely intermediacy of a nucleoside phosphonium salt (I in Scheme 1). Finally, this room temperature reaction appears to be complete at 120 minutes (panel E). Therefore, the plausible mechanism is one which parallels that reported previously for the hypoxanthine nucleosides,¹³ involving formation of a nucleoside phosphonium salt.

Fig. 1.

Monitoring the course of the reaction between 1 and BOP at room temperature using ³¹P{¹H} NMR: (A) BOP in 0.5 mL CD₃CN; (B) after addition of 1 (0.1 M in CD₃CN); (C) immediately after addition of DBU; (D) 60 minutes after addition of DBU; (E) 120 minutes after addition of DBU; (F) pure HMPA in CD₃CN.

Scheme 1.

Plausible reaction pathway and chemical shifts from ³¹P{¹H} NMR

It is also instructive to compare the reactions of 4 with those of 3′,5′-bis-O-(tert-butyldimethylsilyl)-O⁶-[(2,4,6-trimethylphenyl)sulfonyl]-2′-deoxyguanosine (yields shown in parentheses in Table 2).²⁴ In this comparison 4 performs just as well as the nucleoside O6-arylsulfonate, although synthesis and utilization of the O⁶-(benzotriazol-1-yl) guanosine derivatives are generally operationally simpler.

Conclusions

In this paper we have demonstrated that silyl-protected guanosine (1) and 2′-deoxyguanosine (2) can be conveniently converted to the O⁶-(benzotriazol-1-yl) derivatives 3 and 4. Use of DBU as base in CH₃CN as solvent leads to fast conversion in good to high yield. These O⁶-(benzotriazol-1-yl) derivatives are excellent reagents for S_NAr displacement and a variety of C-6 modified 2-amino purine nucleosides can be efficiently synthesized. It appears that amination reactions can be conducted as a 2-step, 1-pot procedure but a similar reaction with an alcohol was not successful. Extensive experimentation has not presently been conducted on a 1-pot synthesis of C-6 ethers via amide group activation by BOP, and work is in progress on this aspect. However, the readily isolated and stable O⁶-(benzotriazol-1-yl) guanosine derivatives undergo efficient conversion to the wide variety of products using relatively simple methods.

Experimental

General experimental considerations

All reactions were carried out in oven-dried glassware. Reactions were monitored on glass-backed TLC plates coated with silica gel (250 μm), containing a fluorescent indicator. Column chromatographic purifications were performed on 200–300 mesh silica gel. CH₃CN and i-Pr₂NEt were distilled over CaH₂, CH₂Cl₂ was distilled over CaCl₂, THF was distilled over LiAlH₄ and then over Na. Commercially available anhydrous DMSO and DME were used without further purification. All other reagents were obtained from commercial sources and used without further purification. ¹H NMR spectra were recorded at 500 MHz and are referenced to residual protonated solvent. Chemical shifts are reported in parts per million (δ) and coupling constants (J) are in hertz. ¹³C NMR spectra were obtained at 125 MHz and are referenced to the solvent resonance. The imidazolyl proton in the nucleoside is labeled H–8 and the sugar protons are numbered 1′-5′ beginning at the anomeric carbon atom and proceeding to the primary carbinol carbon atom.

General procedure for the synthesis of O⁶-(benzotriazol-1-yl)-2′,3′,5′-tri-O-(tert-butyldimethylsilyl)guanosine (3) and O⁶-(benzotriazol-1-yl)-3′,5′-di-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine (4)

In a clean, dry round-bottomed flask equipped with a stirring bar were placed the silylated nucleoside (1 or 2), BOP (2.0 molar equiv), and DBU (2.0 molar equiv) in dry CH₃CN (8.5 mL/mmol). The reaction mixture was flushed with nitrogen gas, stoppered and stirred at room temperature for 1 to 2 h. The reaction progress was monitored by TLC. Upon completion, the mixture was diluted with EtOAc and washed with deionized water containing a small amount of NaCl. The aqueous layer was back extracted (2x) with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered and evaporated to leave a yellowish oil that was dried under oil pump vacuum. Chromatographic purification then provided products 3 and 4.

