Straightforward Synthesis of the Poly(ADP-ribose) Branched Core Structure.

Poly(ADP-ribosyl)ation is a post-translational modification that produces poly(ADP-ribose) with a branched structure every 20-50 units; such branching structure has been previously suggested to be involved in regulating chromatin remodeling. To elucidate its detailed functions, we developed a straightforward method for the synthesis of the poly(ADP-ribose) branched core structure, α-d-ribofuranosyl-(1‴ → 2″)-α-d-ribofuranosyl-(1″ → 2')-adenosine 5',5'',5‴-trisphosphate 1, from 6-chloropurine ribofuranoside 4 in 10 steps and 6.1% overall yield. The structure poses synthetic challenges for constructing iterative α-1,2-cis-glycosidic bonds in the presence of a purine base and the installation of three phosphate groups at primary hydroxyl groups. Iterative glycosidic bonds were formed by α-1,2-cis-selective ribofuranosylation using 2-O-(2-naphthylmethyl)-protected thioglycoside donor 6 and a thiophilic bismuth promoter. After the construction of diribofuranosyl adenosine 5 had been constructed, it was chemo- and regioselectively phosphorylated at a later stage. Subsequent deprotection provided the synthetic target 1.

Introduction

ADP-ribosylation is a post-translational modification (PTM) in which the ADP-ribose moiety is transferred from nicotinamide adenine dinucleotide (NAD+) to a specific amino acid residue, such as arginine, asparagine, aspartate, cysteine, glutamate, lysine, and serine.[1] This PTM plays a pivotal role in a variety of biological processes, including transcriptional regulation,[2] DNA repair,[3] cell differentiation,[4] stress response,[5] apoptosis,[6] proliferation of cancer cells,[7] and viral and bacterial infections[8] (including COVID-19[8c]). Poly(ADP-ribose)polymerase 1 (PARP1), one of the corresponding biosynthetic enzymes, catalyze elongation of the ADP-ribose chain at the 2′ position of the adenosine moiety in a terminal ADP-ribose residue through a 1,2-cis(α)-glycosidic linkage to provide a long-chain poly(ADP-ribose) (PAR) with up to approximately 200 units.[9] The PAR possesses a branched structure every 20–50 units that is linked to the 2″ position of the ribose moiety of an inner ADP-ribose through an α-glycosidic bond (Figure ).[10]

Figure 1

Structure of naturally occurring poly(ADP-ribose).

Chen et al. showed that PARP2, the closet paralogue of PARP1, accelerates the branching formation and suggested that the PAR branching structure is involved in the regulation of chromatin remodeling by the recognition of DNA damage-specific histone chaperone APLF (aprataxin and PNKP like factor).[11] While more than 30 years has elapsed since the discover of the branched structure by Miwa and coworkers,[10a] its detailed functions at the molecular level have not been intensively investigated compared to those of the PAR liner structure. One major obstacle is the availability of homogeneous ADP-ribose molecules with branched structures, which are limited by the heterogeneity of naturally occurring PAR. Therefore, the development of chemical synthetic methods that facilitate the production of highly pure structurally well-defined molecules is a very important objective. Liu et al. reported the chemical synthesis of the PAR branched core structure, α-d-ribofuranosyl-(1‴ → 2″)-α-d-ribofuranosyl-(1″ → 2′)-adenosine 5′,5′′,5‴-trisphosphate 1 (Scheme A).[12] While their synthetic route represents a pioneering chemical study of PAR, the long multistep synthesis and synthetic efficiency remain to be issues that need to be addressed. The longest linear sequence (LLS) in their route to 1 from the commercially available 1,3,5-tri-O-benzoyl-α-d-ribofuranose 2 involved 18 steps, with an overall yield of 4.1% and major drawbacks including several protecting group manipulations for O-glycosylation and phosphorylation, and the late-stage installation of the N-benzoyl-protected adenine base 3. Therefore, we envisaged a straightforward route to shorten the synthesis of 1 and improve the overall yield. Herein, we report the straightforward synthesis of 1 from 6-chloropurine ribofuranoside 4 in 10 steps and 6.1% overall yield (Scheme B). Our synthetic route features α-1,2-cis-selective O-glycosidic bond formation and late-stage chemo- and regioselective phosphorylation of α-d-ribofuranosyl-(1‴ → 2″)-α-d-ribofuranosyl-(1″ → 2′)-adenosine 5.

Scheme 1

Synthetic Outline of Previous Work (A) and This Work (B) in the Synthesis of Diribofuranosyl Adenosine Trisphosphate 1

Results and Discussion

For the improvement of the synthetic efficiency and overall yield of 1, we highlight the following three key points: (1) the use of 6-chloropurine ribofuranoside 4 as the starting material for the synthesis of diribofuranosyl adenosine 5, which avoids the challenging late-stage N-glycosylation of a purine base with trisaccharide building block; (2) the use of a 2-naphthylmethyl (NAP)-protecting group at the 2-position of the ribofuranosyl thioglycoside donor, as Ito and Ishiwata have widely reported that 1,2-cis-selective O-glycosylation can be achieved via intramolecular aglycon delivery (IAD) using 2-O-NAP-protected glycosyl donors;[13] and (3) chemo- and regioselective phosphorylation of fully unprotected substrate 5 to dramatically decrease the number of synthetic steps.

As depicted in Scheme , our efforts commenced with the preparation of the 2-O-NAP-protected ribofuranosyl thioglycoside donor 6. Following the MoO2Cl2-catalyzed thioglycosylation method reported by Chen and coworkers,[14] phenyl β-thioglycoside 8 was obtained from commercially available tetra-acetate 7 in excellent yield. After deacetylation, the resulting triol was subjected to Williamson ether synthesis with NAP bromide and sodium hydride in N,N-dimethylformamide (DMF). The numbers of equivalents of reagents, reaction temperature, and methods to add the NAP bromide were optimized, which enabled regioselective monoalkylation at the 2-position to afford the 2-O-NAP ether 9 in a satisfactory yield of 54%, along with monoalkylated regioisomers and dialkylated compounds. A similar regioselective alkylation strategy has been reported for the secondary 2′-hydroxyl group of ribonucleosides in the presence of a primary 5′-hydroxyl group.[15] Benzylation of the remaining hydroxyl groups provided the desired product 6 in 93% yield.

Scheme 2

Preparation of 2-O-NAP-Protected Ribofuranosyl Thioglycoside 6

We next attempted NAP-mediated IAD to α-1,2-cis-selectively ribofuranosylate 6-chloropurine ribofuranoside acceptor 10 with thioglycoside donor 6 (Scheme ). 2′-Hydroxyl acceptor 10 was readily prepared by reacting commercially available compound 4 with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDSCl2) in the presence of pyridine.[16] The corresponding mixed acetal 11 was formed from 10 and 6 in an acceptable yield of 66% as a single stereoisomer using the previous DDQ procedure.[13] Although the thioglycoside moiety is usually activated with methyl triflate, we used triphenylbismuth ditriflate (Ph3Bi(OTf)2),[17] a thiophilic promoter developed by Pohl’s group, owing to differences in the thioglycoside moiety (methylthio versus phenylthio) and the presence of the purine base. While 11 was observed to be completely converted by TLC, the desired IAD product 12 was not obtained; the reaction provided 10 and a 1,2-O-naphthylidene acetal byproduct derived from the thioglycoside moiety of 11. It is likely that thioglycoside activation using Ph3Bi(OTf)2 and/or the stereochemistry of the mixed acetal 11 is unfavorable for glycosidic bond formation by IAD.

