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Highly Diastereoselective Synthesis of A HCV NS5B Nucleoside Polymerase Inhibitor Yong-Li Zhong, Ed Cleator, Zhijian Liu, Jianguo Yin, William J Morris, Mahbub Alam, Brian Bishop, Aaron M. Dumas, John Edwards, Adrian Goodyear, Peter Mullens, Zhiguo Jake Song, Michael Shevlin, David A. Thaisrivongs, Hongming Li, Edward C. Sherer, Ryan D. Cohen, Jingjun Yin, Lushi Tan, Nobuyoshi Yasuda, John Limanto, Antony Davies, and Kevin R Campos J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02500 • Publication Date (Web): 26 Nov 2018 Downloaded from http://pubs.acs.org on November 26, 2018

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The Journal of Organic Chemistry

Highly Diastereoselective Synthesis of a HCV NS5B Nucleoside Polymerase Inhibitor Yong-Li Zhong,* Ed Cleator,* Zhijian Liu, Jianguo Yin, William J. Morris, Mahbub Alam, Brian Bishop, Aaron M. Dumas, John Edwards, Adrian Goodyear, Peter Mullens, Zhiguo Jake Song, Michael Shevlin, David A. Thaisrivongs, Hongming Li, Edward C. Sherer, Ryan D. Cohen, Jingjun Yin, Lushi Tan, Nobuyoshi Yasuda, John Limanto, Antony Davies, and Kevin R. Campos Process Research and Development, Merck & Co., Inc., P.O. Box 2000, Rahway, New Jersey 07065, USA Supporting Information Placeholder

ABSTRACT: An asymmetric synthesis of HCV NS5B nucleoside polymerase inhibitor (1) is described. This novel route features several remarkably diastereoselective and high yielding transformations, including construction of the all-carbon quaternary stereogenic center at C-2 via a thermodynamic aldol reaction. Subsequent glycosylation reaction with activated uracil via C-1 phosphate, and installation of the cyclic phosphate group using an achiral phosphorous (III) reagent followed by oxidation provides 1.

first example of a diastereoselective open-chain aldol reaction ■ INTRODUCTION to efficiently set a chiral all-carbon quaternary stereogenic Chronic hepatitis C virus (HCV) is a liver disease that has center. Intermediates 2a/2b are readily assembled from cominfected an estimated 130 to 150 million people worldwide as mercially available materials in a three-step sequence. of 2016. While an estimated 500,000 people around the world die annually as a result of HCV-related liver complications, the recent introduction of several novel and effective products Scheme 1. Retrosynthetic Analysis of 1 for the treatment of HCV infections have given patients enhanced treatment options. However, continued HCV research is still needed to develop safer drugs with improved efficacy and simplified treatment regimens. As part of an ongoing drug discovery program in our research laboratories, 1 has been identified as a selective and highly potent inhibitor of HCV NS5B nucleoside polymerase. Herein, we report a novel asymmetric synthesis of this drug candidate. 1 is a structurally complex and functionally dense molecule that presents numerous challenges for chemical synthesis. The medicinal chemistry route to 1 was lengthy (19 steps in the longest linear sequence) and proceeded in approximately 0.4% overall yield.3 This route was deemed unsuitable for scale-up and we set out to evaluate alternative approaches. Our retrosynthetic analysis of 1 is outlined in Scheme 1. 1 would be derived from A by diastereoselective installation of P(III) chemistry followed by oxidation. Intermediate A could be synthesized by the diastereoselective glycosylation of the intermediate B, which could be accessed from the key intermediate 4 in several steps through reduction/activation sequences. Key intermediate 4 could be prepared from the enolate of 2a/2b with chiral aldehyde 3 through diastereoselective aldol reaction to set up the chiral quaternary carbon stereogenic center. To the best of our knowledge, this would represent the ACS Paragon Plus Environment 4

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■ RESULTS AND DISCUSSION Employing modified literature conditions, compounds 2a/2b were prepared in 79% overall yield as a mixture of acetylene and allene tautomers from tert-butyl acrylate and TIPSprotected acetylene in a three-step sequence via bromination/elimination, Sonogashira coupling and NaBH4-mediated reduction.5 Aldehyde 3 was synthesized in a 2-step sequence starting from ≥ 99.0% ee optical pure D-mannitol. With the precursors 2a/2b and 3 in hand, the aldol reaction was initially examined. As highlighted in Table 1, aldol products 7a/7b were undetectable at -78 °C (entry 1). When the reaction was slowly warmed up to -55 °C, 70% conversion was achieved with the desired quaternary carbon center 7a and undesired quaternary carbon center 7b in a ratio of 60/40 (entry 2). Interestingly, a competing retro-aldol reaction was observed after the reaction was warmed to room temperature and agitated for a few hours (entry 3). Only precursor 2a was recovered while aldehyde 3 decomposed. The optimal temperature for the reaction was determined by slowly warming to -20 °C where > 95% conversion of the reaction was achieved (entries 4-12). In the presence of additives such as DMPU or HMPA, the ratio of 7a/7b was increased (entries 5-6). Not surprisingly, employing a Lewis acid, magnesium bromide, lowered selectivity and increased production of the undesired isomer (entry 8). There was no significant difference observed by running the reaction in THF, MeTHF or MTBE or by using alternative bases such as LDA and LTMP (entries 9-11). It Table 1. Selected Results of Screening for the Diastereoselective Aldol Condensation

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more facile retro-aldol. Thus, employing NaHMDS as base and 2 equiv of DMPU as an additive, the aldol reaction was achieved in excellent diastereoselectivity favoring the desired product 7a (entries 12-13).

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Figure 1. 500 MHz H NMR spectra of (a) completely addition of NaHMDS at ̴ - 50 °C. (b) agitate at ̴ - 50 °C for 0.5 h. Samples were taken at -50 °C and quenched to 20 wt% citric acid. All spectra were recorded in CDCl . 1

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Further studies indicated the aldol reaction is an equilibrium process that requires an excess of aldehyde 3 and NaHMDS along with sufficient dilution to achieve both good dr and conversion. The equilibrium of the undesired isomers to 7a occurred between -60 to -50 °C. The order of addition was also determined to be important. The optimal conditions involved adding 1 M NaHMDS (1.85 equiv) last to a degassed mixture of 2a/2b, aldehyde 3 (2 equiv) and DMPU (2 equiv) in THF (~13.5 vol) at -60 to -50 °C over 2 hours (Figure 1). Under the optimized conditions, the aldol products were obtained in 95% assay yield (Scheme 2). Among the four diastereomers, diastereomer 8 was the major component (97%) with the remaining 3% being a combination of the other three diastereomers. The stereochemistry of compound 8 was confirmed by nOe studies of compound 8a, which was prepared by treatment of 8 with 70% TFA aqueous. Scheme 2. Diastereoselective Synthesis of 4

aDetermined by NMR analysis. bRatio of 7a and 7b is indicated to the quaternary center. cRetro-aldol reaction is faster than aldol reaction at room temperature. d1.15 equiv of base was used for the screening reaction.

became clear that the desired 7a is likely a thermodynamically controlled product. It is reasonable that the use of lesscoordinating Na+ counterion, aided by DMPU, may allow for

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The Journal of Organic Chemistry Our next task was to determine the best way to install the uracil unit into compound 4. There is a significant amount of literature available which indicates that diastereoselective glycosylation of similar substrates is often not straightforward. In particular, a substrate with an all-carbon quaternary center at the α-position presents a challenge and is unlikely to benefit from anchimeric assistance. We evaluated functional groups in the compounds 13a-h, which could potentially impact the diastereoselectivity of the glycosylation (Table 2). The substrates 13a-h could be accessed in multiple steps from the lactone diol 4.5b Table 2. Selected Results of Screening for the Diastereoselective Glycosylation 9

aConversion of the glycosylation was typically ≥ 95%. bRatio of the glycosylation products 14a-h determined by HPLC analysis.

We first investigated the leaving group for the glycosylation and found that diphenylphosphate was the best leaving group in terms of reactivity and diastereoselectivity (e.g. Table 2, entries 2 vs. 1). Next, we evaluated the impact of the TIPSsubstituted group connected to the alkyne. Initially, our hypothesis was that the bulky TIPS group might potentially shield the α-face of the furan ring, favoring a β-selective glycosylation. However, experimental results demonstrated that this was not the case. The glycosylation of substrate 13b, in which the alkyne is protected by a TIPS group, was less stereoselective (64:36) than that with substrate 13c (86:14), in which the alkyne is unprotected (entry 3). Contrary to our original thinking, a bulky group on the alkyne is actually detrimental to the formation of the desired anomer. Finally, the impact of the hydroxyl protecting groups on the glycosylation diastereoselectivity was examined. When both C-3 and C-5 hydroxyl groups were protected as benzoyl esters, undesired α-anomer 14d-α was the major glycosylation product (entry 4). Further investigation indicated that the undesired α-anomer 14e-α was the predominant product when C-3 and C-5 hydroxyl group were protected as TBDMS and 4methoxybenzoyl ester, respectively (entry 5). On the other hand, when the hydroxyl group at C-5 was masked as the TBDMS ether and the C-3 hydroxyl was protected as p10

In order to prepare the desired diasteromer, the hydroxyl group of 8 required inversion. After careful consideration of alternative methods to invert the hydroxyl center, we focused our attention on an oxidation/reduction approach. Thus, Swern oxidation of the crude aldol products gave the corresponding ketone 9 in 97% assay yield and 98% dr. Global deprotection and cyclization in the presence of BF3 etherate and ethylene glycol afforded the corresponding ketolactone without any stereochemical erosion. The role of ethylene glycol in the BF3mediated deprotection was situ trapping of the released 3pentanone and led to a completed reaction. Sodium borohydride-mediated reduction presumably directed by the hydroxyl group gave the desired selective reduction, in which the hydride is delivered from the upper face to afford the desired lactone diol 4 as a single diastereomer in 75% overall yield from ketone 9. The absolute configuration of 4 was confirmed by single crystal X-ray analysis. 7

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methoxylbenzoyl ester, the desired β-anomer 14f-β was predominantly formed (entry 6). The selectivity of the glycosylation decreased when the hydroxyl group at C-5 was protected using a more sterically hindered group (e.g. TIPS, entry 7) or the C-3 hydroxyl was masked by an electron deficient ester (entry 8). Alpha approach open

Beta approach open

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of the secondary alcohol in one-pot afforded product 15 in 96% yield. Treatment of 15 with 2.4 equiv of potassium fluoride in the presence of 4.8 equiv of acetic acid removed both silyl groups of 15 in 92% assay yield. Monitoring the reaction by HPLC showed that the TBDMS group was rapidly cleaved prior to the TIPS-protecting group. Nevertheless the primary alcohol was protected again with TBDMS-Cl in the presence of imidazole to give 16 in 96% assay yield. LiAlH(OtBu)3mediated chemoselective reduction of lactone 16 at 0-10 °C afforded the desired lactol 17 in 92% assay yield. Activation of the lactol with diphenyl phosphorochloridate gave the glycosylation precursors 18. The resulting mixture was cooled to -60 °C and 1.3 equiv of bis-TMS uracil 5 was charged. TMSOTf (2.5 equiv) was then slowly added to the solution followed by warming the mixture to -30 °C. HCl-mediated aqueous work up and crystallization, which rejected the αanomer to low levels, was performed in one-pot to give the desired product 19 in 70% isolated yield overall from 17. Scheme 3. Diastereoselective Synthesis of Uracil Ester 19 16

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13f-T

Figure 2. Global minimum conformations for 13e-T and 13f-T indicating preferential approach of alpha or beta, respectively.