O⁶-(Benzotriazol-1-yl)-2′,3′,5′-tri-O-(tert-butyldimethylsilyl)guanosine (3)

Chromatography on a silica gel column using 20% EtOAc in hexanes gave a white solid (65% yield). R_f (silica/20% EtOAc in hexanes) = 0.46; δ_H (500 MHz; CDCl₃) 8.23 (1H, s, H–8), 8.11 (1H, d, J 8.3, Ar–H), 7.54-7.48 (2H, m, Ar–H), 7.44 (1H, dt, J 1.5, 8.3, Ar–H), 5.95 (1H, d, J 4.4, H–1′), 4.75 (2H, s, NH₂), 4.47 (1H, t, J 4.2, H–2′), 4.30 (t, 1H, J 4.2, H–3′), 4.12 (1H, q, J 3.9, H–4′), 4.00 (1H, dd, J 3.4, 11.3, H–5′), 3.80 (1H, dd, J 3.0, 11.3, H–5′), 0.97, 0.93 and 0.84 (27H, 3s, tert-Bu), 0.16, 0.15, 0.11, 0.09, 0.00 and −0.11 (18H, 6s, SiCH₃);δ_C (125 MHz; CDCl₃) 159.6, 159.0, 156.2, 143.7, 140.2, 129.1, 128.7, 124.9, 120.6, 113.3, 109.2, 87.8, 85.9, 77.1, 72.3, 62.9, 26.4, 26.0, 25.9, 18.8, 18.3, 18.2, −4.1, −4.6, −4.4, −4.8, −5.1; HRMS (ESI) calcd for C₃₄H₅₉N₈O₅Si₃ [M + H]⁺: 743.3911, found: 743.3898.

O⁶-(Benzotriazol-1-yl)-3′,5′-di-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine (4)²⁰

Chromatography on a silica gel column using 30% EtOAc in hexanes gave a white solid (85% yield). R_f (silica, 30% EtOAc in hexanes) = 0.24; δ_H (500 MHz; CDCl₃): 8.12 (1H, s, H–8), 8.11 (1H, d overlapping with the singlet at 8.12, J 7.3, Ar–H), 7.54-7.48 (2H, m, Ar–H), 7.44 (1H, t, J 6.8, Ar–H), 6.35 (1H, t, J 6.6, H–1′), 4.76 (2H, s, NH₂), 4.60 (1H, m, H–3′), 4.00 (1H, app q, J 3.4, H–4′), 3.84 (1H, dd, J 3.9, 11.2, H–5′), 3.77 (1H, dd, J 2.9, 11.2, H–5′), 2.57 (1H, app quint, J 6.5, H–2′), 2.40 (1H, ddd, J 3.4, 6.3, 13.7, H–2′), 0.924 and 0.916 (18H, 2s, tert-Bu), 0.108, 0.101, 0.098 (12H, 3s, SiCH₃); δ_C (125 MHz; CDCl₃) 159.7, 158.8, 156.0, 143.6, 140.4, 129.1, 128.8, 124.9, 120.6, 113.7, 109.1, 88.1, 84.3, 72.0, 63.0, 41.4, 26.2, 26.0, 18.7, 18.3, −4.4, −4.5, −5.1, −5.2.

General procedure for the reaction of 3 with amines

In a clean, dry reaction vial equipped with a stirring bar were placed 3 (50.0 mg, 67.3 μmol), amine (4.0 molar equiv) and anhydrous DME (0.5 mL). The reaction mixture was flushed with nitrogen gas, sealed with a Teflon-lined cap and stirred either at room temperature or at 55 °C for 2–20.5 h. Progress of the reaction was monitored by TLC. Upon completion, the mixture was diluted with EtOAc and washed with deionized water containing a small amount of NaCl. The aqueous layer was back extracted (2x) with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered and evaporated. Purification on a silica gel column using 20% EtOAc in hexanes then provided the C-6 amino derivatives 5–8. Additional details and any deviations from this general procedure are noted under the individual compound headings.

2-Amino-9-[2,3,5-tri-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-6-(morpholin-4-yl)purine (5)

Light orange solid, prepared in 87% yield using 3 (70.0 mg, 94.2 μmol) and morpholine (33 μL, 0.38 mmol) in DME (0.7 mL), in a reaction time of 2 h at room temperature. R_f (silica, 5% MeOH in CH₂Cl₂) = 0.46; δ_H (500 MHz; CDCl₃) 7.75 (1H, s, H–8), 5.88 (1H, d, J 5.4, H–1′), 4.61 (1H, t, J 4.7, H–2′), 4.54 (2H, s, NH₂), 4.28 (1H, t, J 3.9, H–3′), 4.21 (4H, br s, morpholinyl–CH₂), 4.08 (1H, app q, J 3.9, H–4′), 3.97 (1H, dd, J 4.4, 11.2, H–5′), 3.80 (4H, t, J 4.9, morpholinyl–CH₂), 3.76 (1H, dd, J 3.0, 11.2, H–5′), 0.935, 0.925 and 0.830 (27H, 3s, tert-Bu), 0.11, 0.106, 0.10, 0.09, −0.03 and −0.13 (18H, 6s, SiCH₃); δ_C (125 MHz; CDCl₃) 159.3, 154.5, 153.3, 135.3, 115.6, 88.0, 85.1, 75.6, 72.2, 67.3, 62.9, 45.7 (br), 26.4, 26.1, 26.0, 18.8, 18.4, 18.2, −4.1, −4.4, −4.5, −4.6, −5.0, −5.1; HRMS (ESI) calcd for C₃₂H₆₃N₆O₅Si₃ [M + H]⁺: 695.4163, found: 695.4173.