Scheme 3

α-1,2-cis-Selective Ribofuranosylation via NAP-Mediated IAD

Based on these results, we examined the direct ribofuranosylation of 3′,5′-TIPDS-protected acceptor 10 with thioglycoside donor 6 using Ph3Bi(OTf)2 (Table ). The corresponding α-1,2-cis product 13 was successfully obtained in good yield and with high stereoselectivity (63%, α/β = 6.1:1) using 3 equivalents of 6 (entry 1). To determine the effect of the 2-O-NAP-protecting group on the coupling yield and stereoselectivity during ribofuranosylation, 10 was coupled with 3 equivalents of the 2-O-benzylated and allylated thioglycosides S1(18) and S2, respectively. Glycosylation of 10 with S1 afforded the desired product 14 in good yield and stereoselectivity although lower than in entry 1 (61%, α/β = 3:1; entry 2), while the use of S2 facilitated complete α-1,2-cis-selective ribofuranosylation to give 15 but with a dramatically lower coupling yield (36%, α only; entry 3). These results indicated that the presence of the 2-O-electron-rich alkyl substituent in the thioglycoside donor, such as the NAP or benzyl group, stabilizes the oxocarbenium intermediate, which facilitates glycosidic bond formation. However, the α-1,2-cis-directing effect of the 2-O-NAP protecting group remains questionable. One possible explanation for the α-1,2-cis-selectivity observed in each entry involves remote stereocontrol by the C3 benzyloxy group. Larsen et al. experimentally showed that a ribofuranosyl oxocarbenium ion prefers to be in a pseudoaxial E3 conformation stabilized by a C3 alkoxy group, which assists α-1,2-cis-selective ribofuranosylation through nucleophilic attack from the inside face of the envelope intermediate.[19] Notably, the anomeric configurations of ribofuranosides 13–15 were easily determined by vicinal 1H NMR coupling constants between the C1 and C2 protons (3JH1,H2) and the 13C NMR chemical shift of the anomeric carbon.[20] The 3JH1,H2 value of an α-1,2-cis-ribofuranosides (4–5 Hz) is larger than its β-1,2-trans stereoisomer (0–2 Hz). The carbon signal of the α-isomer typically appears between 100 and 105 ppm; in contrast, the corresponding β-isomers is downshifted by approximately 5 ppm.

Table 1

Screening for Direct Ribofuranosylation

entry	donor	R	product	yield (%)	α/β ratio
1	6	NAP	13	63	6.1/1
2	S1	Bn	14	61a	3/1b
3	S2	all	15	36a	α only

Including impurities.

α/β ratio was determined based on1H NMR spectrum.

With the appropriate reaction conditions for α-1,2-cis-selective ribofuranosidic bond formation with 6 in hand, we next focused on the synthesis of diribofuranosyl adenosine 5 (Scheme ). The 2″-O-NAP group was oxidatively removed using DDQ to afford glycosyl acceptor 12 in 74% yield. The same ribofuranosylation conditions as shown in entry 1 of Table produced the desired α-1,2-cis product 16 in high coupling yield and stereoselectivity (82%, α/β = 15.7:1), which is ascribable to the difference in acceptor reactivities. The 2″-hydroxyl group of 12 is expected to be more reactive than the 2′-hydroxyl group of 10 because the reactive site is far from the bulky and electron-withdrawing 6-chloropurine base. The use of the more reactive acceptor 12 predominately provided the kinetic α-product via the pseudoaxial E3 conformation. Exposure to the hydrogen fluoride–pyridine complex deblocked the TIPDS group of 16 to give S3 in 89% yield. The following microwave-assisted transformation of the 6-chloropurine base with ammonia–methanol solution proceeded efficiently to afford the diribofuranosyl adenosine structure 17 in 98% yield, after which 5 was synthesized in 87% yield by the hydrogenolysis of 17 using Pearlman’s catalyst to cleave the remaining benzyl-type protecting groups. Notably, our synthetic approach (seven steps in 19.4% overall yield) is five steps shorter than the first reported synthesis[21] and afforded a more than 10-fold improved overall yield (12 steps in 1.6% overall yield).

Scheme 4

Synthesis of Diribofuranosyl Adenosine 5

To complete the synthesis of 1, we chemo- and regioselectively phosphorylated the fully unprotected substrate 5 at its primary hydroxyl groups (Scheme ). Although several regioselective phosphorylation methods using P(V) reagents[22] have been reported, the phosphoramidite method that uses a P(III) reagent,[23] which is widely used in nucleic acid syntheses, was chosen. We hypothesized that the acid used to activate the phosphoramidite reagent protonates the free amino group, thereby decreasing its reactivity. In addition, the secondary hydroxyl groups adjacent or close to the N- and O-glycosidic bonds are expected to be less reactive. According to Haché et al.,[24] the primary alcohol in a 1,2-diol substrate can be regioselectively phosphorylated by phosphitylation with a di(tert-butyl)phosphoramidite reagent ((tBuO)2PNiPr2) and subsequent oxidation. Accordingly, 5 was first phosphitylated using an excess amount of (tBuO)2PNiPr2; while the reaction proceeded only slightly, the predominant introduction of a single phosphite group was confirmed by mass spectrometry. Therefore, we chose to use the dibenzylphosphoramidite reagent ((BnO)2PNiPr2), which is less sterically hindered and more reactive than (tBuO)2PNiPr2; indeed, 5 was phosphitylated slowly when (BnO)2PNiPr2 was added. The addition of 16.5 equivalents of (BnO)2PNiPr2 mainly resulted in triphosphitylation along with some diphosphitylation. The desired 5′,5″,5‴-phosphorylated product 18 was isolated in an acceptable yield of 33% over two steps after oxidation and purification. Finally, hydrogenolysis using Pearlman’s catalyst led to debenzylation and the target PAR branched core structure 1 in an excellent yield of 95%. The 1H NMR spectrum of the synthesized 1 is consistent with that of the naturally occurring 1 reported by Miwa and coworkers.[10b] Although only a slightly upfield shift of 0.1 ppm was observed, this difference is account for the presence or absence of sodium 2,2-dimethyl-2-silapentane-5-sulfonate.

Scheme 5

Chemo- and Regioselective Phosphorylation of 5 to Synthesize 1

Conclusions

We have successfully developed a straightforward method for the synthesis of the PAR branched core structure 1. In this method, α-1,2-cis-selective O-ribofuranosidic bond formation and late-stage chemo- and regioselective phosphorylation of 5 were achieved. In terms of both the number of steps and overall yield, our route to 1 is significantly more efficient compared to that previously reported.[12] Recently, Filippov and coworker have synthesized a branched tri(ADP-ribose) for the first time.[25] Further synthetic efforts on such molecules lead to the detailed elucidation of the functions of the branched structure at the molecular level. Application of this method to synthesis of branched oligo(ADP-ribose) molecules is currently underway in our laboratory.