The mechanism for the glycosylation of substrates 13e and 13f possibly involves the anchimeric assistance from the C-5 and C-3 ester moiety resulting in the formation of the bicyclic oxocarbenium ions 13e-T and 13f-T, respectively (Figure 2). These intermediates presumably would form when both 13e and 13f were treated with TMSOTf, which triggers the loss of the phosphate leaving group. The addition of TMS uracil to these intermediates would then occur preferentially from the α-face (13e-T) or the β-face (13f-T) to give α- or β-anomers, respectively. To better understand the driving force for this selectivity, density functional theory calculations were performed. An exploration of conformational space was undertaken through a combination of conformer sampling algorithms, molecular mechanics calculations, and density functional theory as described previously. For each molecule, 240 conformers were geometry optimized using B3LYP/6-31G**. Secondary calculations using M062X/6-31+G**, B3LYP-D3 dispersion correction, ωB97XD, or implicit solvation in tetrahydrofuran consistently ranked the conformers depicted in Figure 2 as the lowest in free energy. Minima were confirmed via frequency calculations and all calculations were run using Gaussian09. It is apparent from Figure 2 that the beta position of 13e-T is blocked by a charge-charge interaction of the 5’ ester protecting group stabilizing the carbocation at position C1’ (1.53 Å). Alternatively, for 13f-T, the alpha position is blocked by this same protecting group interaction with C1’ (1.51 Å), this time preferentially exposing the beta face of the carbocation. Many low-lying conformers for either 13e-T or 13f-T maintain the same protecting group interaction while sampling conformational variability of the methoxyphenyl or TBDMS groups. Any conformations of 13e-T or 13f-T where the protecting group has rotated away from blocking the carbocation in order to free up either face are at least 5.0 kcal/mol above the global minima. We note that the calculated selectivity is larger than experimentally determined which might come into better agreement with more detailed modeling using explicit solvent. With the optimized glycosylation conditions in hand, the synthesis of intermediate 19 was investigated from lactone diol 4 (Scheme 3). Selective TBDMS-protection on the primary hydroxyl group of lactone diol 4, followed by esterification 12

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Finally, installation of chiral phosphate (V) moiety to 19 was required to complete the synthesis of 1. Typically, there are two approaches to convert a 1,3-diol to a cyclic phosphate, direct reaction of a 1,3-diol with a P(V) reagent or a 2-step sequence involving reaction of a 1,3-diol with a P(III) reagent and then oxidation. Initially, we began our investigation exploring the direct installation of P(V). Unfortunately, the undesired diastereomer was the major product in all reactions, with limited amounts of the desired product 1 being generated. In the best scenario, around 30% of the desired product 1 was obtained. At this point, we turned our attention to the P(III) chemistry. Thus, saponification of ester 19 afforded the corresponding uracil diol in 95% yield (Scheme 4, approach-1). Treatment of the uracil diol with N,N,N’,N’tetraisopropylphosphorodiamidite 6 in the presence of 4,5dicyanoimidazole (DCI), followed by heating the reaction mixture at 65-70 °C afforded the desired cyclic phosphite 20 in 95:5 dr. Addition of the oxidant mixture (I2/pyridine/water) to the reaction stream afforded moderate to low yields of 1 ( 99 A% conversion). The reaction mixture was cooled to room temperature. MTBE (500 mL) was added and the resulting mixture was stirred for 10 min. After phase separation, the organic layer was washed with 25 wt% Na2S2O3 (300 mL x 2) and brine (200 mL x 1). The organic layer was concentrated and azotropically dried by MTBE at 35-45 °C under vacuum and dark (product was sensitive to light) to give crude desired product as an oil (76.83 g, > 99% HPLC assay yield). KF of the crude product < 350 ppm. An analytical sample for the compound was obtained as light yellow oil by concentration to remove the solvent. 1H NMR (CDCl3, 500 MHz) δ 7.32 (s, 1 H), 6.51 (s, 1 H), 1.51 (s, 9 H). 13C{1H} NMR (CDCl3, 125 MHz) δ 161.3, 138.5, 99.7, 83.2, 27.8. tert-butyl 2-methyl-4-(triisopropylsilyl)but-3-ynoate (2a) and tert-butyl 2-methyl-4-(triisopropylsilyl)buta-2,3-dienoate (2b). A: Sonogashira coupling reaction: To a 500 mL threeneck round bottom (cover with aluminum foil), equipped with an overhead stirrer, a thermocouple, and a nitrogen inlet, was charged tert-butyl 2-iodoacrylate (50.00 g, 0.197 mol, KF < 350 ppm) and toluene (175 mL, 3.5 vol). PdCl2(PPh3)2 (4.14 g, 5.9 mmol) and CuI (1.124 g, 5.9 mmol) were added, respectively. To the resulting mixture was slowly added neat trisoproylsilylacetylene (50.1 mL, 0.216 mol) at 22-27 °C over 3 h (slightly exothermic reaction) at a water batch 22-27 °C. The reaction mixture was stirred at 22-27 °C for 2 h (> 99 A% conversion). The resulting slurry mixture was then filtered to remove TEA salt. The solid was washed with toluene (50 ml x 1, 1 vol). The desired cross-coupling product [tert-butyl 2methylene-4-(trimethylsilyl)but-3-ynoate] in the combined filtrates was assayed against to standard (51.66 g, 85% assay yield). The solution was directly used for the next step without purification needed. B. Reduction: To a 2 L three-neck round bottom, equipped with an overhead stirrer, a thermocouple, and a nitrogen inlet, was charged EtOH (144 mL, 2.4 vol) and 21 wt% NaOEt in EtOH (1.46 mL, 3.90 mmol). The mixture was cooled to -20 °C. NaBH4 (5.16 g, 0.137 mol) was added in one portion. The mixture was stirred at around -20 °C for 10 min. Then, crude tert-butyl 2-methylene-4-(trimethylsilyl)but-3-ynoate 2-2 in toluene solution (340.3 g, 17.56 wt% ) was slowly added at 20 to -10 °C over 1 h. After the addition was complete, the resulting mixture was stirred at -20 to -10 °C for 0.5-1 h (100% conversion). Heptanes (300 mL, 5 vol) was charged at 20 to -10 °C. 2 N HCl (205 mL) was slowly added dropwise at -20 to 0 °C. The resulting reaction mixture was stirred at 15-20 °C for 0.5 h. The reaction mixture was filtered through solka flock (9 g, 15 wt%) to remove palladium black, rinsed with heptanes (60 mL x 1). The filtrates were phase separation. The organic layer was washed with 2 N HCl (120 mL x 1), 10% NaHCO3 (120



mL x 1), water (120 mL x 1). The organic layer was azotropically dried at 45-55 °C and removed EtOH by heptanes and adjusted to around 160 mL. The resulting mixture as a slurry was aged at rt for 0.5-1 h and was filtered. The solid (catalyst from the Sonogashira reaction) was rinsed with heptanes (30 mL, 0.5 vol). The combined filtrates were concentrated by distillation under reduced pressure to low volume (113.9 g, 42.84 wt%, assay yield 94%), which was used for the aldol reaction without purification needed. The water content (KF) for the solution was typical < 500 ppm. An analytical sample for compound 2a was obtained by silica gel chromatography using EtOAc:hexanes as eluent (EtOAc/hexanes = 0-5%) as colorless oil: 1H NMR (CDCl3, 500 MHz) δ 3.31 (q, J = 7.1 Hz, 1 H), 1.46 (s, 9 H), 1.38 (d, J = 7.1 Hz, 3 H), 1.06 (m, 21 H). 13C NMR (CDCl3, 125 MHz) δ 170.3, 106.3, 82.3, 81.2, 34.3, 27.9, 18.6, 18.0, 11.2. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C18H36O2Si 311.2401; found 311.2408. General screening procedure (Table 1) for aldol reaction: A. preparation of LDA or LTMP: To a solution of amines (diisopropylamine or TMP, 3.75 mmol) in solvents (THF, 2MeTHF or MTBE, 10.0 mL) was added dropwise 2.5 M nBuLi in hexanes (3.59 mmol, 1.44 mL) at < -50 °C. The reaction mixture was stirred at -40 to -30 °C for 1 h. B. General procedure for aldol reaction: To a LDA or LTMP or commercial available NaHMDS in indicated solvent (3.59 mmol) was charged additive (0-2.0 equiv) at -78 °C and stirred for 5 min. The substrates 2a/2b (3.12 mmol total) was charged at < -70 °C. The resulting mixture was stirred at < -78 °C for 10-30 min. Then aldehyde 3 (0.740 g, 4.68 mmol) was added dropwise and was stirred at -78 °C. The mixture was slowly warmed to -20 °C over 2.5 h. 15 wt% citric acid aqueous (8.8 g) was added dropwise at -20 °C and the mixture was stirred at 0-10 °C for 10 min. Phase separation, the organic layer was concentrated to give crude products 8, and other 3 diastereomers 7a-b, 7b-a and 7b-b. The ratio of 7a (sum of 8 and 7ab) and 7b (sum of 7b-a and 7b-b) was measured by 1H NMR. Analytical samples for 8, 7a-b, 7b-a and 7b-b were obtained by silica gel chromatography using EtOAc:hexanes as eluent ((EtOAc/hexanes = 0-10%). tert-butyl (R)-2-((R)-((R)-2,2-diethyl-1,3-dioxolan-4yl)(hydroxy)methyl)-2-methyl-4-(triisopropylsilyl)but-3-ynoate (8): 8 was obtained as colorless oil compound. [a]25D – 27.0 (c 0.20, CH2Cl2). 1H NMR (CDCl3, 400 MHz) δ 4.65 (t, J = 7.6 Hz, 1 H), 4.03 (t, J = 7.1 Hz, 1 H), 3.88 (t, J = 8.2 Hz, 1 H), 3.52 (d, J = 12.0, 1 H), 3.49 (d, J = 12.0, 1 H), 1.61 (m, 7 H), 1.47 (s, 9 H), 1.07 (m, 21 H), 0.89 (m, 6 H); 13C{1H} NMR (CDCl3, 100 MHz) δ 171.6, 113.2, 107.0, 85.7, 82.1, 77.2, 75.8, 75.7, 67.2, 47.3, 30.0, 29.4, 27.8, 25.0, 18.5, 11.2, 8.0, 7.9 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C26H48NaO5Si 491.3163; found 491.3159. tert-butyl (S)-2-((S)-((R)-2,2-diethyl-1,3-dioxolan-4yl)(hydroxy)methyl)-2-methyl-4-(triisopropylsilyl)but-3-ynoate (7b-b): 7b-b was obtained as a colorless oil compound. [a]25D – 8.4 (c 0.32, CH2Cl2). 1H NMR (CDCl3, 400 MHz) δ 4.26 (dt, J = 8.2, 6.4 Hz, 1 H), 4.12 (dd, J = 8.2, 6.2 Hz, 1 H), 3.94 (m, 1 H), 2.67 (d, J = 5.6, 1 H), 1.61 (m, 4 H), 1.51 (s, 3 H), 1.47 (s, 9 H), 1.08 (m, 21 H), 0.89 (m, 6 H); 13C{1H} NMR (CDCl3, 100 MHz) δ 171.3, 113.1, 110.0, 106.4, 85.7, 82.2, 76.0, 67.9, 49.8, 29.8, 29.5, 27.8, 21.8, 18.5, 11.2, 8.1, 8.0 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C26H48NaO5Si 491.3163; found 491.3160. tert-butyl (S)-2-((R)-((R)-2,2-diethyl-1,3-dioxolan-4yl)(hydroxy)methyl)-2-methyl-4-(triisopropylsilyl)but-3-ynoate