9-[2,3,5-Tri-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-N⁶,N⁶-diethyl-2,6-diaminopurine (6)

Yellow solid, prepared in 69% yield using Et₂NH (28 μL, 0.27 mmol) in a reaction time of 19 h at room temperature. R_f (silica, 20% EtOAc in hexanes) = 0.47; δ_H (500 MHz; CDCl₃) 7.68 (1H, s, H–8), 5.85 (1H, d, J 5.4, H–1′), 4.68 (1H, t, J 4.7, H–2′), 4.48 (2H, br s, NH₂), 4.29 (1H, t, J 4.2, H–3′), 4.07 (1H, app q, J 3.9, H–4′), 3.96 (1H, dd, J 4.4, 11.0, H–5′), 3.90 (4H, br, NCH₂), 3.75 (1H, dd, J 3.4, 11.0, H–5′), 1.23 (6H, t, J 7.0, CH₃) 0.93, 0.92 and 0.82 (27H, 3s, tert-Bu), 0.103, 0.097, 0.090, −0.03 and −0.15 (18H, 5s, SiCH₃); δ_C (125 MHz; CDCl₃) 159.4, 154.4, 152.8, 135.2, 115.5, 88.1, 85.0, 75.0, 72.2, 62.9, 42.8 (br), 26.3, 26.1, 26.0, 18.8, 18.4, 18.2, 13.8, −4.1, −4.4, −4.5, −4.8, −5.0, −5.1; HRMS (ESI) calcd for C₃₂H₆₅N₆O₄Si₃ [M + H]⁺: 681.4370, found: 681.4380.

2-Amino-9-[2,3,5-tri-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-6-(4-methylpiperazin-1-yl)purine (7)

White solid, prepared in 82% yield using 1-methylpiperazine (30 μL, 0.27 mmol) in a reaction time of 3.5 h at 55 °C. R_f (silica, 20% EtOAc in hexanes) = 0.05; δ_H (500 MHz; CDCl₃) 7.74 (1H, s, H–8), 5.86 (1H, d, J 5.4, H–1′), 4.61 (1H, t, J 4.7, H–2′), 4.51 (2H, s, NH₂), 4.29 (1H, t, J 4.2, H–3′), 4.24 (4H, br s, piperazinyl–CH₂), 4.08 (1H, app q, J 3.9, H–4′), 3.99 (1H, dd, J 4.4, 11.2, H–5′), 3.78 (1H, dd, J 3.0, 11.2, H–5′), 2.51 (4H, br s, piperazinyl–CH₂), 2.34 (3H, s, CH₃), 0.93, 0.925 and 0.830 (27H, 3s, tert-Bu), 0.103, 0.096, 0.087, −0.03 and −0.13 (18H, 5s, SiCH₃); δ_C (125 MHz; CDCl₃) 159.3, 154.5, 153.3, 135.2, 115.7, 88.1, 85.1, 75.4, 72.2, 62.9, 55.4, 46.5, 45.0 (br), 26.4, 26.1, 26.0, 18.8, 18.4, 18.2, −4.1, −4.4, −4.5, −4.6, −5.0, −5.1; HRMS (ESI) calcd for C₃₃H₆₆N₇O₄Si₃ [M + H]⁺: 708.4479, found: 708.4486.

9-[2,3,5-tri-O-(tert-butyldimethylsilyl)-β-D-ribofuranosyl]-N⁶-[(1S)-phenylethyl]-2,6-diaminopurine (8)