Experimental Section

General Methods

All chemicals were purchased from commercial suppliers and used without further purification. Dry solvents for reaction media (DMF, MeOH, pyridine, THF, and toluene) were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan) and used without purification. Microwave irradiation experiments were carried out using Initiator+ Eight (Biotage, Uppsala, Sweden) in sealed reaction vials without pressure. 6-Chloropurine ribofuranoside 4 and tetra-O-acetyl-β-d-ribofuranose 7 were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Phenyl 2,3,5-tri-O-acetyl-1-thio-β-d-ribofuranoside 8 and 6-chloropurine 3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-β-d-ribofuranoside 10 were prepared using previously reported methods.[16] Phenyl 2,3,5-tri-O-benzyl-1-thio-β-d-ribofuranoside S1 and phenyl 2-O-allyl-3,5-tri-O-benzyl-1-thio-β-d-ribofuranoside S2 were prepared using conventional methods (see the Supporting Information). TLC analyses were performed using TLC plates (silica gel 60F254 on a glass plate; Merck KGaA, Darmstadt, Germany). Compound detection was carried out either by exposure to UV light (2536 Å) or by soaking in H2SO4 solution (10% in EtOH) or phosphomolybdic acid solution (20% in EtOH) followed by heating. Flash column chromatography separations were performed by using silica gel (80 mesh and 300 mesh; Fuji Silysia Co., Aichi, Japan) or Biotage Isolera equipped with Biotage SNAP Ultra Silica Cartridges (10, 25, 50, 100, and 340 g). The quantity of silica gel was typically 100 to 200 times the weight of the crude sample. Sephadex LH-20 (Cytiva, Marlborough, MA, USA) was used for size-exclusion chromatography. Solvent systems for chromatography were specified in v/v ratios. 1H and 13C NMR spectra were recorded using an Avance III 500 spectrometer (Bruker, Billerica, MA, USA). Chemical shifts in 1H NMR spectra were expressed in ppm (δ) relative to the Me4Si signal (0.00 ppm). Chemical shifts in 13C NMR spectra were reported relative to the residual solvent signals (CDCl3, 77.16 ppm; CD3OD, 49.00 ppm). The data are presented as follows: chemical shift multiplicity (s = singlet, d = doublet, dd = doublet of doublets, td = triplet of doublets, ddd = doublet of doublet of doublets, t = triplet, q = quartet, m = multiplet), coupling constant (Hz), and integration. Structural assignments were performed using additional information from the 2D NMR (COSY) experiment. High-resolution mass spectrometry (ESI-TOF MS) was performed using a Bruker micrOTOF mass spectrometer (Bruker, Billerica, MA, USA). Optical rotations were measured with a SEPA-500 automatic polarimeter (Horiba, Kyoto, Japan).

Experimental Details

α-d-Ribofuranosyl-(1‴ → 2″)-α-d-ribofuranosyl-(1″ → 2′)-adenosine 5′,5′′,5‴-trisphosphate (1)

To a solution of 18 (13.1 mg, 9.98 μmol) in THF/H2O (2:1, 0.50 mL), Pd(OH)2/C (20% loading: 13.5 mg) was added at room temperature. After stirring for 1.5 h at room temperature under a H2 gas atmosphere, the reaction mixture was filtered through a membrane filter and lyophilized to yield 1 (7.3 mg, 95%) as white powder. The physical characteristics of 1 was consistent with the reported values.[10b,12]

α-d-Ribofuranosyl-(1‴ → 2″)-α-d-ribofuranosyl-(1″ → 2′)-adenosine (5)

To a solution of 17 (143 mg, 0.139 mmol) in THF/H2O (2:1, 4.2 mL), Pd(OH)2/C (20% loading: 188 mg) was added at room temperature. The reaction was monitored by TLC (CHCl3/MeOH = 10:1 and CHCl3/MeOH/H2O = 5:6:2). After stirring for 5 h at room temperature under a H2 gas atmosphere, Pd(OH)2/C (20% loading: 94.0 mg) was re-added to the reaction mixture. After stirring for additional 12 h, Pd(OH)2/C (20% loading: 94.0 mg) was added again. After stirring for additional 13 h, the reaction mixture was filtered through a pad of Celite, and the pad was rinsed with MeOH. The combined filtrate was concentrated in vacuo. The resulting residue was purified using reverse-phase silica gel column chromatography (H2O only) to yield 5 (63.0 mg, 87%) as white powder. The physical characteristics of 5 was consistent with the reported values.[21]

Phenyl 3,5-Di-O-benzyl-2-O-(2-naphthylmethyl)-1-thio-β-d-ribofuranoside (6)

To a solution of 9 (2.44 g, 6.38 mmol) in DMF (25.5 mL), NaH (2.28 g, 19.1 mmol) was added at 0 °C. After stirring for 30 min, BnBr (0.760 mL, 19.1 mmol) was added at 0 °C. The reaction was monitored by TLC (n-hexane/EtOAc = 1:1). After stirring for 17 h at room temperature, the reaction was quenched with MeOH. The reaction mixture was diluted with CHCl3 and washed with water and brine. The organic layer was dried over Na2SO4, filtered off, and co-evaporated with toluene. The resulting residue was purified using flash silica gel column chromatography (n-hexane/EtOAc = 17:3) to yield 6 (3.34 g, 93%) as a colorless syrup: [α]D25–23.4 (c 0.9, CHCl3); 1H NMR (500 MHz, CDCl3): δ 7.76–7.05 (m, 22 H, Ar), 5.47 (d, 1 H, J1,2 = 4.0 Hz, H-1), 4.78 (d, 1 H, Jgem = 11.0 Hz, CH2Ar), 4.70 (d, 1 H, Jgem = 12.0 Hz, CH2Ar), 4.57–4.47 (m, 4 H, CH2Ar), 4.32 (dd, 1 H, J3,4 = 4.5 Hz, J4,5 = 10.0 Hz, H-4), 3.95 (m, 2 H, H-2, H-3), 3.54 (m, 2 H, H-5a, H-5b); 13C{1H} NMR (125 MHz, CDCl3): δ 138.2137.7, 135.0, 133.6, 133.3, 133.2, 132.4, 128.9, 128.4, 128.4, 128.3, 128.1, 128.0, 128.0, 127.8, 127.7, 127.6, 127.6, 127.0, 126.2, 126.1, 88.9, 82.0, 80.4, 77.6, 77.4, 76.9, 73.4, 72.3, 70.3; HRMS (ESI) m/z [M + Na]+ calcd for C34H36O4S, 585.2070; found [M + Na]+, 585.2068.