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(7b-a): 7b-a was obtained as a colorless oil compound. [a]25D – 0.54 (c 0.37, CH2Cl2). 1H NMR (CDCl3, 400 MHz) δ 4.44 (ddd, J = 8.7, 6.4, 2.2 Hz, 1 H), 4.07 (m, 1 H), 3.98 (dd, J = 8.7, 2.2 Hz, 1 H), 3.82 (t, J = 8.4 Hz, 1 H), 2.84 (d, J = 8.7 Hz, 1 H), 1.65 (m, 4 H), 1.47 (s, 9 H), 1.44 (s, 3 H), 1.06 (s, 21 H), 0.90 (m, 6 H); 13C{1H} NMR (CDCl3, 100 MHz) δ 170.4, 113.4, 107.1, 85.0, 81.7, 75.0, 73.4, 67.8, 48.8, 29.9, 27.8, 18.8, 18.6, 11.1, 8.1, 8.0 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C26H48NaO5Si 491.3163; found 491.3166. tert-butyl (R)-2-((S)-((R)-2,2-diethyl-1,3-dioxolan-4yl)(hydroxy)methyl)-2-methyl-4-(triisopropylsilyl)but-3-ynoate (7a-b): 7a-b was obtained as colorless oil compound. [a]25D – 0.48 (c 0.63, CH2Cl2). 1H NMR (CDCl3, 400 MHz) δ 4.35 (m, 1 H), 4.13 (m, 1 H), 4.06 (m, 1 H), 3.95 (t, J = 8.2 Hz, 1 H), 2.25 (d, J = 6.4 Hz, 1 H), 1.62 (m, 4 H), 1.47 (s, 9 H), 1.45 (s, 3 H), 1.07 (m, 21 H), 0.90 (m, 6 H); 13C{1H} NMR (CDCl3, 100 MHz) δ 170.3, 112.3, 106.1, 85.9, 81.9, 76.8, 75.2, 66.1, 48.2, 29.7, 29.4, 27.8, 21.8, 18.6, 11.1, 8.0, 7.9 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C26H48NaO5Si 491.3163; found 491.3163. (3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methyl-3((triisopropylsilyl)ethynyl)dihydrofuran-2(3H)-one (8a). To a 70% TFA aqueous (0.28 mL) was charged dropwise compound 8 (200 mg, 0.427 mmol) at room temperature. The resulting mixture was stirred at rt for 2 h. The mixture was diluted with IPAc (10 mL). The resulting solution was washed with 10% NaHCO3 (5 mL x 2). The organic layer was concentrated to give crude product, which was purified by silica gel chromatography using EtOAc:hexanes as eluent (EtOAc/hexanes = 10-50%) to afford desired product 8a as colorless oil compound. [a]25D – 12.3 (c 0.15, CH2Cl2). 1H NMR (CDCl3, 500 MHz) δ 4.78 (q, J = 4.1 Hz, 1 H), 4.48 (d, J = 3.3 Hz, 1 H), 4.16 (m, 2 H), 3.77 (brs, 1 H), 2.72 (brs, 1 H), 1.52 (s, 3 H), 1.04 (m, 21 H); 13C{1H} NMR (CDCl3, 125 MHz) δ 174.8, 103.4, 86.4, 79.7, 78.0, 60.8, 46.6, 18.5, 16.6, 11.03 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C17H31O4Si 327.1991; found 327.1988. tert-butyl (R)-2-((R)-2,2-diethyl-1,3-dioxolane-4-carbonyl)2-methyl-4-(triisopropylsilyl)but-3-ynoate (9). A. Aldol reaction: To a 5 L three-neck round bottom, equipped with an overhead stirrer, a thermocouple, and a nitrogen inlet, was charged a THF solution of the 2a/2b (508.0 g at 25.0 wt% = 127.0 g assay, 0.409 mol) and THF (110 mL). DMPU (105.0 g, 0.818 mol) was added. Aldehyde 3 (129.4 g, 0.818 mol) in THF (265 mL) was charged. The reaction mixture was degassed by vacuum/nitrogen purges for three times and then cooled to -60 °C. A 1 M solution of NaHMDS in THF (760 mL, 0.76 mol) was charged to the mixture over 2 h while maintaining the internal temperature in the range -55 to -50 °C. After completely addition, the reaction mixture was agitated at -55 to -50 °C for 10-30 min. To another 10 L threeneck round bottom, equipped with an overhead stirrer, a thermocouple, and a nitrogen inlet, was charged a 20 wt% citric acid aqueous solution (2.0 L) and heptane (1.5 L). The mixture was cooled to 0 °C. The bath in the 5 L vessel was transferred into the 10 L quench vessel while maintaining internal temperature below 5 °C. Charge THF (60 mL) to the 5 L vessel and transfer this to the 10 L quench vessel as a rinse. The reaction mixture was warmed to room temperature and then allows the layers to separate. The organic layer was washed with water (890 mL x 3). The organic was azeotropically dried under vacuum to low volume to give crude aldol products 7a/7b (531.0 g, 34.3 wt% of product 8 by NMR assay, 531.0 g x 34.3 wt% = 182.1 g of product 8, 95% yield, ratio of 8 with


Page 7 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


other 3 isomers = 97 : 3). The KF for the solution was typical < 500 ppm. The crude product was diluted with dichloromethane (400 mL). The resulting solution was directly used for the oxidation without purification needed. B. Oxidation: To a 5 L three-neck round bottom, equipped with an overhead stirrer, a thermocouple, and a nitrogen inlet, was charged dichloromethane (2.0 L) and oxalyl chloride (100.2 mL, 1.17 mol). The solution was cooled to -60 °C. To another 1 L three-neck round bottom, equipped with an overhead stirrer, a thermocouple, and a nitrogen inlet, was charged dichloromethane (400 mL) and DMSO (481 mL, 1.95 mol). The mixture was stirred and cooled to ~ 0°C. The DMSO in dichloromethane solution was then added dropwise to above 5 L vessel solution while maintaining the internal temperature at -60 and -50°C. This was followed by dichloromethane (100 mL) wash. The mixture in the 5 L vessel was then stirred at 55°C for 15 min. The above crude aldol products 7a/7b (531.0 g, 34.3 wt% by of product 8 by NMR assay, 531.0 g x 34.3 wt% = 182.1 g of product 8, 0.389 mol) in dichloromethane solution was added dropwise to above 5 L vessel solution while maintaining the internal temperature at -50 and -60°C. This was followed by a dichloromethane (100 mL) wash. After completely addition, the mixture in the 5 L vessel was then stirred at - 55 °C for 2 h. Hünig’s base (388.8 mL, 1.95 mol) was added dropwise to above 5 L vessel solution while maintaining the internal temperature at -50 and -60°C. This was followed by a dichloromethane (100 mL) wash. The reaction mixture was stirred at -55°C for 15 min. and then warmed to 0 °C over 1.5 h. The reaction was quenched by transferring into the vigorously stirred 10 wt% ammonium chloride solution (2.6 L) while maintaining the temperature below 20°C. The phases were separated and the organic layer washed with water (1.2 L x 2). The organic layer was concentrated to a low volume below 30 °C under reduced pressure. The desired keto ester 9 was obtained as a pale orange solution (437.0 g, 40.3 wt% by NMR assay, 437.0 g x 40.3 wt% = 176.1 g of product 9, 97% yield, dr 98:2). The KF for the solution was typical < 500 ppm. The crude product 9 was directly used in the next step with purification needed. An analytical sample for 9 was obtained as colorless oil by silica gel chromatography using EtOAc:hexanes as eluent (EtOAc/hexanes = 0-10%). [a]25D + 84.0 (c 0.22, CH2Cl2). 1H NMR (CDCl3, 400 MHz) δ 5.20 (t, J = 7.4 Hz, 1 H), 4.39 (dd, J = 8.3, 7.6 Hz, 1 H), 3.88 (dd, J = 8.3, 7.3 Hz, 1 H), 1.75 (m, 2 H), 1.66 (m, 2 H), 1.58 (s, 3 H), 1.46 (s, 9 H), 1.08 (m, 21 H), 0.93 (m, 6 H); 13C{1H} NMR (CDCl3, 100 MHz) δ 200.5, 166.7, 115.2, 103.4, 88.5, 83.4, 78.9, 68.2, 54.8, 29.4, 28.8, 27.6, 21.5, 18.6, 11.2, 8.2, 7.9 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C26H46NaO5Si 489.3007; found 489.3008. (3R,4S,5R)-4-hydroxy-5-(hydroxymethyl)-3-methyl-3((triisopropylsilyl)ethynyl)dihydro-furan-2(3H)-one (4). To a 5 L three-neck round bottom, equipped with an overhead stirrer, a thermocouple, and a nitrogen inlet, was charged above crude product 9 (437.0 g, 40.3 wt% by NMR assay, 437.0 g x 40.3 wt% = 176.1 g of product 9, 0.377 mol), followed by THF (1.25 L) and ethylene glycol (92.8 mL). The reaction mixture was stirred at 20 °C. Then, BF3•OEt2 (102.6 mL, 0.943 mol) was added dropwise. The resulting reaction mixture was warmed to 50 °C. The mixture was stirred overnight at 50 °C (17 h total reaction time) at which point it was complete by HPLC and assayed for ~80% yield of 10. The mixture was cooled to -18 °C. To another 2 L three-neck round bottom, equipped with an overhead stirrer, a thermocouple, and a nitrogen inlet, was charged ethanol (1.08 L), followed by NaOEt (2.0 g) and the