Light yellow solid, prepared in 92% yield using (S)-(−)-α-methylbenzylamine (35 μL, 0.27 mmol) in a reaction time of 20.5 h at room temperature. R_f (silica, 20% EtOAc in hexanes) = 0.13; δ_H (500 MHz; CDCl₃) 7.85 (1H, s, H–8), 7.42 (2H, d, J 7.3, Ar–H), 7.31 (2H, t, J 7.3, Ar–H), 7.22 (1H, t, J 7.3, Ar–H), 6.53 (1H, br s, NH), 5.84 (1H, d, J 4.4, H–1′), 5.50 (1H, br s, NCH), 4.71 (2H, br s, NH₂), 4.53 (1H, t, J 4.4, H–2′), 4.28 (1H, t, J 3.9, H–3′), 4.07 (1H, app q, J 3.9, H–4′), 3.99 (1H, dd, J 4.4, 11.5, H–5′), 3.78 (1H, dd, J 3.0, 11.5, H–5′), 1.60 (3H, d, J 6.8, CH₃), 0.94, 0.92 and 0.83 (27H, 3s, tert-Bu), 0.119, 0.114, 0.090, 0.00 and −0.11 (18H, 5s, SiCH₃); proton assignments were made by ¹H-¹H COSY of this compound at 60 °C, where the NH sharpened to a br d (5.97 ppm, J 7.8), the NCH became a sharper br m (5.56 ppm) and the CH₃ d sharpened (1.61 ppm, J 7.3); δ_C (125 MHz; CDCl₃) 160.0, 154.5,144.5, 136.4, 128.7, 127.2, 126.5, 115.0, 88.4, 84.9, 75.7, 71.8, 62.6, 26.4, 26.1, 26.0, 22.9, 18.8, 18.4, 18.2, −4.1, −4.4, −4.5, −4.6, −5.1; HRMS (ESI) calcd for C₃₆H₆₅N₆O₄Si₃ [M + H]⁺: 729.4370, found: 729.4374.

General procedure for the reaction of 3 with alcohols

In a clean, dry reaction vial equipped with a stirring bar were placed 3 and Cs₂CO₃ (2.0 molar equiv) in the appropriate alcohol as solvent. The reaction mixture was flushed with nitrogen gas, sealed with a Teflon-lined cap and stirred at 55 °C for 2–20 h. Progress of the reaction was monitored by TLC. Upon completion, the mixture was diluted with EtOAc and washed with deionized water containing a small amount of NaCl. The aqueous layer was back extracted (2x) with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered and evaporated. Purification on a silica gel column using 20% EtOAc in hexanes then provided the guanosine O6-alkyl ethers 9–11. Additional details are listed under the specific compound headings.

2′,3′,5′-Tri-O-(tert-butyldimethylsilyl)-O⁶-methylguanosine (9)

Light orange solid, prepared in 95% yield using 3 (45.3 mg, 61.0 μmol) and Cs₂CO₃ (42.9 mg, 0.131 mmol) in MeOH (0.45 mL), in a reaction time of 2 h at 55 °C. R_f (silica, 20% EtOAc in hexanes) = 0.11; δ_H (500 MHz; CDCl₃) 7.98 (1H, s, H–8), 5.90 (1H, d, J 4.9, H–1′), 4.79 (2H, s, NH₂), 4.50 (1H, t, J 4.4, H–2′), 4.28 (1H, t, J 4.2, H–3′), 4.08 (1H, app q, J 3.0, H–4′), 4.05 (3H, s, OCH₃), 3.97 (1H, dd, J 3.9, 11.2, H–5′), 3.77 (1H, dd, J 2.4, 11.2, H–5′), 0.94, 0.92 and 0.82 (27H, 3s, tert-Bu), 0.12, 0.11, 0.09, 0.08, −0.04 and −0.16 (18H, 6s, SiCH₃); δ_C (125 MHz; CDCl₃) 161.7, 159.5, 153.8, 138.2, 116.2, 88.0, 85.4, 76.4, 72.1, 62.8, 54.0, 26.4, 26.1, 26.0, 18.8, 18.4, 18.2, 14.8, −4.1, −4.4, −4.5, −4.7, −5.1; HRMS (ESI) calcd for C₂₉H₅₈N₅O₅Si₃ [M + H]⁺: 640.3741, found: 640.3745.

2′,3′,5′-Tri-O-(tert-butyldimethylsilyl)-O⁶-ethylguanosine (10)