Phenyl 2-O-(2-Naphthylmethyl)-1-thio-β-d-ribofuranoside (9)

To a solution of 8 (12.9 g, 35.0 mmol) in MeOH (70.0 mL), NaOMe (1 m solution in MeOH: 3.5 mL) was added at room temperature. The reaction was monitored by TLC (n-hexane/EtOAc = 1:1). After stirring for 1 h at room temperature, the reaction was neutralized with Muromac (H+ form) at 0 °C. The reaction mixture was evaporated in vacuo and exposed to high vacuum for 2 h. The resulting residue was dissolved in DMF (70.0 mL). To the solution, NaH (1.40 g, 35.0 mmol) was added at 0 °C. After stirring for 30 min, NAPBr (7.70 g, 35.0 mmol) was added at 0 °C. The reaction was monitored by TLC (n-hexane/EtOAc = 1:1). After stirring for 17 h at 0 °C, the reaction was quenched with MeOH. The reaction mixture was diluted with CHCl3 and washed with water and brine. The organic layer was dried over Na2SO4, filtered off, and co-evaporated with toluene. The resulting residue was purified using flash silica gel column chromatography (n-hexane/EtOAc = 9:1) to yield 9 (6.78 g, 54%) as a colorless syrup: [α]D25–23.9 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): δ 7.86–7.25 (m, 12 H, Ar), 5.42 (d, 1 H, J1,2 = 4.5 Hz, H-1), 4.90 (d, 1 H, Jgem = 12.0 Hz, CH2Ar), 4.80 (d, 1 H, Jgem = 12.0 Hz, CH2Ar), 4.20 (m, 1 H, H-3), 4.07 (m, 2 H, H-2, H-4), 3.81 (m, 1 H, H-5a), 3.63 (m, 1 H, H-5b), 2.65 (d, 1 H, J3-OH,3 = 6.5 Hz, 3-OH), 1.75 (dd, 1 H, J5-OH,5a = J5-OH,5b = 4.4 Hz, 5-OH); 13C{1H} NMR (125 MHz, CDCl3): δ 134.1, 133.2, 132.9, 132.4, 132.3, 132.1, 131.9, 131.6, 129.1, 128.9, 128.6, 128.0, 127.8, 127.2, 126.4, 126.3, 125.8, 88.4, 85.9, 82.4, 73.1, 70.6, 62.3; HRMS (ESI) m/z [M + Na]+ calcd for C22H22O4S, 405.1131; found [M + Na]+, 405.1132.

2-Naphthaldehyde [6-Chloropurine 3″,5″-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-β-d-ribofuranosid-2″-yl] (Phenyl 3′,5′-di-O-benzyl-1-thio-β-d-ribofuranoside) Acetal (11)

To a solution of 6 (117 mg, 0.210 mmol) and 10 (84.0 mg, 0.160 mmol) in CH2Cl2 (3.2 mL), MS4Å (320 mg) was added at room temperature. After stirring for 30 min at room temperature, DDQ (50 mg, 0.220 mmol) was added at 0 °C. The reaction was monitored by TLC (Toluene/EtOAc = 3:1). After stirring for 8 h at room temperature, the reaction mixture was filtered through a pad of Celite, and the pad was rinsed with CHCl3. The combined filtrate was washed with sat. aq. NaHCO3, water, and brine. The organic layer was dried over Na2SO4, filtered off, and concentrated in vacuo. The resulting residue was purified using size-exclusion chromatography on Sephadex LH-20 (CHCl3/MeOH = 1:1) to yield 11 (single isomer: 115 mg, 66%) as a colorless syrup: [α]D25–60.2 (c 1.3, CHCl3); 1H NMR (500 MHz, CDCl3): δ 8.38, 8.20 (2 s, 2 H, H-2, H-8), 7.90–7.07 (m, 22 H, Ar), 6.25 (s, 1 H, ArCH<), 6.16 (s, 1 H, H-1′), 5.87 (d, 1 H, J1″,2″ = 3.0 Hz, H-1″), 4.71 (t, 1 H, J2″,3″ = 4.2 Hz, H-2″), 4.60 (d, 1 H, Jgem = 12.0 Hz, CH2Ar), 4.53 (m, 2 H, CH2Ar), 4.45 (m, 2 H, H-2′, H-3′), 4.40 (d, 1 H, J3′,4′ = 4.5 Hz, H-4′), 4.34 (td, 1 H, J4″,5″a = J4″,5″b = 4.5 Hz, J3″,4″ = 5.7 Hz, H-4″), 4.27 (d, H, Jgem = 9.0 Hz, CH2Ar), 4.21 (d, 1 H, Jgem = 13.5 Hz, H-5′a), 4.11 (t, 1 H, H-3″), 3.99 (dd, 1 H, J4′,5′b = 1.9 Hz, H-5′b), 3.66–3.58 (m, 2 H, H-5″a, H-5″b), 1.15–0.73 (m, 28 H, Pr); 13C{1H} NMR (125 MHz, CDCl3): δ 151.6, 150.7, 150.3143.5, 138.2, 137.4, 134.1, 133.8, 132.8, 132.2, 131.3, 129.0, 128.5, 128.4, 128.3, 127.8, 127.7, 127.6, 127.3, 126.7, 126.3, 124.7, 103.6, 90.0, 89.1, 81.9, 81.8, 80.2, 78.3, 76.2, 73.4, 72.3, 70.4, 68.5, 59.6, 17.5, 17.3, 17.1, 16.9, 13.3, 13.0, 12.8; HRMS (ESI) m/z [M + Na]+ calcd for C58H69ClN4O4SSi2, 1111.3905; found [M + Na]+, 1111.3908.

6-Chloropurine 3″,5″-Di-O-benzyl-α-d-ribofuranosyl-(1″ → 2′)-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-β-d-ribofuranoside (12)

To a solution of 13α (164 mg, 0.170 mmol) in CH2Cl2/H2O (20:1, 3.3 mL), DDQ (75.0 mg, 0.330 mmol) was added at 0 °C. The reaction was monitored by TLC (n-hexane/EtOAc = 1:1). After stirring for 3.5 h at room temperature, the reaction mixture was diluted with CHCl3 and washed with sat. aq. NaHCO3 and brine. The organic layer was dried over Na2SO4, filtered off, and concentrated in vacuo. The resulting residue was purified using flash silica gel column chromatography (n-hexane/EtOAc = 7:3) to yield 12 (107 mg, 74%) as a colorless syrup: [α]D25 + 39.5 (c 1.1, CHCl3); 1H NMR (500 MHz, CDCl3): δ 8.71, 8.50 (2 s, 2 H, H-2, H-8), 7.36–7.24 (m, 10 H, Ar), 6.17 (s, 1 H, H-1′), 5.51 (d, 1 H, J2″,3″ = 4.5 Hz, H-1″), 4.65 (d, 1 H, Jgem = 12.0 Hz, CH2Ar), 4.60–4.50 (m, 4 H, H-2′, H–4′, CH2Ar), 4.43 (d, 1 H, Jgem = 12.0 Hz, CH2Ar), 4.37 (dd, 1 H, J3″,4″ = 4.0 Hz, J4″,5″a = J4″,5″b = 7.5 Hz, H-4″), 4.29 (d, 1 H, Jgem = 13.5 Hz, H-5′a), 4.26–4.21 (m, 2 H, H-2″, H-3′), 4.02 (dd, 1 H, J4′,5′b = 2.5 Hz, H-5′b), 3.89 (m, 1 H, H-3″), 3.46–3.40 (m, 2H, H-5″a, H-5″b), 3.18 (d, 1 H, J2″-OH,H-2″ = 11.5 Hz, 2″-OH) 1.15–0.83 (m, 28 H, Pr); 13C{1H} NMR (125 MHz, CDCl3): δ 152.0, 151.0, 150.5, 143.0, 138.0, 137.9, 132.5, 128.4, 128.3, 127.8, 127.7, 127.6, 101.2 (C-1″), 89.2, 82.3, 81.9, 79.1, 75.9, 73.5, 72.8, 72.1, 69.7, 67.6, 59.4, 17.5, 17.4, 17.3, 17.0, 16.9, 14.1, 13.5, 12.9, 12.7; HRMS (ESI) m/z [M + Na]+ calcd for C44H61ClN4O9Si2, 863.3245; found [M + Na]+, 863.3243.