solution was cooled to below 9 °C. To this mixture was added NaBH4 (22.8 g, 0.603 mol). The solution was stirred for 30 min. until a colourless solution was obtained. The resulting solution was cooled to 0 °C. Using a positive pressure of nitrogen, the cooled solution of sodium borohydride was added to the above cold solution of 10 in 5 L vessel at a rate such that the internal temperature of did not exceed -10 °C. After completely addition, the reaction mixture was stirred at -10 °C for 0.5 h. The reduction is addition controlled and is typically complete at the end of the addition. The reaction mixture was warmed to ~30 °C and distilled under reduced pressure to ~ 1.1 L. To the vessel was then charged with water (865 mL) and MTBE (450 mL). The contents were mixed well and the phases were separated. The aqueous layer was back to extracted with MTBE (560 mL x 1). The combined organic layers were filtered through sequential 1.0 and 0.1 µm in-line filters to remove the black precipitate. The solid was washed with MTBE (250 mL). The organic solution was washed with 5 wt% NaCl aqueous solution (135 mL x 1) and water (135 mL x 1). The organic solution was distilled to ~ 460 mL under vacuum while maintaining the internal temperature below 40 °C. The organic layer was diluted with heptane (1.08 L). It was then distilled to ~ 420 mL under reduced pressure while maintaining the internal temperature below 40 °C. At this point, 1H NMR indicated the mixture contained only heptane. The heptane distillate contains a significant amount of water and dries the mixture effectively. No particular target for water content was needed to achieve good crystallization. While keeping the internal temperature of the mixture near 40 °C, it was diluted with heptane to bring the volume to ~ 670 mL. The solution was cooled to 30 °C, seeded with 4 (0.10 g), then cooled to 0 °C. The resulting slurry was stirred for 16 h at 0 °C. The crystalline solid was collected by filtration, washed with heptane (160 mL). The solid was dried in a vacuum oven at 45 °C until a constant weight was achieved (total drying time 4 h) to give desired product 4 (92.2 g, 75% yield overall) was as white crystalline solid, m.p. 102.0-102.8°C. [a]25D + 13.1 (c 1.3, CH2Cl2). 1H NMR (CD3CN, 400 MHz) δ 4.26 (ddd, J = 8.5, 4.4, 2.3 Hz, 1 H), 4.00 (dd, J = 8.5, 6.4 Hz, 1 H), 3.90 (m, 2 H), 3.68 (ddd, J = 12.9, 6.6, 4.4 Hz, 1 H), 3.21 (dd, J = 6.6, 5.3 Hz, 1 H), 1.47 (s, 3 H), 1.08 (m, 21 H); 13C{1H} NMR (CD3CN, 100 MHz) δ 174.2, 118.3, 103.6, 88.5, 83.9, 74.9, 60.3, 47.1, 21.4, 18.9, 11.9 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C17H31O4Si 327.1991; found 327.1992. (2R,3S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4methyl-5-oxo-4-((triisopropylsilyl)-ethynyl)tetrahydrofuran-3yl 4-methoxybenzoate (15). To a 10 L three-neck round bottom, equipped with an overhead stirrer, a thermocouple, and a nitrogen inlet, was charged lactone diol 4 (192.3 g, 0.589 mol), followed by 4-dimethylaminopyridine (195.8 g, 1.60 mol). DMF (1.63 L) was then charged and the resulting solution was cooled to 10 °C. TBDMSCl (135.9 g, 0.902 mol) was dissolved in THF (334 mL) and the resulting solution then charged to above 10 L vessel while maintaining internal temperature < 25 °C. The reaction mixture was stirred at 20-25 °C for 0.5 h. The batch was then cooled to 10 °C and 4methoxybenzoyl chloride (109.4 g, 0.641 mol) added while maintaining internal temperature < 25 °C. The reaction mixture was then stirred for 15 h at 20-25 °C. The batch was then cooled to 10 °C. A solution of 5 % NaHCO3 (1.00 L) was charged to above 5 L vessel over at least 5 min. while maintaining internal temperature < 25 °C. MTBE (2.00 L) was then charged. The mixture was stirred thoroughly then allowed to separate and the lower aqueous layer removed. The organic layers were washed with 10% citric acid then 5% sodium chlo-



ride aqueous solution. The aqueous layer from the 5% sodium chloride wash was back extracted with 2-MeTHF (1.00 L x 2). The combined organic layers were then concentrated to approx. 400 mL using partial vacuum, diluted with THF (1.08 L) then concentrated to approx. 800 mL to give the bis-protected lactone 15 in THF solution (657.5 g, 49.4 wt% by HPLC assay, 657.5 g x 49.4 wt% = 324.8 g of product 15, 96% yield, water concentration = 0.15 wt%). The crude product 15 was directly used in the next step with purification needed. An analytical sample for 15 was obtained as colorless oil by silica gel chromatography using EtOAc:hexanes as eluent (EtOAc/hexanes = 0-20%). [a]25D + 51.5 (c 0.54, CH2Cl2). 1H NMR (CDCl3, 500 MHz) δ 8.05 (d, J = 8.9 Hz, 2 H), 6.91 (d, J = 8.9 Hz, 2 H), 5.45 (d, J = 6.7 Hz, 1 H), 4.60 (dt, J = 6.4, 2.8 Hz, 1 H), 4.00 (dd, J = 6.4, 2.8 Hz, 1 H), 3.88 (s, 3 H), 3.85 (m, 1 H), 1.71 (s, 3 H), 0.99 (m, 19 H), 0.88 (s, 9 H), 0.08 (s, 3 H), 0.07 (s, 3 H); 13C{1H} NMR (CDCl3, 125 MHz) δ 172.9, 165.1, 163.9, 132.1, 121.4, 113.6, 101.3, 88.1, 80.8, 74.9, 61.3, 55.5, 45.5, 25.8, 23.2, 18.5, 18.3, 11.1, -5.5, -5.4 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C31H51O6Si2 575.3224; found 575.3248. (2R,3S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4ethynyl-4-methyl-5-oxotetrahydro-furan-3-yl 4methoxybenzoate (16). To a 10 L three-neck round bottom, equipped with an overhead stirrer, a thermocouple, and a nitrogen inlet, was charged the solution of 15 in THF solution (657.5 g, 49.4 wt% of compound 15, water concentration = 0.15 wt%, 0.565 mol), DMF (1.62 L) and acetic acid (155.7 mL). The mixture was stirred at room temperature. Potassium fluoride (78.7 g) and water ( (8.7 g, 0.483 mol) were charged, respectively. The reaction mixture was heated to 80 °C and stirred at 80 °C for 13 h. The system was then cooled to below 20 °C. The reaction was diluted with 2-MeTHF (1.73 L) and then 10% lithium chloride solution (2.57 L) was slowly charged while maintaining the batch temperature below 20 °C. The phases were separated and the aqueous layer back extracted with 2-MeTHF (1.73 L). The two organic layers were combined and washed with 8% sodium bicarbonate solution (0.935 L) and then water (0.935 L). The organic solution was azotropically dried at around 30 °C under reduced pressure to a low volume (398.6 g, 39.7 wt% by HPLC assay, 92% yield). The KF for the solution was typical < 500 ppm. To a new 10 L three-neck round bottom, equipped with an overhead stirrer, a thermocouple, and a nitrogen inlet, was charged DMAP (101.6 g, 0.832 mol) followed by DMF (1.19 L). The resulting mixture was stirred at room temperature to become homogenous solution. A solution of TBDMSCl (117.5 g, 0.780 mol) in 2-MeTHF (80 mL) was added through a dropping funnel while maintaining the temperature at 20-25 °C. The reaction mixture was stirred at 20-25 °C for 16 h. MTBE (1.58 L) was added followed by a solution of 20% NaCl (1.58 L) while maintaining the temperature at 20-25 °C. The mixture was thoroughly agitated and the layers separated. The aqueous solution was back extracted with MTBE (1.58 L x 1). The combined organic layers were washed with 15% citric acid aqueous (0.800 L x 1) and water (1.58 L x 3). The organic solution was then azeotropically dried and concentrated under vacuum while maintaining internal temperature below 35 °C. The resulting crude product was diluted with 2MeTHF (1595.1 g, 13.1 wt% assay by HPLC, 1595.1 g x 13.1 wt% = 209.0 g of compound 16, 96% yield). The KF for the solution was typical < 400 ppm. An analytical sample for 16 was obtained as colorless oil by silica gel chromatography using EtOAc:hexanes as eluent (EtOAc/hexanes = 0-20%). [a]25D + 40.8 (c 0.38, CH2Cl2). 1H NMR (CDCl3, 500 MHz) δ

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8.05 (d, J = 8.9 Hz, 2 H), 6.95 (d, J = 8.8 Hz, 2 H), 5.47 (d, J = 5.9 Hz, 1 H), 4.60 (dt, J = 5.9, 2.7 Hz, 1 H), 4.00 (dd, J = 11.8, 2.6 Hz, 1 H), 3.89 (m, 4 H), 2.39 (s, 1 H), 1.73 (s, 3 H), 0.89 (s, 9 H), 0.09 (s, 3 H), 0.08 (s, 3 H); 13C{1H} NMR (CDCl3, 125 MHz) δ 173.1, 165.2, 164.0, 132.0, 121.3, 113.8, 81.4, 78.4, 74.9, 74.8, 61.4, 55.5, 44.5, 25.8, 23.1, 18.3, -5.4, -5.5 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C22H31O6Si 419.1890; found 419.1896. (2R,3S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4ethynyl-5-hydroxy-4-methyltetra-hydrofuran-3-yl 4methoxybenzoate (17). To a new 10 L three-neck round bottom, equipped with an overhead stirrer, a thermocouple, and a nitrogen inlet, was charged above compound 16 in 2-MeTHF solution ((1595.1 g, 13.1 wt% assay by HPLC, 1595.1 g x 13.1 wt% = 209.0 g of compound 16, 0.499 mol). The solution was cooled to -1 °C. A 30% solution of Li(OtBu)3AlH in THF (845.7 g, 0.998 mol) was slowly charged over 1.5 h while maintaining internal temperature below 3 °C. The resulting mixture was stirred at -1 to 1 °C for 16 h. The batch was cooled to < -5 °C. A solution of 15% citric acid made from citric acid (1.72 L) was charged to the batch over 0.5 h while maintaining internal temperature below 10 °C. The batch was then warmed to 20 °C and the layers separated. The lower aqueous layer was extracted with EtOAc (1.05 L x 2). The combined organic layers were then washed with 15% citric acid prepared each time from citric acid (1.05 L x 2) and water (0.89 L x 1). The organics were then washed with 4.5% sodium bicarbonate (1.05 L x 2) and water (1.05 L x 2). The organic solution was then azeotropically dried by 2-MeTHF and the concentrated product was diluted with THF to obtain 17 in THF solution (1520.5 g, 12.7 wt% assay by HPLC, 1520.5 g x 12.7 wt% = 193.1 g of compound 17, 92% yield). The KF of the solution was typical < 75 ppm. An analytical sample for 17 was obtained as colorless oil by silica gel chromatography using EtOAc:hexanes as eluent (EtOAc/hexanes = 0-20%). [a]25D + 37.7 (c 0.94, CH2Cl2). 1H NMR (CDCl3, 500 MHz) δ 8.06 (dd, J = 10.7, 8.9 Hz, 2 H), 6.94 (dd, J = 8.9, 5.2 Hz, 2 H), 5.38 (d, J = 6.0 Hz, 0.59 H), 5.28 (d, J = 5.2 Hz, 0.41 H), 5.22 (d, J = 9.3 Hz, 0.59 H), 4.34 (m, 1 H), 3.88 (m, 5 H), 3.20 (d, J = 7.6 Hz, 0.41 H), 2.39 (s, 0.41 H), 2.34 (s, 0.59 H), 1.48 (s, 1.23 H), 1.43 (s, 1.77 H), 0.96 (s, 5.31 H), 0.90 (s, 3.69 H), 0.20 (s, 1.77 H), 0.17 (s, 1.77 H), 0.09 (s, 1.23 H), 0.08 (s, 1.23 H); 13C{1H} NMR (CDCl3, 125 MHz) δ 165.7, 165.5, 163.7, 163.6, 131.9, 131.9, 122.1, 121.9, 113.7, 103.1, 102.5, 84.2, 83.0, 82.7, 81.3, 77.7, 76.9, 75.3, 72.0, 62.7, 55.4, 49.0, 48.9, 25.9, 25.8, 22.9, 19.8, 18.4, 18.3, -5.4, -5.5, -5.6 ppm. HRMS (ESI-TOF) m/z: [M + H ]+ Calcd for C22H33O6Si 421.2046; found 421.2042. (2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-4-ethynyl-2-(hydroxymethyl)-4-methyltetrahydrofuran-3-yl 4-methoxybenzoate (19). To a 10 L three-neck round bottom, equipped with an overhead stirrer, a thermocouple, and a nitrogen inlet, was charged above compound 17 in THF solution ((1520.5 g, 12.7 wt% assay by HPLC, 1520.5 g x 12.7 wt% = 193.1 g of compound 17, 0.459 mol). The solution was cooled to -78 °C. To this solution was added NaHMDS (505.1 mL, 0.505 mol) while maintaining internal temperature below -20 °C. Phosphoryl chloride (135.6 g, 0.505 mol) was then slowly added while maintaining internal temperature below -20 °C. The reaction mixture was re-cooled to less than -70 °C. After stirring for 15 min. the batch was sampled to check for conversion to the phosphonate 18 (typical > 97% conversion). Then, bis-TMS uracil 5 (344.8 g, 44.4 wt % of 5 solution in THF, 344.8 g x 44.4% = 153.1 g of 5, kg of active, 0.597 mol) followed by the cautious addition of TMS-OTf ( 254.8 g, 1.15