White solid, prepared in 71% yield using 3 (50.0 mg, 67.3 μmol) and Cs₂CO₃ (43.8 mg, 0.134 mmol) in EtOH (0.45 mL), in a reaction time of 2.5 h at 55 °C. R_f (silica, 20% EtOAc in hexanes) = 0.20; δ_H (500 MHz; CDCl₃) 7.98 (1H, s, H–8), 5.91 (1H, d, J 4.9, H–1′), 4.76 (2H, br s, NH₂), 4.55 (2H, q, J 7.0, OCH₂), 4.52 (1H, t, J 4.4, H–2′) 4.29 (1H, t, J 4.3, H–3′), 4.09 (1H, app q, J 2.9, H–4′), 3.99 (1H, dd, J 3.4, 11.5, H–5′), 3.76 (1H, dd, J 3.0, 11.5, H–5′), 1.45 (3H, t, J 7.0, CH₃), 0.95, 0.93 and 0.83 (27H, 3s, tert-Bu), 0.13, 0.12, 0.095, 0.09, −0.03 and −0.15 (18H, 6s, SiCH₃); δ_C (125 MHz; CDCl₃) 161.5, 159.5, 153.9, 138.0, 116.1, 88.0, 85.4, 76.3, 72.2, 62.8, 26.4, 26.1, 26.0, 18.8, 18.4, 18.2, 14.8, −4.1, −4.4, −4.5, −4.7, −5.1; HRMS (ESI) calcd for C₃₀H₆₀N₅O₅Si₃ [M + H]⁺: 654.3897, found: 654.3890.

O⁶-Allyl-2′,3′,5′-tri-O-(tert-butyldimethylsilyl)guanosine (11)

Orange solid, prepared in 88% yield using 3 (70.0 mg, 94.2 μmol) and Cs₂CO₃ (61.4 mg, 0.188 mmol) in allyl alcohol (0.7 mL), in a reaction time of 20 h at room temperature. R_f (silica, 20% EtOAc in hexanes) = 0.31; δ_H (500 MHz; CDCl₃) 8.00 (1H, s, H–8), 6.15 (1H, m, =CH), 5.93 (1H, d, J 4.6, H–1′), 5.45 (1H, dd, J 1.5, 18.6, =CH_trans), 5.29 (1H, dd, J 1.5, 11.7, =CH_cis), 5.02 (2H, d, J 5.9, OCH₂), 4.79 (2H, s, NH₂), 4.54 (1H, t, J 4.7, H–2′), 4.31 (1H, t, J 3.4, H–3′), 4.11 (1H, app q, J 3.9, H–4′), 4.01 (1H, dd, J 3.9, 11.2, H–5′), 3.80 (1H, dd, J 2.4, 11.2, H–5′), 0.97, 0.95 and 0.85 (27H, 3s, tert-Bu), 0.15, 0.14, 0.12, 0.11, 0.00 and −0.13 (18H, 6s, SiCH₃); δ_C (125 MHz; CDCl₃) 161.0, 159.4, 154.1, 138.1, 133.1, 118.4, 116.0, 87.9, 85.4, 76.4, 72.2, 67.4, 62.8, 26.4, 26.1, 26.0, 18.8, 18.4, 18.2, −4.1, −4.4, −4.5, −4.7, −5.11, −5.14; HRMS (ESI) calcd for C₃₁H₆₀N₅O₅Si₃ [M + H]⁺: 666.3897, found: 666.3912.

S⁶-Benzyl-2′,3′,5′-tri-O-(tert-butyldimethylsilyl)thioguanosine (12)

In a clean, dry reaction vial equipped with a stirring bar were placed 3 (50.0 mg, 67.0 μmol), Cs₂CO₃ (43.8 mg, 0.134 mmol) and dry DME (0.5 mL). Benzyl mercaptan (16.0 μL, 0.134 mmol) was added, the reaction mixture was flushed with nitrogen gas, sealed with a Teflon-lined cap and allowed to stir at room temperature for 24 hrs. The mixture was diluted with EtOAc and washed with deionized water containing a small amount of NaCl. The aqueous layer was back extracted (2x) with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered and evaporated. Purification on a silica gel column using 20% EtOAc in hexanes then provided 12 as a light yellow solid (89% yield). R_f (silica, 10% EtOAc in hexanes) = 0.47; δ_H (500 MHz; CDCl₃) 7.98 (1H, s, H–8), 7.43 (2H, d, J 7.3, Ar–H), 7.31-7.21 (3H, m, Ar–H), 5.90 (1H d, J 4.9, H–1′), 4.81 (s, 2H, NH₂), 4.57 (2H, AB_quart, J 13.7, SCH₂), 4.54 (1H, t, J 4.4, H–2′), 4.29 (t, 1H, J 4.2, H–3′), 4.10 (1H, app q, J 2.9, H–4′), 3.97 (1H, dd, J 3.9, 11.2, H–5′), 3.77 (1H, dd, J 2.9, 11.2, H–5′), 0.93, 0.927 and 0.83 (27H, 3s, tert-Bu), 0.12, 0.11, 0.010, 0.09, −0.02, −0.16 (18H, 5s, SiCH₃); δ_C (125 MHz; CDCl₃) 161.1, 159.0, 150.8, 139.0, 138.1, 129.3, 128.7, 127.4, 126.2, 88.1, 85.3, 76.1, 72.1, 62.7, 32.9, 26.4, 26.1, 26.0, 18.8, 18.4, 18.2, −4.1, −4.4, −4.5, −4.7, −5.0, −5.1; HRMS (ESI) calcd for C₃₅H₆₂N₅O₄SSi₃ [M + H]⁺: 732.3825, found: 732.3831.