6-Chloropurine 3″,5″-Di-O-benzyl-2″-O-(2-naphthylmethyl)-d-ribofuranosyl-(1″ → 2′)-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-β-d-ribofuranoside (13)

To a solution of 6 (2.08 g, 3.70 mmol) and 10 (648 mg, 1.23 mmol) in (CH2Cl)2 (74.0 mL), MS4Å (7.40 g) and Ph3Bi(OTf)2 (5.45 g, 7.38 mmol) were added at room temperature. The reaction was monitored by TLC (n-hexane/EtOAc = 3:1). After stirring for 24 h at 40 °C, the reaction was quenched with NEt3. The reaction mixture was filtered through a pad of Celite, and the pad was rinsed with CHCl3. The combined filtrate was concentrated in vacuo. The resulting residue was purified using size-exclusion chromatography on Sephadex LH-20 (CHCl3/MeOH = 1:1) and flash silica gel column chromatography (n-hexane/EtOAc = 7:3) to yield 13α (648 mg, 54%) as a yellow syrup along with 13β (113 mg, 9%) as a yellow syrup.

13α: [α]D25 + 39.8 (c 1.1, CHCl3); 1H NMR (500 MHz, CDCl3): δ 8.57, 8.26 (2 s, 2 H, H-2, H-8), 7.79–7.16 (m, 22 H, Ar), 6.01 (s, 1 H, H-1′), 5.44 (d, 1 H, J1″,2″ = 2.5 Hz, H-1″), 4.89 (d, 1 H, Jgem = 12.0 Hz, CH2Ar), 4.80 (d, 1 H, CH2Ar), 4.69 (d, 1 H, Jgem = 13.0 Hz, CH2Ar), 4.63–4.55 (m, 3 H, CH2Ar, H-2′, H-4′), 4.47–4.32 (m, 4 H, CH2Ar, H-3′, H-4″), 4.23 (d, 1 H, Jgem = 13.5 Hz, H-5′a), 4.01–3.95 (m, 3 H, H-2″, H-3″, H-5′b), 3.43–3.36 (m, 2 H, H-5a″, H-5b″), 1.13–0.88 (m, 28 H, Pr); 13C{1H} NMR (125 MHz, CDCl3) δ 151.9, 150.9, 150.5, 143.6, 138.6, 138.0, 135.3, 133.3, 133.2, 132.5, 128.5, 128.5, 128.4, 128.4, 128.1, 127.9, 127.8, 127.8, 127.7, 127.7, 127.0, 127.0, 126.4, 126.3, 126.0, 101.0 (C-1″), 89.6, 82.3, 81.8, 79.6, 78.7, 75.0, 73.6, 73.2, 72.2, 69.9, 67.9, 59.6, 31.1, 17.6, 17.5, 17.2, 17.2, 17.1, 17.1, 13.6, 13.1, 13.0, 12.9; HRMS (ESI) m/z [M + Na]+ calcd for C44H61ClN4O9Si2, 1003.3871; found [M + Na]+, 1003.3869.

13β: [α]D25–13.9 (c 1.6, CHCl3); 1H NMR (500 MHz, CDCl3): δ 8.59, 7.97 (2 s, 2 H, H-2, H-8), 7.84–7.18 (m, 22 H, Ar), 6.01 (s, 1 H, H-1′), 5.45 (s, 1 H, H-1″), 4.95 (dd, 1 H, J2′,3′ = 5.1 Hz, J3′,4′ = 5.2 Hz, H-3′), 4.76 (d, 1 H, Jgem = 12.0 Hz, CH2Ar), 4.70 (d, 1 H, H-2′), 4.63 (d, 1 H, Jgem = 13.0 Hz, CH2Ar), 4.47 (m, 3 H, CH2Ar), 4.34 (q, 1 H, J3″,4″ = J4″,5″a = J4″,5″b = 3.5 Hz, H-4″), 4.27 (td, 1 H, J4′,5′a = J4′,5′b = 4.5 Hz, H-4′), 4.11 (m, 2 H, H-3″, H-5′a), 4.00 (m, 2 H, H-2″, H-5′b), 3.74 (dd, 1 H, Jgem = 10.9 Hz, H-5″a), 3.58 (dd, 1 H, H-5″b), 1.10–0.88 (m, 28 H, Pr); 13C{1H} NMR (125 MHz, CDCl3): δ 151.9, 151.2, 150.8, 144.7, 138.0, 135.3, 133.4, 133.2, 132.6, 128.6, 128.5, 128.5, 128.4, 128.1, 128.0, 127.9, 127.9, 127.9, 127.8, 127.6, 126.8, 126.3, 126.2, 125.9, 106.2 (C-1″), 89.6, 81.7, 81.1, 79.6, 78.8, 77.7, 73.3, 72.7, 72.3, 70.1, 69.9, 60.1, 17.6, 17.4, 17.4, 17.3, 17.3, 17.2, 13.5, 13.1, 12.9; HRMS (ESI) m/z [M + Na]+ calcd for C44H61ClN4O9Si2, 1003.3871; found [M + Na]+, 1003.3874.

6-Chloropurine 2″,3″,5″-Tri-O-benzyl-d-ribofuranosyl-(1″ → 2′)-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-β-d-ribofuranoside (14)

To a solution of S1 (168 mg, 0.324 mmol) and 10 (57.1 mg, 0.108 mmol) in (CH2Cl)2 (6.5 mL), MS4Å (648 mg) and Ph3Bi(OTf)2 (478 mg, 0.648 mmol) were added at room temperature. The reaction was monitored by TLC (n-hexane/EtOAc = 3:1). After stirring for 12 h at 40 °C, the reaction was quenched with NEt3. The reaction mixture was filtered through a pad of Celite, and the pad was rinsed with CHCl3. The combined filtrate was concentrated in vacuo. The resulting residue was purified using size-exclusion chromatography on Sephadex LH-20 (CHCl3/MeOH = 1:1) and flash silica gel column chromatography (n-hexane/EtOAc = 4:1) to yield a mixture of 14α and 14β (67.4 mg containing impurities, 61%, α/β = 3:1) as a colorless syrup. 14α and 14β were partially separated by flash silica gel column chromatography (n-hexane/EtOAc = 5:1) in pure form.