Page 9 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mol) while maintaining internal temperature below -60 °C. The reaction mixture was stirred for 0.5 h at -60 °C then warmed to -30 °C over 1 h. The reaction mixture was slowly added to a cooled (-2 °C) mixture of EtOAc (2.01 L) and 8 % aqueous NaHCO3 (2.01 L). After phase separation, the aqueous layer was back extracted with EtOAc (1.00 L x 1). The combined organic layers were washed with water (2.01 L x 1). The organic solution was concentrated to around 0.86 L. Methanol (2.01 L) was charged to the vessel and the contents were distilled to a final volume of around 1.41 L. The contents of the vessel were cooled to 20 °C and conc. HCl (46.1 mL) was added. The reaction mixture was stirred at 20 °C for a minimum of 1 h during which time the product precipitated. The crystalline solid was collected by filtration, rinsed with methanol (250 mL). The crystalline solid was dried in a tray dryer at 40 °C with an N2 sweep. The final solid was obtained as a white crystalline solid (128.7 g, 70% overall yield), m.p. 246.5-247.5 °C. [a]25D + 18.3 (c 0.18, THF). 1H NMR (DMSO-d6, 400 MHz) δ 11.51 (S, 1 H), 8.12 (d, J = 8.2 Hz, 1 H), 8.03 (m, 2 H), 7.13 (m, 2 H), 6.26 (s, 1 H), 5.76 (d, J = 8.1 Hz, 1 H), 5.41 (t, J = 5.2 Hz, 1 H), 5.32 (d, J = 7.8 Hz, 1 H), 4.29 (dt, J = 7.7, 2.6 Hz, 1 H), 3.89 (s, 3 H), 3.86 (m, 1 H), 3.68 (m, 1 H), 3.44 (s, 1 H), 1.25 (s, 3 H);. 13C{1H} NMR (DMSO-d6, 100 MHz) δ 164.7, 163.8, 163.1, 150.7, 140.4, 131.8, 121.3, 114.4, 101.9, 89.3, 82.8, 81.5, 76.7, 74.7, 58.8, 55.8, 45.5, 20.1 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C20H21N2O7 401.1349; found 401.1353. Chemistry for the preparation of substrates 13a-e and 13g-h (Table 2) from compound 4 (see Supporting Information for the reaction Schemes). General procedure for the LiAlH(OtBu)3-mediated reduction of lactones to lactols: Lactone (4.00 mmol, 1 equiv) was dissolved in THF (23 mL) and the resulting solution cooled to 0 °C. 1M LiAlH(OtBu)3 in THF solution(8.00 mL, 8.00 mmol, 2.0 equiv) was added dropwise over 10 minutes while maintaining the temperature at 0-5 °C. The solution was agitated at 0-5 °C for 3 hours. To the reaction mixture was charged EtOAc (100 mL), followed by 5% aqueous citric acid (100 mL) below 10 °C. The organic phase was separated and washed brine (100 mL x 2). The organic layer was dried by Na2SO4, evaporated under reduced pressure. The residue was applied onto a silica gel column with EtOAc/petroleum ether (1:10) to afford the corresponding lactol. General procedure for the phosphorylation of lactols to the corresponding phosphates (13a-e and 13g-h): To 50 mL round bottom was charged lactol (0.274 mmol) and THF (1.5 mL) and the resulting solution was cooled to -15 °C. 1 M NaHMDS in THF solution (302 µl, 0.302 mmol, 1.1 equiv) was slowly added at -15 °C. The resulting solution was stirred for 10 minutes. Then, diphenyl phosphorochloridate (81.0 mg, 0.302 mmol, 1.1 equiv) was slowly added over 30 minutes. The reaction mixture was stirred at -15 to -10 °C for about 30 minutes and LCMS analysis showed the conversion of lactol to the corresponding phosphate intermediates. The reaction mixture was then cooled to -30 °C and was directly used for the glycosylation. General procedure for the glycosylation of phosphates 13bh. To a crude solution of 13b-h (0.274 mmol) in THF solution (1.5 mL) was charged 1.4 equiv of 2,4bis((trimethylsilyl)oxy)pyrimidine 5 (98 mg, 0.384 mmol) while maintaining the temperature below -30 °C. The reaction mixture was cooled to -65 °C and 2.3 equiv of trimethylsilyl trifluoromethanesulfonate (140 mg, 0.631 mmol) was added dropwise over 30 minutes at -65 °C. The resulting solution

was then slowly warmed to -10 °C over about 25 hours. The LCMS monitor response is complete. To the reaction mixture was added 5% aqueous sodium bicarbonate (6 mL) followed by ethyl acetate (7 mL); the mixture was then warmed to ambient temperature. The organic layer was separated and the aqueous layer extracted with ethyl acetate (15 mL) and the subsequently combined organic layers were washed with 5% brine (40 mL). The solution was concentrated under vacuum. The crude products were analyzed by HPLC to obtain the ratio of β/α, which showed in the Table 2 in text. Analytical samples for 14c-e-b/14g-h-b and 14c-e-a/14g-h-a were obtained by silica gel chromatography using EtOAc:petroleum ether as eluent (EtOAc:petroleum = 10-80%). (3R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4hydroxy-3-methyl-3 ((triisopropylsilyl)ethynyl)dihydrofuran2(3H)-one (13a). To a 100 mL round bottom flask was charged compound 4 (2.00 g, 6.1 mmol, 1 equiv), DMF (30 mL) and imidazole (1.00 g, 14.6 mmol, 2.4 equiv). The resulting solution was cooled to 15 °C and tertbutyldimethylsilylchloride (1.20 g, 7.93 mmol, 1.3 equiv) was added in one portion. The resulting mixture was stirred for about 2 hours at room temperature. To this reaction mixture was added EtOAc (100 mL) and water (100 mL). The organic layer was separated and washed with 5% brine (100 mL x 2). The solution was concentrated to give desired compound 13a1 (2.20 g, 83% yield ) as a yellow oil. The compound 13a-1 was used direct in the next step without purification. (2R,3S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4methyl-5-oxo-4-((triisopropylsilyl)ethynyl)tetrahydrofuran-3yl benzoate (13a-2). To a 100 mL round bottom flask was charged compound 13a-1 (5.00 g, 11.3 mmol, 1 equiv) and DCM (50 mL). To the resulting solution was added 4dimethylaminopyridine (0.12 g, 1.13 mmol, 0.1 equiv) and triethylamine (1.95 g, 19.2 mmol, 1.7 equiv) followed by benzoyl chloride (2.38 g, 17.0 mmol, 1.5 equiv) at 10 °C. The resulting mixture was stirred overnight at room temperature. To the reaction mixture was charged 5% aqueous NaHCO3 (100 mL) and the mixture stirred for 5 minutes. The organic layer was separated and washed with 5% aqueous citric acid (100 mL), followed by 5% brine (100 mL). The organic solution was concentrated to give desired compound 13a-2 (4.00 g, 65% yield) as yellow oil. [a]25D + 2.2 (c 0.94, CHCl3). 1H NMR (CDCl3, 300 MHz) δ 8.12 - 8.02 (m, 2 H), 7.63 - 7.50 (m, 1 H), 7.41 (t, J = 7.7 Hz, 2 H), 5.46 (d, J = 6.7 Hz, 1 H), 4.59 (dt, J = 6.3, 2.8 Hz, 1 H), 3.99 (dd, J = 11.9, 2.7 Hz, 1 H), 3.84 (dd, J = 11.9, 2.9 Hz, 1 H), 1.70 (s, 3 H), 0.94 (s, 20 H), 0.86 (s, 9 H), 0.079 (s, 3 H), 0.069 (s, 3 H); 13C{1H} NMR (CDCl3, 75 MHz) δ 172.8, 165.4, 133.6, 130.01, 129.0, 128.4, 101.2, 88.3, 77.5, 77.2, 77.0, 76.6, 61.2, 45.6, 25.8, 23.3, 18.4, 18.1, 11.0, -5.4, -5.5 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C30H49O5Si2 545.3118; found: 545.3118. (2R,3S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-5hydroxy-4-methyl-4((triisopropylsilyl)ethynyl)tetrahydrofuran-3-yl benzoate (13a3). 13a-3 was prepared by a standard LiAlH(OtBu)3-mediated reduction procedure as colorless oil (1.31 g, 60% yield). [a]20D + 69.6 (c 0.84, CHCl3). 1H NMR (CDCl3, 300 MHz) δ 8.09 (ddd, J = 14.0, 8.4, 1.5 Hz, 2 H), 7.55 (ddd, J = 7.4, 6.2, 4.5 Hz, 1 H), 7.40 (td, J = 7.5, 7.1, 1.4 Hz, 2 H), 5.36 (dd, J = 20.0, 6.0 Hz, 1 H),5.06 (dd, J = 20.0, 6.0 Hz, 1 H), 4.32 (td, J = 6.0, 2.5 Hz, 1 H), 3.91 - 3.75 (m, 2 H), 1.44 (d, J = 17.6 Hz, 3 H), 1.08-0.86 (m, 30 H), 0.15 (d, J = 7.9 Hz, 3 H), 0.05 (s, 3 H); 13C{1H} NMR (CDCl3, 75 MHz) δ 166.1, 165.6, 133.3, 133.2, 130.0, 129.9, 129.7, 129.5, 128.3, 128.2, 108.1, 104.4,