9-[3,5-Di-O-(tert-butyldimethylsilyl)-2-deoxy-β-D-ribofuranosyl]-6-(morpholin-4-yl)purine (13)²⁴

One-pot procedure

In a clean, dry, round-bottomed flask equipped with a stirring bar were placed 2 (70.0 mg, 0.141 mmol) and BOP (0.125 g, 0.283 mmol) in anhydrous CH₃CN (1.20 mL). DBU (47.3 μL, 0.316 mmol) was added to the stirring mixture, the flask was flushed with nitrogen gas and stoppered. After being stirred at room temperature for 1 h TLC showed consumption of 2. At this time morpholine (49.7μl, 0.568 mmol) was added. The flask was again flushed with nitrogen gas, stoppered and the mixture was allowed to stir at room temperature for an additional 2 hours at which time the reaction was complete as assessed by TLC. The mixture was diluted with EtOAc and washed with deionized water containing a small amount of NaCl. The aqueous layer was back extracted (2x) with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered and evaporated to leave a yellowish oil. Purification on a silica gel column using 30% EtOAc in hexanes then provided 13 as a yellow solid (91% yield). R_f (silica, 30% EtOAc in hexanes) = 0.11; δ_H (500 MHz; CDCl₃) 7.72 (1H, s, H–8), 6.31 (1H, t, J 6.8, H–1′), 4.60 (2H, s, NH₂), 4.57 (1H, m, H–3′), 4.21 (4H, br s, morpholinyl CH₂), 3.96 (1H, app q, J 4.1, H–4′), 3.79 (4H, t, J 4.4, morpholinyl–CH₂), 3.76 (1H, dd, J 4.6, 11.1, H–5′), 3.75 (1H, dd, J 3.6, 11.1, H–5′), 2.55 (1H, app quint, J 6.8, H–2′), 2.33 (1H, ddd, J 3.4, 6.0, 13.0, H–2′), 0.91 and 0.90 (18H, 2s, tert-Bu), 0.10, 0.07 and 0.06 (12H, 3s, SiCH₃); δ_C (125 MHz; CDCl₃) 159.4, 154.4, 153.1, 134.6, 115.4, 87.8, 83.6, 72.4, 67.3, 63.2, 45.7 (br), 40.9, 26.2, 26.0, 18.7, 18.3, −4.4, −4.5, −5.1, −5.2.

General procedure for the reaction of 4 with amines

In a clean, dry reaction vial equipped with a stirring bar were placed 4 (50.0 mg, 81.6 μmol), amine (4.0 molar equiv) and anhydrous DME (0.5 mL). The reaction mixture was flushed with nitrogen gas, sealed with a Teflon-lined cap and stirred at room temperature for 4–22 h (see details under specific compound headings). Progress of the reaction was monitored by TLC. Upon completion, the mixture was diluted with EtOAc and washed with deionized water containing a small amount of NaCl. The aqueous layer was back extracted (2x) with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered and evaporated. Purification on a silica gel column using 30% EtOAc in hexanes then provided the C-6 amino derivatives 14 and 15. Additional details and any deviations from this general procedure are noted under the individual compound headings.

9-[3,5-Di-O-(tert-butyldimethylsilyl)-2-deoxy-β-D-ribofuranosyl]-N⁶,N⁶-dimethyl-2,6-diaminopurine (14)²⁴

Light yellow solid, prepared in 83% yield using a 40 wt% solution of Me₂NH in water (37.3 μL, 0.331 mmol) in a reaction time of 2 h. R_f (silica, 5% MeOH in CH₂Cl₂) = 0.43;δ_H (500 MHz; CDCl₃) 7.69 (1H, s, H–8), 6.30 (1H, t, J 6.1, H–1′), 4.67 (2H, s, NH₂), 4.56 (1H, m, H–3′), 3.95 (1H, app q, J 4.0, H–4′), 3.78-3.71 (2H, m, H–5′), 3.43 (6H, br s, NCH₃), 2.54 (1H, app quint, J 6.6, H–2′), 2.32 (1H, ddd, J 3.4, 6.0, 13.1, H–2′), 0.90 and 0.896 (18H, 2s, tert-Bu), 0.09, 0.06 and 0.05 (12H, 3s, SiCH₃); δ_C (125 MHz; CDCl₃) 159.4, 155.5, 152.7, 134.2, 115.7, 87.8, 83.5, 72.4, 63.2, 40.8, 38.5 (br), 26.2, 26.0, 18.7, 18.3, −4.4, −4.5, −5.1, −5.2.