14α: [α]D25 + 5.32 (c 0.5, CHCl3); 1H NMR (500 MHz, CDCl3): δ 8.66, 8.27 (2 s, 2 H, H-2, H-8), 7.32–7.19 (m, 15 H, Ar), 6.09 (s, 1 H, H-1′), 5.48 (d, 1 H, J1″,2″ = 3.9 Hz, H-1″), 4.69–4.58 (m, 6 H, H-2′, H-3′, CH2Ar), 4.48 (d, 1 H, Jgem = 12.1 Hz, CH2Ar), 4.44 (dd, 1H, J3″,4″ = 4.0 Hz, H-4″), 4.40 (d, 1 H, Jgem = 12.1 Hz, CH2Ar), 4.30 (d, 1 H, J3′,4′ = 9.2 Hz, H-4′), 4.22 (d, 1 H, Jgem = 13.4 Hz, H-5′a), 4.00 (dd, 1 H, J4′,5′b = 2.5 Hz, H-5′b), 3.92 (m, 2 H, H-2″, H-3″), 3.45–3.38 (m, 2 H, H-5″a, H-5″b), 1.10–0.98 (m, 28 H, Pr); 13C{1H} NMR (125 MHz, CDCl3): δ 152.0, 151.1, 150.6, 143.7, 138.6, 138.0, 137.8, 132.6, 128.5, 128.5, 128.4, 128.1, 128.1, 127.9, 127.8, 127.8, 127.7, 101.1 (C-1″), 89.7, 82.5, 81.8, 79.6, 78.8, 77.7, 75.0, 73.6, 72.8, 72.3, 70.0, 68.1, 59.7, 17.6, 17.5, 17.5, 17.3, 17.2, 17.2, 17.1, 13.6, 13.1, 13.0, 12.9; HRMS (ESI) m/z [M + Na]+ calcd for C48H63ClN4O9Si2, 953.3714; found [M + Na]+, 953.3715.

14β: [α]D25–13.2 (c 0.6, CHCl3); 1H NMR (500 MHz, CDCl3): δ 8.60, 7.98 (2 s, 2 H, H-2, H-8), 7.36–7.19 (m, 15 H, Ar), 6.01 (s, 1 H, H-1′), 5.41 (s, 1 H, H-1″), 4.95 (dd, 1 H, J3′,4′ = 4.2 Hz, J2′,3′ = 5.2 Hz, H-3′), 4.74 (d, 1 H, Jgem = 12.1 Hz, CH2Ar), 4.69 (d, 1 H, H-2′), 4.62–4.45 (m, 5 H, CH2Ar), 4.30 (ddd, 1 H, J4″,5″a = 3.5 Hz, J4″,5″b = 4.0 Hz, J3″,4″ = 4.5 Hz, H-4″), 4.25 (ddd, J4′,5′a = 4.6 Hz, 1H, H-4′), 4.11 (near d, 1 H, Jgem = 13.2 Hz, H-5′a), 4.05 (d, 1 H, H-3″), 4.00 (m, 2 H, H-2″, H-5′b), 3.73 (dd, 1 H, Jgem = 10.9 Hz, H-5″a), 3.58 (dd, 1 H, H-5″b), 1.12–0.97 (m, 28 H, Pr); 13C{1H} NMR (125 MHz, CDCl3): δ 151.9, 151.2, 150.9, 144.7, 138.0, 138.0, 137.8, 132.6, 128.6, 128.1, 128.0, 127.9, 127.9, 127.6, 106.1 (C-1″), 89.6, 81.7, 81.1, 79.6, 78.8, 77.6, 73.3, 72.6, 72.1, 70.1, 69.6, 60.1, 17.6, 17.4, 17.4, 17.3, 17.2, 13.5, 13.1, 12.9; HRMS (ESI) m/z [M + Na]+ calcd for C48H63ClN4O9Si2, 953.3714; found [M + Na]+, 953.3713.

6-Chloropurine 3″,5″-Di-O-benzyl-2″-O-allyl-α-d-ribofuranosyl-(1″ → 2′)-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-β-d-ribofuranoside (15)

To a solution of S2 (142 mg, 0.307 mmol) and 10 (54.1 mg, 0.102 mmol) in (CH2Cl)2 (6.1 mL), MS4Å (610 mg) and Ph3Bi(OTf)2 (453 mg, 0.614 mmol) were added at room temperature. The reaction was monitored by TLC (n-hexane/EtOAc = 3:1). After stirring for 4 h at 40 °C, the reaction was quenched with NEt3. The reaction mixture was filtered through a pad of Celite, and the pad was rinsed with CHCl3. The filtrate was concentrated in vacuo. The resulting residue was purified using size-exclusion chromatography on Sephadex LH-20 (CHCl3/MeOH = 1:1) and flash silica gel column chromatography (n-hexane/EtOAc = 9:2) to yield 15 (45.7 mg containing impurities, 36%, α only) as a colorless syrup. 15 was partially separated by flash silica gel column chromatography (n-hexane/EtOAc = 4:1) in pure form: [α]D25 + 1.62 (c 1.1, CHCl3); 1H NMR (500 MHz, CDCl3): δ 8.69, 8.36 (2 s, 2 H, H-2, H-8), 7.34–7.23 (m, 10 H, Ar), 6.18 (s, 1 H, H-1′), 5.92–5.86 (m, 1 H, CH2CH=CH2), 5.52 (d, 1 H, J1″,2″ = 4.5 Hz, H-1″), 5.25 (near d, 1 H, CH2CH=CH2), 5.16 (near d, 1 H, CH2CH=CH2), 4.67–4.49 (m, 5 H, H-2′, H-4′, CH2Ar), 4.42 (m, 2 H, H-4″, CH2Ar), 4.32 (d, 1 H, J3′,4′ = 9.0 Hz, H-3′), 4.24 (d, 1 H, Jgem = 13.5 Hz, H-5′a), 4.17–4.10 (m, 2 H, CH=CH2), 4.02–3.88 (m, 3 H, H-2″, H-3″, H-5′), 3.45–3.37 (m, 2 H, H-5″), 1.25–0.91 (m, 28 H, Pr); 13C{1H} NMR (125 MHz, CDCl3): δ 151.9, 151.0, 150.6, 143.5, 138.4, 137.9, 134.5, 132.5, 128.4, 128.3, 128.0, 127.7, 127.6, 101.1 (C-1″), 89.6, 82.3, 81.7, 79.7, 78.8, 74.8, 73.5, 72.1, 72.0, 69.8, 67.8, 59.5, 17.5, 17.4, 17.3, 17.1, 17.0, 16.9, 13.5, 13.0, 12.9, 12.7; HRMS (ESI) m/z [M + Na]+ calcd for C44H61ClN4O9Si2, 903.3558; found [M + Na]+, 903.3559.