103.6, 102.2, 89.3, 84.2, 82.8, 82.0, 76.6, 62.7, 62.6, 50.5, 49.9, 29.7, 25.9, 25.8, 25.8, 22.9, 20.0, 18.6, 18.5, 18.5, 18.4, 18.3, 11.2, 11.0, -5.4, -5.5, -5.6 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C30H51O5Si2 547.3275; found: 547.3269. (2R,3S,4R)-5-acetoxy-2-(((tertbutyldimethylsilyl)oxy)methyl)-4-methyl-4((triisopropylsilyl)ethynyl)tetrahydrofuran-3-yl benzoate (13a). To a solution of 13a-3 (200 mg, 0.366 mmol) in THF (2.0 mL) was added 1 M NaHMDS in THF solution (0.402 mL, 0.402 mmol) at -15 °C. The resulting solution was stirred for 10 minutes. Then, acetyl chloride (31.6 mg, 0.402 mmol) was slowly added over 30 minutes. The reaction mixture was stirred at -15 to -10 °C for about 30 minutes and LCMS analysis showed the full conversion of lactol to the corresponding ester intermediates 13a. The reaction mixture was then cooled to -30 °C and was directly used for the glycosylation. (2R,3S,4R,5R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-5(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methyl-4((triisopropylsilyl)ethynyl)tetrahydrofuran-3-yl benzoate (14ab) and (2R,3S,4R,5S)-2-(((tert-butyldimethylsilyl)oxy)methyl)5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-methyl-4((triisopropylsilyl)ethynyl)tetrahydrofuran-3-yl benzoate (14aa). To a crude solution of 13a (0.366 mmol) in THF solution (2 mL) was charged 2,4-bis((trimethylsilyl)oxy)pyrimidine 5 (131 mg, 0.512 mmol) while maintaining the temperature below -30 °C. The reaction mixture was cooled to -65 °C and trimethylsilyl trifluoromethanesulfonate (187 mg, 0.841 mmol) was added dropwise over 30 minutes while maintaining the temperature at -65 °C . The resulting solution was then slowly warmed to 65 °C over about 25 hours. The LCMS monitor response is complete. To the reaction mixture was added 5% aqueous sodium bicarbonate (6 mL) followed by ethyl acetate (7 mL); the mixture was then warmed to ambient temperature. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (15 mL x 1). The subsequently combined organic layers were washed with 5% brine (40 mL x 1). The solution was concentrated under vacuum. The crude products were analyzed by HPLC to obtain the ratio of 14a-b/14a-a = 45:55. Analytical samples for 14a-b and 14a-a were obtained as colorless oil by silica gel chromatography using EtOAc:petroleum ether as eluent (10-60%). Compound 14a-b was obtained as colorless oil (14 mg, 6% yield). [a]25D – 13.6 (c 1.2, CHCl3). 1H NMR (CDCl3, 300 MHz) δ 8.43 (s, 1 H), 8.20 - 8.03 (m, 3 H), 7.58 (td, J = 7.2, 1.4 Hz, 1 H), 7.41 (t, J = 7.7 Hz, 2 H), 6.41 (s, 1 H), 5.71 (d, J = 8.1 Hz, 1 H), 5.44 (d, J = 8.6 Hz, 1 H), 4.42 - 4.31 (m, 1 H), 4.10 (dd, J = 12.1, 2.0 Hz, 1 H), 3.76 (dd, J = 12.1, 1.8 Hz, 1 H), 1.31 (s, 3 H), 1.03 (s, 21 H), 0.91 (s, 9 H), 0.07 (d, J = 2.1 Hz, 6 H); 13C{1H} NMR (75 MHz, CDCl3) δ 165.3, 162.7, 150.1, 140.3, 133.6, 130.0, 129.1, 128.4, 106.1, 102.1, 90.8, 87.0, 81.5, 73.3, 60.4, 48.1, 25.86, 20.3, 18.6, 18.5, 11.1, -5.6, -5.7 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C34H53N2O6Si2 641.3442; found: 641.3434. Compound 14a-a was obtained as colorless oil (47 mg, 20% yield). [a]25D – 10.5 (c 0.55, CHCl3). 1H NMR (CDCl3, 300 MHz) δ 8.45 (s, 1 H), 8.10 - 8.01 (m, 2 H), 7.75 (d, J = 8.2 Hz, 1 H), 7.57 (t, J = 7.4 Hz, 1 H), 7.41 (t, J = 7.7 Hz, 2 H), 6.13 (s, 1 H), 5.69 (d, J = 8.2 Hz, 1 H), 5.55 (d, J = 6.4 Hz, 1 H), 4.43 (dt, J = 6.6, 3.3 Hz, 1 H), 3.93 - 3.76 (m, 2 H), 1.62 (s, 3 H), 0.91 (d, J = 14.4 Hz, 30 H), 0.06 (d, J = 2.8 Hz, 6 H); 13 C{1H} NMR (CDCl3, 75 MHz) δ 165.4, 162.7, 150.2, 140.9, 133.6, 130.0, 129.1, 128.4, 103.4, 101.1, 90.3, 83.1, 62.8, 49.6, 25.8, 24.1, 18.4, 18.3, 11.0, -5.4, -5.5 ppm. HRMS (ESI-

Page 10 of 18

TOF) m/z: [M + H]+ Calcd for C34H53N2O6Si2 641.3442; found: 641.3434. (2R,3S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-5((diphenoxyphosphoryl)oxy)-4-methyl-4((triisopropylsilyl)ethynyl)tetrahydrofuran-3-yl benzoate (13b). 13b was prepared by the general phosphorylation procedure and in one-pot for the next step. Based on the general procedure for the glycosylation of phosphates, 13b was converted to products 14a-a and 14a-b. (2R,3S,4R)-4-ethynyl-2-(hydroxymethyl)-4-methyl-5oxotetrahydrofuran-3-yl benzoate (13c-1). To a 50 mL round bottom flask was charged compound 13a-2 (3.00 g, 5.51 mmol) and THF (30 ml). The resulting solution was cooled to -78 °C and acetic acid (0.827 g, 13.8 mmol) was added followed by TBAF (13.76 ml, 13.76 mmol) over 30 minutes. The resulting solution was stirred and slowly warmed to rt over 20 hours. The LCMS monitor response is complete. The mixture was cooled to 0 °C, quenched with saturated NH4Cl (40 mL). The mixture was extracted with EtOAc (40 mL x 2). The organic layers was dried with anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column with EtOAc/petroleum ether (1:10-1:5) to give compound 13c-1 (1.00 g, 63 % yield) as colorless oil. [a]25D + 48.8 (c 0.77, CHCl3). 1H NMR (CDCl3, 300 MHz) δ 8.14 8.03 (m, 2 H), 7.68 - 7.55 (m, 1 H), 7.53 - 7.40 (m, 2 H), 5.43 (d, J = 7.2 Hz, 1 H), 4.62 (ddd, J = 7.2, 3.3, 2.5 Hz, 1 H), 4.06 (dd, J = 13.1, 2.6 Hz, 1 H), 3.83 (dd, J = 13.1, 3.3 Hz, 1 H), 2.45 (s, 1 H), 1.69 (s, 3 H). 13C{1H} NMR (CDCl3, 75 MHz) δ 172.4, 165.8, 134.0, 130.9, 130.0, 128.7, 128.6, 81.0, 75.2, 74.8, 65.6, 60.4, 44.4, 22.6 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C15H15O5 275.0919; found: 275.0935. (2R,3S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4ethynyl-4-methyl-5-oxotetrahydrofuran-3-yl benzoate (13c-2). To a 50 mL round bottom flask was charged 13c-1 (1.00 g, 3.61 mmol) and DMF (15 mL). The resulting solution was cooled to 5 °C. Imidazole (0.589 g, 8.65 mmol) and tertbutylchlorodimethylsilane (0.707 g, 4.69 mmol) was added, respectively while maintaining the temperature at 5 °C. The resulting solution was stirred and slowly warmed to rt over about 16 hours. The LCMS monitor response is complete. The reaction mixture was quenched with saturated NH4Cl (40 mL) and was extracted by EtOAc (40 mL x 2). The organic layers were dried with anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by a silica gel column with EtOAc/petroleum ether (1:10-1:5) to give compound 13c-2 (1.00 g, 70 % yield) as colorless oil. [a]25D + 55.9 (c 0.70, CHCl3). 1H NMR (CDCl3, 300 MHz) δ 8.13 - 8.03 (m, 2 H), 7.65 - 7.53 (m, 1 H), 7.52 - 7.39 (m, 2 H), 5.48 (d, J = 5.9 Hz, 1 H), 4.60 (dt, J = 5.7, 2.7 Hz, 1 H), 3.99 (dd, J = 11.9, 2.7 Hz, 1 H), 3.86 (dd, J = 11.9, 2.8 Hz, 1 H), 2.38 (s, 1 H), 1.71 (s, 3 H), 0.87 (s, 9 H), 0.06 (d, J = 3.7 Hz, 6 H). 13C{1H} NMR (CDCl3, 75 MHz) δ 173.0, 165.6, 133.7, 129.9, 129.1, 128.7, 128.6, 81.3, 78.3, 75.2, 75.0, 61.4, 44.6, 25.8, 23.2, 18.4, -5.4, -5.5 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C21H29O5Si 389.1784; found: 389.1795. (2R,3S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4ethynyl-5-hydroxy-4-methyltetrahydrofuran-3-yl benzoate (13c-3). 13c-3 was prepared by a standard LiAlH(OtBu)3mediated reduction procedure as colorless oil (0.937 g , 60% yield). [a]25D + 33.7 (c 1.1, CHCl3). 1H NMR (CDCl3, 300 MHz) δ 8.15 - 8.03 (m, H), 7.63 - 7.53 (m, H), 7.46 - 7.40 (m, H), 5.39 (d, J = 6.0 Hz, H), 5.32 - 5.18 (m, H), 4.40 - 4.27 (m, H), 3.94 - 3.75 (m, H), 2.35 (d, J = 12.0 Hz, H), 1.44 (d, J = 13.3 Hz, H), 0.90 (d, J = 17.8 Hz, H), 0.16 (d, J = 8.7 Hz, H),


Page 11 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.06 (d, J = 1.3 Hz, H); 13C{1H} NMR (CDCl3, 75 MHz) δ 166.0, 165.8, 133.4, 133.2, 129.9, 129.8, 129.8, 129.5, 128.5, 128.4, 103.1, 102.5, 84.0, 82.9, 82.7, 81.29, 78.0, 75.4, 72.2, 62.8, 62.7, 49.0, 49.0, 25.9, 25.8, 25.7, 23.0, 19.8, 18.4, 18.4, 5.4, -5.4, -5.6 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C21H31O5Si 391.1941; found: 391.1945. (2R,3S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-5((diphenoxyphosphoryl)oxy)-4-ethynyl-4methyltetrahydrofuran-3-yl benzoate (13c). 13c was prepared by the general phosphorylation procedure and in one-pot for the next step. (2R,3S,4R,5R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-5(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-ethynyl-4methyltetrahydrofuran-3-yl benzoate (14c-β) and (2R,3S,4R,5S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-5-(2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-ethynyl-4methyltetrahydrofuran-3-yl benzoate (14c-α). Based on the general procedure for the glycosylation of phosphates, 13c was converted to products 14c-a and 14c-b. Compound 14c-b was obtained as a white solid (4.0 mg, 3% yield), m.p. 180182 °C. [a]25D -14.4 (c 0.42, CHCl3). 1H NMR (CDCl3, 300 MHz) δ 9.13 (s, 1 H), 8.15 - 8.02 (m, 3 H), 7.65 - 7.52 (m, 1 H), 7.45 (dd, J = 8.3, 6.9 Hz, 2 H), 6.41 (s, 1 H), 5.73 (d, J = 8.2 Hz, 1 H), 5.44 (d, J = 8.2 Hz, 1 H), 4.34 (dt, J = 8.3, 1.9 Hz, 1 H), 4.08 (dd, J = 12.1, 2.1 Hz, 1 H), 3.77 (dd, J = 12.0, 1.9 Hz, 1 H), 2.45 (s, 1 H), 1.31 (s, 3 H), 0.91 (s, 9 H), 0.08 (d, J = 1.7 Hz, 6 H); 13C{1H} NMR (100 MHz, CDCl3) δ 165.3, 163.0, 150.4, 140.2, 133.6, 129.9, 129.1, 128.6, 102.2, 90.2, 82.3, 81.5, 74.4, 73.7, 60.5, 47.1, 25.9, 20.0, 18.4, -5.6, -5.7 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C25H33N2O6Si 485.2108; found: 485.2114. Compound 14c-a was obtained as a white solid (29 mg, 22% yield), m.p. 85.0-86.0 °C. [a]25D – 6.7 (c 0.16, CHCl3). 1 H NMR (CDCl3, 300 MHz) δ 8.10 - 8.00 (m, H), 7.72 (d, J = 8.2 Hz, H), 7.64 - 7.52 (m, H), 7.44 (dd, J = 8.5, 7.0 Hz, H), 6.16 (s, H), 5.73 (d, J = 8.2 Hz, H), 5.55 (d, J = 6.3 Hz, H), 4.42 (dt, J = 6.4, 3.2 Hz, H), 3.85 (qd, J = 11.4, 3.2 Hz, H), 2.43 (s, H), 1.59 (s, H), 0.89 (s, H), 0.06 (d, J = 3.2 Hz, H); 13 C{1H} NMR (100 MHz, CDCl3) δ 165.5, 163.3, 150.5, 140.7, 133.6, 129.8, 129.2, 128.6, 101.1, 90.2, 83.0, 80.2, 63.0, 48.84, 25.9, 23.2, 18.3, -5.4, -5.5 ppm. HRMS (ESITOF) m/z: [M + H]+ Calcd for C25H33N2O6Si 485.2108; found: 485.2126. ((2R,3S,4R)-3-(benzoyloxy)-4-methyl-5-oxo-4((triisopropylsilyl)ethynyl)tetrahydrofuran-2-yl)methyl benzoate (13d-1). To a 100 mL round bottom flask was charged compound 4 (2.00 g, 6.10 mmol, 1 equiv) , DCM (20 mL), 4dimethylaminopyridine (70 mg, 0.60 mmol, 0.1 equiv) and triethylamine (2.10 g, 20.7 mmol, 3.4 equiv). Then, benzoyl chloride (2.56 g, 18.3 mmol, 3.0 equiv) was slowly added while maintaining the temperature at 10 °C. The resulting mixture was stirred overnight at room temperature. 5% Aqueous NaHCO3 (50 mL) was charged at rt and the mixture was stirred for 5 minutes. The solution was extracted by DCM (40 mL x 2). The organic layers were washed with 5% aqueous citric acid (30 mL x 1), followed by 5% brine (30 mL x 1), and then dried by Na2SO4. The organic layers were concentrated under reduced pressure. The residue was purified a silica gel column with EtOAc/ petroleum ether (1:10) to afford compound 13d-1 (2.40 g, 73 % yield) as white solid, m.p. 102.0103.0 °C. [a]25D + 15.6 (c 0.62, CHCl3). 1H NMR (CDCl3, 300 MHz) δ 8.09 (dd, J = 8.2, 1.4 Hz, 2 H), 8.04 - 7.94 (m, 2 H), 7.66 - 7.48 (m, 2 H), 7.41 (dt, J = 13.6, 7.7 Hz, 4 H), 5.46 (d, J = 7.9 Hz, 1 H), 4.93 (ddd, J = 8.1, 5.1, 3.4 Hz, 1 H), 4.75 (dd,