N⁶-Benzyl-9-[3,5-di-O-(tert-butyldimethylsilyl)-2-deoxy-β-D-ribofuranosyl]purine (15)²⁴

Light yellow solid, prepared in 81% yield using benzylamine (35.6 μL, 0.33 mmol) in a reaction time of 4 h. R_f (silica, 30% EtOAc in hexanes) = 0.08; δ_H (500 MHz; CDCl₃) 7.76 (1H, s, H–8), 7.37-7.27 (5H, m, Ar–H), 6.30 (1H, t, J 6.8, H–1′), 5.89 (1H, br s, NH), 4.79 (2H, s, NH₂), 4.71 (2H, br s, NCH₂), 4.58 (1H, m, H–3′), 3.97 (1H, app q, J 3.4, H–4′), 3.81 (1H, dd, J 4.4, 11.0, H–5′), 3.75 (1H, dd, J 2.9, 11.0, H–5′), 2.59 (1H, app quint, J 6.8, H–2′), 2.34 (1H, ddd, J 3.7, 6.0, 13.0, H–2′), 0.90 and 0.896 (18H, 2s, tert-Bu), 0.10, 0.08 and 0.07 (12H, 3s, SiCH₃); δ_C (125 MHz; CDCl₃) 160.0, 155.18, 139.0, 136.0, 128.8, 128.0, 127.6, 115.1, 87.9, 83.8, 72.3, 63.1, 44.7 (br), 41.0, 26.2, 26.0, 18.7, 18.3, −4.4, −4.5, −5.10, −5.2.

General procedure for the reaction of 4 with alcohols

In a clean, dry reaction vial equipped with a stirring bar were placed 4 (50.0 mg, 81.6 μmol) and Cs₂CO₃ (53.1 mg, 0.163 mmol) in the appropriate alcohol as solvent (0.5 mL). The reaction mixture was flushed with nitrogen gas, sealed with a Teflon-lined cap and stirred for 4–7 hours at room temperature. Progress of the reaction was monitored by TLC. Upon completion, the mixture was diluted with EtOAc and washed with deionized water containing a small amount of NaCl. The aqueous layer was back extracted (2x) with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered and evaporated. Purification on a silica gel column using 30% EtOAc in hexanes then provided the 2′-deoxyguanosine O6-alkyl ethers 16 and 17. Additional details are listed under the specific compound headings.

3′,5′-Di-O-(tert-butyldimethylsilyl)-O⁶-methyl-2′-deoxyguanosine (16)²⁴

Light yellow solid, prepared in 79% yield using MeOH, in a reaction time of 4 h. R_f (silica, 5% MeOH in CH₂Cl₂) = 0.41; δ_H (500 MHz; CDCl₃) 7.91 (1H, s, H–8), 6.32 (1H, t, J 6.4, H–1′), 4.83 (2H, s, NH₂), 4.59 (1H, m, H–3′), 4.08 (3H, s, OCH₃), 3.97 (1H, app q, J 3.9, H–4′), 3.81 (1H, dd, J 4.4, 11.2, H–5′), 3.75 (1H, dd, J 3.4, 11.2, H–5′), 2.57 (1H, app quint, J 6.3, H–2′), 2.34 (1H, ddd, J 3.8, 6.1, 13.0, H–2′), 0.912 and 0.910 (18H, 2s, tert-Bu), 0.10, 0.08 and 0.07 (12H, 3s, SiCH₃); δ_C (125 MHz; CDCl₃) 161.8, 159.5, 153.7, 137.8, 116.2, 87.9, 83.9, 72.1, 63.1, 54.1, 41.2, 26.2, 26.0, 18.7, 18.3, −4.4, −4.5, −5.1, −5.2.