6-Chloropurine 3‴,5‴-Di-O-benzyl-2‴-O-(2-naphthylmethyl)-d-ribofuranosyl-(1‴ → 2″)-3″,5″-di-O-benzyl-α-d-ribofuranosyl-(1″ → 2′)-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-β-d-ribofuranoside (16)

To a solution of 6 (523 mg, 0.930 mmol) and 12 (265 mg, 0.310 mmol) in (CH2Cl)2 (6.2 mL), MS4Å (620 mg) and Ph3Bi(OTf)2 (1.44 g, 1.86 mmol) were added at room temperature. The reaction was monitored by TLC (n-hexane/EtOAc = 2:1). After stirring for 9 h at 40 °C, the reaction was quenched with NEt3. The reaction mixture was filtered through a pad of Celite, and the pad was rinsed with CHCl3. The combined filtrate was concentrated in vacuo. The resulting residue was purified using size-exclusion chromatography on Sephadex LH-20 (CHCl3/MeOH = 1:1) and flash silica gel column chromatography (n-hexane/EtOAc = 7:3) to yield 16α (310 mg, 77%) as a yellow syrup along with 16β (21.0 mg, 5%) as a yellow syrup.

16α: [α]D25 + 66.6 (c 1.3, CHCl3); 1H NMR (500 MHz, CDCl3): δ 8.64, 8.41 (2 s, 2 H, H-2, H-8), 7.78–7.14 (m, 27 H, Ar), 6.09 (s, 1 H, H-1′), 5.69 (d, 1 H, J1″,2″ = 4.1 Hz, H-1″), 5.56 (d, 1 H, J1‴,2‴ = 4.2 Hz, H-1‴), 5.06–5.00 (m, 2 H, CH2Ar), 4.74 (m, 2 H, CH2Ar), 4.60–4.35 (m, 11 H, H-2′, H-2″, H-4′, H-4″, H-4‴, CH2Ar), 4.12 (m, 1 H, H-2‴), 4.04 (m, 2 H, H-3″, H3‴), 3.80 (m, 1 H, H-3′), 3.67–3.61 (m, 2 H, H-5′a, H-5‴a), 3.54 (dd, 1 H, J4′,5′b = 4.2 Hz, Jgem = 10.6 Hz, H-5′b), 3.74 (m, 2 H, H-5″a, H-5″b), 3.36 (m, 1 H, J4‴,5‴b = 2.3 Hz, Jgem = 13.7 Hz, H-5‴b), 1.02–0.81 (m, 28 H, Pr); 13C{1H} NMR (125 MHz, CDCl3): δ 152.0, 150.9, 150.4, 143.1, 139.6, 138.6, 138.2, 138.0, 136.0, 133.2, 133.0, 132.5, 128.5, 128.5, 128.4, 128.0, 128.0, 127.9, 127.8, 127.7, 127.7, 127.6, 127.0, 126.3, 126.3, 126.1, 125.9, 100.6 (C-1″), 100.5 (C-1‴), 89.4, 82.6, 82.1, 81.8, 79.9, 78.7, 76.5, 76.0, 75.5, 73.7, 73.6, 72.8, 72.5, 72.4, 70.1, 69.9, 67.3, 59.1, 17.6, 17.5, 17.4, 17.2, 17.1, 17.1, 17.0, 13.5, 13.0, 12.9, 12.9; HRMS (ESI) m/z [M + Na]+ calcd for C62H80ClN4O12Si2, 1315.5232; found [M + Na]+, 1315.5235.

16β: [α]D25 + 35.9 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): δ 8.52, 8.37 (2 s, 2 H, H-2, H-8), 7.79–7.11 (m, 27 H, Ar), 6.02 (s, 1 H, H-1′), 5.39 (d, 1 H, J1″,2″ = 4.3 Hz, H-1″), 5.25 (s, 1 H, H-1‴), 4.73 (m, 2 H, CH2Ar), 4.57–4.37 (m, 12 H, H-2′, H-4′, H-4″, H-4‴, CH2Ar), 4.27 (near d, 1 H, J2′,3′ = 9.4 Hz, H-3′), 4.21–4.15 (m, 2 H, H-2″, H-5′a), 4.00–3.93 (m, 4 H, H-2‴, H-3″, H-3‴, H-5′b), 3.58 (m, 2 H, H-5″a, H-5″b), 3.41 (m, 2 H, H-5‴a, H-5‴b), 1.10–0.87 (m, 28 H, Pr); 13C{1H} NMR (125 MHz, CDCl3): δ 152.0, 150.9, 150.5, 143.3, 138.7, 138.1, 138.0, 137.8, 135.5, 133.4, 133.2, 132.6, 128.5, 128.4, 128.3, 128.3, 128.3, 128.0, 127.8, 127.8, 127.7, 127.6, 127.3, 126.7, 126.3, 126.0, 125.9, 105.7 (C-1‴), 101.7 (C-1″), 89.2, 82.4, 81.6, 80.7, 80.1, 78.1, 77.7, 76.4, 74.6, 73.7, 73.1, 72.5, 72.4, 72.0, 71.5, 67.7, 59.6, 17.7, 17.5, 17.5, 17.2, 17.1, 17.0, 13.6, 13.1, 13.0, 12.8; HRMS (ESI) m/z [M + Na]+ calcd for C62H80ClN4O12Si2, 1315.5232; found [M + Na]+, 1315.5236.

6-Chloropurine 3‴,5‴-Di-O-benzyl-2‴-O-(2-naphthylmethyl)-α-d-ribofuranosyl-(1‴ → 2″)-3″,5″-di-O-benzyl-α-d-ribofuranosyl-(1″ → 2′)-β-d-ribofuranoside (S3)

To a solution of 16α (112 mg, 86.0 μmol) in pyridine (0.86 mL), HF·pyridine (86 μL) was added at 0 °C. The reaction was monitored by TLC (CHCl3/MeOH = 10:1). After stirring for 6 h at room temperature, sat. aq. NaHCO3 was carefully added to the reaction mixture at 0 °C. The mixture was diluted with CHCl3 and washed with sat. aq. NaHCO3 and brine. The organic layer was dried over Na2SO4, filtered off, and concentrated in vacuo. The resulting residue was purified using flash silica gel column chromatography (CHCl3/MeOH = 99:1) to yield S3 (80.7 mg, 89%) as a yellow syrup: [α]D25 + 42.1 (c 0.5, CHCl3); 1H NMR (500 MHz, CDCl3): δ 8.67, 7.90 (2 s, 2 H, H-2, H-8), 7.82–7.14 (m, 27 H, Ar), 5.80 (d, 1 H, J1′,2′ = 7.0 Hz, H-1′), 5.14 (m, 2 H, H-1″, H-1‴), 5.01 (d, 1 H, Jgem = 12.1 Hz, CH2Ar), 5.00–4.89 (m, 3 H, H-2′, H-4‴, CH2Ar), 4.77 (d, 1 H, Jgem = 11.1 Hz, CH2Ar), 4.65 (d, 2 H, CH2Ar), 4.56–4.40 (m, 6 H, CH2Ar, 5′-OH), 4.37–4.28 (m, 4 H, H-3′, H-4′, H-4″, H-2‴), 4.23 (brs, 1 H, 3′-OH), 4.05 (d, 1 H, J2‴,3‴ = 5.1 Hz, H-3‴), 3.98 (m, 2 H, H-2″, H-3″), 3.86 (d, 1 H, Jgem = 12.7 Hz, H-5‴a), 3.68 (m, 1 H, 5‴b), 3.52–3.44 (m, 2 H, H-5′a, H-5′b), 3.40–3.33 (m, 2 H, H-5″a, H-5″b); 13C{1H} NMR (125 MHz, CDCl3): δ 152.0, 151.5, 150.6, 145.5, 138.2, 138.0, 137.9, 137.8, 135.3, 133.3, 133.3, 133.2, 128.5, 128.5, 128.4, 128.3, 128.1, 128.1, 127.8, 127.8, 127.8, 127.7, 127.6, 127.5, 127.1, 126.4, 126.4, 126.2, 101.1, 100.1, 89.5, 87.7, 83.7, 82.4, 78.5, 78.4, 78.3, 77.4, 77.0, 76.4, 73.6, 73.5, 72.9, 72.7, 72.3, 70.2, 70.0, 63.0; HRMS (ESI) m/z [M + Na]+ calcd for C59H59ClN4O12, 1073.3710; found [M + Na]+, 1073.3710.