J = 12.5, 3.4 Hz, 1 H), 4.57 (dd, J = 12.6, 5.2 Hz, 1 H), 1.74 (s, 3 H), 1.01 (s, 21 H); 13C{1H} NMR (CDCl3, 75 MHz) δ 171.6, 165.8, 165.3, 133.9, 133.4, 130.1, 129.7, 129.2, 128.5, 128.43, 100.1, 89.1, 75.6, 62.5, 45.3, 23.0, 18.5, 11.0 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C31H39O6Si 535.2516; found: 535.2509. ((2R,3S,4R)-3-(benzoyloxy)-4-ethynyl-4-methyl-5oxotetrahydrofuran-2-yl)methyl benzoate (13d-2). To a 100 mL round bottom was charged compound 13d-1 (3.00 g, 5.60 mmol, 1 equiv) and THF (30 mL). The resulting solution was cooled to 5 °C and acetic acid (0.84 g, 14.0 mmol, 2.5 equiv) was added. 1 M TBAF in THF solution (14.0 ml, 14.0 mmol, 2.5 equiv) was added over 30 minutes while maintaining the temperature at 5 °C. The resulting solution was stirred for about 20 hours at room temperature. The mixture was quenched with NH4Cl saturated aqueous (50 mL), then was extracted with EtOAc(40 mL x 2). The organic layers were dried by Na2SO4, evaporated under reduced pressure. The residue was purified by a silica gel column with EtOAc/petroleum ether (1:10) to afford compound 13d-2 (1.50 g, 71%) as a white solid, m.p. 122.0-123.0 °C. [a]25D + 2.2 (c 1.1, CDCl3). 1H NMR (CDCl3, 300 MHz) δ 8.12 - 8.02 (m, 2 H), 8.02 - 7.92 (m, 2 H), 7.66 - 7.44 (m, 3 H), 7.48 - 7.32 (m, 4 H), 5.45 (d, J = 7.5 Hz, 1 H), 4.92 (ddd, J = 7.8, 5.2, 3.4 Hz, 1 H), 4.72 (dd, J = 12.5, 3.4 Hz, 1 H), 4.55 (dd, J = 12.5, 5.3 Hz, 1 H), 2.47 (s, 1 H), 1.71 (s, 3 H); 13C{1H} NMR (CDCl3, 75 MHz) δ171.6, 165.8, 165.4, 134.0, 133.5, 130.0, 129.7, 129.1, 128.6, 128.5, 75.6, 75.4, 62.6, 44.2, 22.7 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C22H19O6 379.1181; found: 379.1192. ((2R,3S,4R)-3-(benzoyloxy)-4-ethynyl-5-hydroxy-4methyltetrahydrofuran-2-yl)methyl benzoate (13d-3). 13d-3 was prepared by a standard LiAlH(OtBu)3-mediated reduction procedure as a white solid (, 66% yield), m. p. 98.7-100.0 °C. [a]25D – 3.0 (c 0.98, CHCl3). 1H NMR (CDCl3, 300 MHz) δ 8.16 - 7.96 (m, 4 H), 7.65 - 7.30 (m, 6 H), 5.61 - 5.18 (m, 2 H), 4.85 - 4.40 (m, 3 H), 3.92 - 3.13 (m, 1 H), 2.41 (d, J = 26.3 Hz, 1 H), 1.50 (d, J = 13.2 Hz, 3 H); 13C{1H} NMR (CDCl3, 75 MHz) δ166.5, 166.2, 165.9, 165.8, 133.5, 133.2, 133.2, 129.9, 129.9, 129.7, 129.7, 129.6, 129.4, 129.3, 128.50, 128.4, 128.3, 102.6, 102.4, 83.1, 80.6, 79.7, 79.0, 78.3, 77.9, 75.9, 72.6, 65.4, 64.1, 48.8, 47.6, 23.3, 19.2 ppm. HRMS (ESITOF) m/z: [M + Na]+ Calcd for C22H20NaO6 403.1152; found: 403.1158. ((2R,3S,4R)-3-(benzoyloxy)-4-ethynyl-5-hydroxy-4methyltetrahydrofuran-2-yl)methyl benzoate (13d). 13d was prepared by the general phosphorylation procedure and in onepot for the next step. ((2R,3S,4R,5R)-3-(benzoyloxy)-5-(2,4-dioxo-3,4dihydropyrimidin-1(2H)-yl)-4-ethynyl-4methyltetrahydrofuran-2-yl)methyl benzoate (14d-β) and ((2R,3S,4R,5S)-3-(benzoyloxy)-5-(2,4-dioxo-3,4dihydropyrimidin-1(2H)-yl)-4-ethynyl-4methyltetrahydrofuran-2-yl)methyl benzoate (14d-α). Based on the general procedure for the glycosylation of phosphates, 13d was converted to products 14d-a and 14d-b. Compound 14d-b was obtained as a white solid (6.5 mg, 5% yield), m.p. 120.0-121.0 °C. [a]25D + 6.2 (c 1.1, CDCl3). 1H NMR (CDCl3, 300 MHz) δ 9.10 (s, 1 H), 8.05 (m, 4 H), 7.66 - 7.53 (m, 3 H), 7.45 (td, J = 7.6, 1.6 Hz, 4 H), 6.43 (s, 1 H), 5.48 (dd, J = 8.2, 2.1 Hz, 1 H), 5.37 (d, J = 7.6 Hz, 1 H), 4.91 - 4.78 (m, 1 H), 4.65 – 4.49 (m, 2 H), 2.49 (s, 1 H), 1.34 (s, 3 H); 13C{1H} NMR (100 MHz, CDCl3) δ 166.0, 165.6, 162.6, 150.2, 139.3, 133.9, 133.6, 130.0, 129.6, 129.4, 128.8, 128.7, 128.6, 102.5,



90.1, 81.5, 78.9, 75.4, 75.0, 62.00, 46.7, 20.1 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C26H23N2O7 475.1505; found: 475.1507. Compound 14d-a was obtained as a white solid (7.8 mg, 6% yield), m.p. 117.0-118.0 °C. [a]25D – 5.7 (c 0.83, CDCl3). 1 H NMR (CDCl3, 300 MHz) δ 9.23 (d, J = 21.7 Hz, 1 H), 8.04 (ddd, J = 16.4, 8.2, 1.4 Hz, 4 H), 7.74 (d, J = 8.2 Hz, 1 H), 7.66 – 7.51 (m, 2 H), 7.44 (dt, J = 17.2, 7.7 Hz, 4 H), 6.26 (s, 1 H), 5.78 (d, J = 8.2 Hz, 1 H), 5.57 (d, J = 7.3 Hz, 1 H), 4.77 (ddd, J = 7.2, 5.1, 3.7 Hz, 1 H), 4.68 (dd, J = 12.2, 3.8 Hz, 1 H), 4.55 (dd, J = 12.2, 5.1 Hz, 1 H), 2.54 (s, 1 H), 1.64 (s, 3 H), 1.26 (s, 1 H); 13C{1H} NMR (100 MHz, CDCl3) δ 166.1, 165.4, 163.2, 150.5, 140.5, 133.9, 133.4, 129.9, 129.7, 129.3, 128.7, 128.6, 128.5, 101.4, 89.6, 79.5, 78.4, 63.7, 48.54, 29.7, 22.9 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C26H23N2O7 475.1505; found: 475.1518. ((2R,3S,4R)-3-hydroxy-4-methyl-5-oxo-4((triisopropylsilyl)ethynyl)tetrahydrofuran-2-yl)methyl 4methoxybenzoate (13e-1). To a 50 ml round bottom flask was charged compound 4 (3.00 g, 9.19 mmol) and DCM (60 mL). The resulting solution was cooled to 5 °C and N,Ndimethylpyridin-4-amine (0.112 g, 0.919 mmol) and triethylamine (1.40 g, 13.8 mmol) was added, respectively. Then, 4methoxybenzoyl chloride (1.88 g, 11.0 mmol) was charged while maintaining the temperature at around 5 °C. The resulting solution was stirred at 5-10 °C for 16 hours. The LCMS monitor response is complete. The resulting solution was quenched with saturated NH4Cl (40 mL) and was extracted by EtOAc (40 mL x 2). The organic layers were dried by Na2SO4 and was concentrated under reduced pressure. The residue was purified by a silica gel column with EtOAc/petroleum ether (1:10-1:5) to give compound 13e-1 (2.50 g, 59 % yield) as colorless oil. [a]23D + 16.2 (c 0.64, CHCl3). 1H NMR (CDCl3, 300 MHz) δ 8.03 - 7.94 (m, 2 H), 6.97 - 6.88 (m, 2 H), 4.73 (dd, J = 12.5, 2.4 Hz, 1 H), 4.59 - 4.39 (m, 2 H), 3.95 (t, J = 8.5 Hz, 1 H), 3.87 (s, 3 H), 1.59 (s, 3 H), 1.07 (d, J = 2.2 Hz, 22 H); 13C{1H} NMR (CDCl3,75 MHz) δ 171.6, 165.8, 163.8, 131.8, 121.6, 113.8, 99.4, 91.6, 80.5, 75.2, 61.82, 55.4, 46.2, 21.2, 18.5, 17.9, 11.4, 11.0 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C25H37O6Si 461.2359; found: 461.2376. ((2R,3S,4R)-4-ethynyl-3-hydroxy-4-methyl-5oxotetrahydrofuran-2-yl)methyl 4-methoxybenzoate (13e-2). To a 50 ml round bottom flask was charged compound 13e-1 (1.00 g, 2.17 mmol) and THF (10 mL). The resulting solution was cooled to -78 °C and acetic acid (0.163 g, 2.71 mmol) was added followed by 1 M TBAF in THF solution (2.71 mL, 2.71 mmol) over 30 minutes. The resulting solution was slowly warmed to rt over 20 hours. The LCMS monitor response is complete. The mixture was quenched with saturated NH4Cl (40 mL) and extracted with EtOAc (40 mL x 2). The organic layers were dried by Na2SO4 and was concentrated under reduced pressure. The residue was purified by a silica gel column with EtOAc/petroleum ether (1:10-1:5) to give compound 13e-2 (450 mg, 68 % yield) as a white solid, m.p. 140.0-140.5 °C. [a]23D + 29.9 (c 0.54, CHCl3). 1H NMR (CDCl3, 300 MHz) δ 8.03 - 7.92 (m, 2 H), 6.97 - 6.86 (m, 2 H), 4.79 - 4.65 (m, 1 H), 4.61 - 4.47 (m, 2 H), 3.96 (d, J = 7.8 Hz, 1 H), 3.86 (s, 3 H), 2.58 (s, 1 H), 1.59 (s, 3 H); 13C{1H} NMR (CDCl3, 75 MHz) δ 171.5, 166.1, 163.8, 131.9, 121.4, 113.8, 80.3, 77.2, 76.9, 75.6, 61.9, 55.5, 44.9, 20.8 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C16H17O6 305.1025; found: 305.1042. ((2R,3S,4R)-3-((tert-butyldimethylsilyl)oxy)-4-ethynyl-4methyl-5-oxotetrahydrofuran-2-yl)methyl 4-methoxybenzoate (13e-3). To 25 mL round bottom flask was charged compound