O⁶-Allyl-3′,5′-di-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine (17)²⁴

Light yellow solid, prepared in 82% yield using allyl alcohol, in a reaction time of 7 h. R_f (silica, 5% MeOH in CH₂Cl₂) = 0.59; δ_H (500 MHz; CDCl₃) 7.90 (1H, s, H–8), 6.32 (1H, t, J 6.8, H–1′), 6.12 (1H, m, =CH), 5.42 (1H, dd, J 1.5, 18.5, =CH_trans), 5.26 (1H, dd, J 1.5, 11.7, =CH_cis), 5.01 (2H, d, J 5.4, OCH₂), 4.81 (2H, s, NH₂), 4.59 (1H, m, H–3′), 3.97 (1H, app q, J 3.3, H–4′), 3.81 (1H, dd, J 4.4, 11.1, H–5′), 3.75 (1H, dd, J 2.9, 11.1, H–5′), 2.57 (1H, app quint, J 6.0, H–2′), 2.35 (1H, ddd, J 3.7, 6.1, 13.1, H–2′), 0.913 and 0.910 (18H, 2s, tert-Bu), 0.10, 0.08 and 0.07 (12H, 3s, SiCH₃); δ_C (125 MHz; CDCl₃) 161.1, 159.4, 154.0, 137.8, 133.0, 118.4, 116.2, 87.9, 83.9, 72.2, 67.5, 63.1, 41.2, 26.2, 26.0, 18.7, 18.3, −4.4, −4.5, −5.1, −5.2.

General procedure for the reaction of 4 with phenols

In a clean, dry reaction vial equipped with a stirring bar were placed 4 (50.0 mg, 81.6 μmol), the appropriate phenol (2 molar equiv) and Cs₂CO₃ (53.1 mg, 0.163 mmol) in anhydrous DME (0.5 mL). The reaction mixture was flushed with nitrogen gas, sealed with a Teflon-lined cap and stirred at 60 °C for 2–2.5 hours. Progress of the reaction was monitored by TLC. Upon completion, the mixture was diluted with EtOAc and washed with deionized water containing a small amount of NaCl. The aqueous layer was back extracted (2x) with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered and evaporated. Purification on a silica gel column using 30% EtOAc in hexanes then provided the 2′-deoxyguanosine O6-aryl ethers 18 and 19.

3′,5′-Di-O-(tert-butyldimethylsilyl)-O⁶-phenyl-2′-deoxyguanosine (18)²⁴

Yellowish solid, prepared in 89% yield using phenol (15.3 mg, 0.163 mmol), in a reaction time of 2.5 h. R_f (silica, 5% MeOH in CH₂Cl₂) = 0.26; δ_H (500 MHz; CDCl₃) 8.01 (1H, s, H–8), 7.40 (2H, t, J 7.8, Ar–H), 7.25-7.23 (3H, m, Ar–H), 6.34 (1H, t, J 6.4, H–1′), 4.82 (2H, s, NH₂), 4.60 (1H, m, H–3′), 3.99 (1H, app q, J 3.4, H–4′), 3.83 (1H, dd, J 3.9, 11.2, H–5′), 3.77 (1H, dd, J 2.9, 11.2, H–5′), 2.58 (1H, app quint, J 6.8, H–2′), 2.37 (1H, ddd, J 3.9, 6.4, 13.1, H–2′), 0.92 and 0.91 (18H, 2s, tert-Bu), 0.10 and 0.09 (12H, 2s, SiCH₃); δ_C (125 MHz; CDCl₃) 160.7, 159.4, 154.7, 152.8, 138.7, 129.5, 125.5, 122.2, 116.2, 88.0, 84.0, 72.1, 63.1, 41.3, 26.3, 26.0, 18.7, 18.3, −4.4, −4.5, −5.1, −5.2.

3′,5′-Di-O-(tert-butyldimethylsilyl)-O⁶-(4-chlorophenyl)-2′-deoxyguanosine (19)²⁴

Yellow solid, prepared in 88% yield using 4-chlorophenol (21.0 mg, 0.163 mmol), in a reaction time of 2 h. R_f (silica, 5% MeOH in CH₂Cl₂) = 0.28; δ_H (500 MHz; CDCl₃) 8.02 (1H, s, H–8), 7.36 (2H, J 8.8, Ar–H), 7.19 (2H, d, J = 8.8, Ar H), 6.34 (1H, t, J 6.4, H–1′), 4.79 (2H, s, NH₂), 4.61 (1H, m, H–3′), 3.99 (1H, app q, J 3.9, H–4′), 3.84 (1H, dd, J 4.4, 11.2, H–5′), 3.77 (1H, dd, J 2.9, 11.2, H–5′), 2.58 (1H, app quint, J 6.8, H–2′), 2.38 (1H, ddd, J 3.9, 5.9, 13.2, H–2′), 0.923 and 0.920 (18H, 2s, tert-Bu), 0.11 and 0.10 (12H, 2s, SiCH₃); δ_C (125 MHz; CDCl₃) 160.3, 159.3, 154.9, 151.3, 138.9, 130.8, 129.6, 123.6, 116.1, 88.0, 84.0, 72.1, 63.0, 41.3, 26.2, 26.0, 18.7, 18.3, −4.4, −4.5, −5.1, −5.2.