3‴,5‴-Di-O-benzyl-2‴-O-(2-naphthylmethyl)-α-d-ribofuranosyl-(1‴ → 2″)-3″,5″-di-O-benzyl-α-d-ribofuranosyl-(1″ → 2′)-adenosine (17)

To a solution of S3 (56.3 mg, 54.0 μmol) in THF (1.08 mL), NH3 (2 m solution in MeOH: 2.14 mL) was added at room temperature. The reaction was monitored by TLC (CHCl3/MeOH = 10:1). After stirring for 1 h at 160 °C under microwave irradiation, the reaction mixture was diluted with CHCl3 and washed with water and brine. The organic layer was dried over Na2SO4, filtered off, and concentrated in vacuo. The resulting residue was purified using flash silica gel column chromatography (CHCl3/MeOH = 19:1) to yield 17 (54.3 mg, 98%) as a colorless syrup: [α]D25 + 21.7 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): δ 8.25, 7.87 (2 s, 2 H, H-2, H-8), 7.79–7.10 (m, 27 H, Ar), 6.36 (d, 1 H, J5′-OH,5′a = J5′-OH,5′b = 10.7 Hz, 5′-OH), 6.15 (brs, 2 H, NH2), 5.78 (d, 1 H, J1′,2′ = 7.3 Hz, H-1′), 5.13 (m, 2 H, H-1″, H-1‴), 5.02–4.92 (m, 3 H, H-2′, CH2Ar), 4.76–4.58 (m, 4 H, CH2Ar), 4.52–4.39 (m, 5 H, CH2Ar, 3′-OH), 4.35–4.27 (m, 5 H, H-3′, H-3″, H-4″, H-4‴, H-2‴), 4.05 (d, 1 H, J2‴,3‴ = 5.4 Hz H-3‴), 3.97 (m, 2 H, H-2″, H-4′), 3.88 (d, 1 H, Jgem = 12.4 Hz, H-5′a), 3.71 (m, 1 H, H-5′b), 3.51–3.43 (m, 2 H, H-5″a, H-5″b), 3.42–3.35 (m, 2 H, H-5‴a, H-5‴b); 13C{1H} NMR (125 MHz, CDCl3): δ 156.1, 152.5, 148.6, 140.7, 138.3, 138.1, 137.9, 137.8, 135.4, 133.3, 133.2, 128.8, 128.4, 128.3, 128.2, 128.1, 127.8, 127.7, 127.7, 127.5, 127.1, 126.4, 126.3, 126.1, 121.1, 100.9, 100.2, 89.3, 88.0, 83.7, 82.2, 78.7, 78.4, 78.2, 77.4, 76.8, 76.3, 73.5, 73.5, 73.5, 72.9, 72.8, 72.7, 70.2, 69.9, 63.3 HRMS (ESI) m/z [M + Na]+ calcd for C59H61N5O12, 1054.4209; found [M + Na]+, 1054.4208.

α-d-Ribofuranosyl-(1‴ → 2″)-α-d-ribofuranosyl-(1″ → 2′)-adenosine 5′,5′′,5‴-tris(dibenzyl phosphate) (18)

To a solution of 5 (19.7 mg, 37.1 μmol) in DMF (1.5 mL), MS4Å (150 mg) and 1H-tetrazole (52.0 mg, 741 μmol) were added at room temperature. After stirring for 4 h, dibenzyl N,N-diisopropylphosphoramidite (55.4 μL, 167 mmol) was added at room temperature. The reaction was monitored by TLC (CHCl3/MeOH = 4:1). After stirring for 1.5 h at room temperature, dibenzyl N,N-diisopropylphosphoramidite (112 μL, 334 mmol) was re-added to the reaction mixture. After stirring for additional 1 h, dibenzyl N,N-diisopropylphosphoramidite (112 μL, 334 mmol) was added again. After stirring for additional 1 h, BuOOH (5.5 m solution in decane: 111 μL) was added to the reaction mixture at 0 °C. After stirring for 1 h at room temperature, the reaction was quenched with sat. aq. Na2S2O3. The reaction mixture was filtered through a pad of Celite, and the pad was rinsed with CHCl3. The combined filtrate was washed with sat. aq. NaHCO3 and brine. The organic layer was dried over Na2SO4, filtered off, and concentrated in vacuo. The resulting residue was purified using flash silica gel column chromatography (CHCl3/MeOH = 5:1) to yield 18 (16.0 mg, 33%) as a colorless syrup: [α]D25 + 53.2 (c 0.5, CHCl3); 1H NMR (500 MHz, CDCl3): δ 8.27, 8.11 (2 s, 2 H, H-2, H-8), 7.32–7.26 (m, 30 H, Ar), 6.13 (d, 1 H, J1′,2′ = 5.0 Hz, H-1′), 5.81 (brs, 2 H, NH2), 5.03–4.96 (m, 13 H, H-1″, CH2Ar), 4.80 (d, 1 H, J1‴,2‴ = 5.5 Hz, H-1‴), 4.59 (brs, 1 H, H-2′), 4.35 (brs, 1 H, H-3′), 4.27–4.12 (m, 6 H, H-3″, H-3‴, H-4′, H-4″, H-4‴, H-5′a), 4.00–3.95 (m, 7 H, H-2″, H-2‴, H-5′b, H-5″a, H-5″b, H-5‴a, H-5‴b), 1.88 (brs, 4 H, 3′-OH, 3″-OH, 2‴-OH, 3‴-OH); 13C{1H} NMR (125 MHz, CDCl3): δ 155.7, 153.1, 149.4, 139.4, 135.7, 135.7, 135.7, 135.7, 135.6, 128.8, 128.8, 128.1, 128.1, 120.1, 101.8, 100.8, 86.9, 83.9, 83.6, 79.6, 78.8, 72.5, 70.9, 70.7, 70.5, 69.8, 69.8, 69.8, 69.7, 69.6, 67.2, 67.0; 31P{1H} NMR (200 MHz, CDCl3): δ 1.40, 1.36; HRMS (ESI) m/z [M + Na]+ calcd for C62H68N5O21P3, 1334.3512; found [M + Na]+, 1334.3510.