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13e-2 (750 mg, 2.47 mmol) and DMF (7.5 mL). The resulting solution was cooled to 5 °C. Imidazole (408 mg, 5.99 mmol) and tert-butylchlorodimethylsilane (505 mg, 3.35 mmol) were added respectively while maintaining the temperature at 5 °C. The resulting solution was slowly warmed to 45 °C over 16 hours. The LCMS monitor response is complete. The resulting solution was quenched with sat NH4Cl (40 mL) and extracted by EtOAc (40 mL x 2). The organic layers were dried by Na2SO4, concentrated under reduced pressure. The residue was purified by a silica gel column with EtOAc/petroleum ether (1:10-1:5) to afford compound 13e-3 (0.82 g, 80 % yield) as colorless oil. [a]24D + 84.5 (c 2.8, CHCl3). 1H NMR (CDCl3, 300 MHz) δ 8.00 - 7.89 (m, 2 H), 6.96 - 6.84 (m, 2 H), 4.68 (dd, J = 12.8, 2.1 Hz, 1 H), 4.56 (ddd, J = 8.3, 4.9, 2.1 Hz, 1 H), 4.37 (dd, J = 12.7, 4.9 Hz, 1 H), 4.01 (d, J = 8.3 Hz, 1 H), 3.84 (s, 3 H), 2.37 (s, 1 H), 1.54 (s, 3 H), 0.91 (s, 9 H), 0.09 (d, J = 6.8 Hz, 6 H); 13C{1H} NMR (CDCl3, 75 MHz) δ 172.3, 165.6, 163.8, 131.8, 121.6, 113.8, 80.1, 75.2, 61.3, 55.5, 45.2, 29.7, 25.6, 21.7, 18.0, -4.2, -4.7 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C22H30O6Si 419.1890; found: 419.1898. ((2R,3S,4R)-3-((tert-butyldimethylsilyl)oxy)-4-ethynyl-5hydroxy-4-methyltetrahydrofuran-2-yl)methyl 4methoxybenzoate (13e-4). 13e-4 was prepared by a general LiAlH(OtBu)3-mediated reduction procedure as colorless oil (1.06 g, 60%). [a]25D + 78.6 (c 0.58, CHCl3). 1H NMR (CDCl3, 300 MHz) δ 8.00 (dq, J = 9.7, 2.9, 2.4 Hz, 2 H), 6.98 6.84 (m, 2 H), 5.31- 5.03 (s, 1 H), 4.64 - 4.48 (m, 1 H), 4.43 4.20 (m, 2 H), 3.96 - 3.82 (m, 4 H), 2.41-2.23 (s, 1 H), 1.37 (s, 3 H), 0.92 (d, J = 2.8 Hz, 9 H), 0.11 (dd, J = 10.9, 4.1 Hz, 6 H); 13C{1H} NMR (CDCl3, 75 MHz) δ 166.2, 166.0, 163.6, 163.5, 131.7, 131.7, 122.3, 122.1, 113.7, 102.4, 102.3, 84.0, 82.3, 81.6, 81.3, 78.52, 75.6, 72.1, 64.2, 63.7, 55.4, 55.4, 49.6, 48.1, 25.8, 25.7, 25.6, 22.4, 19.1, 18.0, -4.3, -4.4, -4.6 ppm. HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C22H32NaO6Si 443.1860; found: 443.1879. ((2R,3S,4R)-3-((tert-butyldimethylsilyl)oxy)-5((diphenoxyphosphoryl)oxy)-4-ethynyl-4methyltetrahydrofuran-2-yl)methyl 4-methoxybenzoate (13e). 13e was prepared by the standard phosphorylation procedure and in one-pot for the next step. ((2R,3S,4R,5S)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-4-ethynyl-3-hydroxy-4-methyltetrahydrofuran-2-yl)methyl 4-methoxybenzoate (14e-α): Based on the general procedure for the glycosylation of phosphates, 13e was converted to products 14e-a and 14e-b. Compound 14e-a was obtained as colorless oil (56.4 g, 40% yield). [a]25D – 38.8 (c 1.2, CHCl3). 1 H NMR (CDCl3, 300 MHz) δ 9.28 (s, 1 H), 8.08 - 7.98 (m, 2 H), 7.77 (dd, J = 8.2, 1.7 Hz, 1 H), 7.04 - 6.94 (m, 2 H), 6.10 (d, J = 1.8 Hz, 1 H), 5.75 (dd, J = 8.4, 2.2 Hz, 1 H), 4.67 (dd, J = 12.4, 2.5 Hz, 1 H), 4.46 (ddd, J = 7.5, 4.3, 2.0 Hz, 1 H), 4.36 (dd, J = 12.5, 4.4 Hz, 1 H), 4.16 - 4.07 (m, 1 H), 3.90 (d, J = 1.7 Hz, 3 H), 2.43 (d, J = 1.7 Hz, 1 H), 1.55 (s, 3 H), 0.95 (d, J = 1.9 Hz, 9 H), 0.14 (dd, J = 10.5, 1.8 Hz, 6 H); 13C{1H} NMR (CDCl3, 100 MHz) δ 165.9, 163.7, 163.3, 150.7, 141.1, 131.7, 121.8, 113.9, 101.1, 89.4, 82.3, 80.4, 62.7, 55.5, 49.3, 25.6, 23.0, 18.0, -4.3, -4.5 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C26H35N2O7Si 515.2213; found: 515.2225. (2R,3S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4methyl-5-oxo-4-((triisopropylsilyl)ethynyl)tetrahydrofuran-3yl 4-methoxybenzoate (13g-1). To a 100 mL round bottom flask was charged 13a-1 (5.00 g, 11.3 mmol, 1 equiv) and DCM (50 mL). The resulting solution was cooled to 10 °C. DMAP (0.137 g, 1.10 mmol, 0.1 equiv) and triethylamine (2.85 g, 28.2 mmol, 2.5 equiv) were added, respectively.


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Then, 4-methoxybenzoyl chloride (2.88 g, 17.0 mmol, 1.5 equiv) was added while maintaining the temperature at 10 °C. The resulting solution was stirred for about 16 hours at room temperature. The solution was quenched with saturated NH4Cl (40 mL), and extracted by DCM (40 mL x 2). The organic layers were combined and dried by Na2SO4, and concentrated under reduced pressure. The residue was purified by a silica gel column with EtOAc/petroleum ether (1:20) to afford 13g-1 (5.10 g, 78 % yield) as a white solid, m.p. 102.5-103.0 °C. [a]25D – 2.0 (c 0.88, CDCl3). 1H NMR (CDCl3, 300 MHz) δ 8.07 - 7.96 (d, 2 H), 6.93 - 6.82 (d, 2 H), 5.42 (d, J = 6.7 Hz, 1 H), 4.57 (dt, J = 6.4, 2.8 Hz, 1 H), 3.97 (dd, J = 11.9, 2.6 Hz, 1 H), 3.83 (s, 3 H), 1.67 (s, 3 H), 0.94 (s, 20 H), 0.85 (s, 9 H), 0.04 (d, J = 3.4 Hz, 6 H). 13C{1H} NMR (CDCl3, 75 MHz) δ 172.8, 165.1, 163.9, 132.1, 121.4, 113.6, 101.3, 88.1, 80.8, 74.8, 61.3, 55.5, 45.5, 25.8, 23.2, 18.5, 18.3, 11.0, -5.4, -5.5 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C31H51O6Si2 575.3224; found: 575.3219. (2R,3S,4R)-4-ethynyl-2-(hydroxymethyl)-4-methyl-5oxotetrahydrofuran-3-yl 4-methoxybenzoate (13g-2). To a 100 mL round bottom flask was charged 13g-1 (5.10 g, 8.90 mmol, 1 equiv) and THF (50 mL). The resulting solution was cooled to 5 °C and acetic acid (1.34 g, 22.3 mmol, 2.5 equiv) was added followed by 1 M TBAF in THF solution (22.3 ml, 22.3 mmol, 2.5 equiv) over 30 minutes. The resulting solution was stirred for about 20 hours at room temperature. The mixture was quenched with saturated NH4Cl (100 mL), then extracted by EtOAc (50 mL x 2). The organic layers were dried by Na2SO4, concentrated under reduced pressure. The residue was purified by a silica gel column with EtOAc/petroleum ether (1:10) to afford compound 13g-2 (1.80 g, 66% yield) as colorless oil. [a]25D – 8.0 (c 1.2, CDCl3). 1H NMR (CDCl3, 300 MHz) δ 8.09 - 7.98 (m, 2 H), 7.00 0 - 6.88 (m, 2 H), 5.42 (d, J = 6.9 Hz, 1 H), 4.62 (dt, J = 6.5, 3.0 Hz, 1 H), 4.05 (dd, J = 13.1, 2.6 Hz, 1 H), 3.86 (s, 4 H), 3.82 (d, J = 3.3 Hz, 1 H), 2.86 (s, 1 H), 1.68 (s, 3 H), 1.25 (s, 0.2 H), 0.85 (d, J = 6.9 Hz, 0.2 H); 13C{1H} NMR (CDCl3, 75 MHz) δ 172.7, 165.6, 164.1, 132.1, 120.9, 113.9, 81.3, 75.1, 74.6, 60.4, 55.54, 44.4, 29.7, 22.5, 21.0, 14.2 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C16H17O6 305.1025; found: 305.1033. (2R,3S,4R)-4-ethynyl-4-methyl-5-oxo-2(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-3-yl 4methoxybenzoate (13g-3). To a 100 mL round bottom flask was charged compound 13g-2 (5.00 g, 16.4 mmol, 1 equiv) and DCM (50 mL). Imidazole (2.36 g, 39.4 mmol, 2.4 equiv) was added, and then followed by TIPSCl (4.20 g, 21.3 mmol, 1.3 equiv). The resulting solution was stirred for about 16 hours at room temperature. The reaction mixture was quenched with saturated NH4Cl (40 mL), and extracted by DCM (40 mL x 2). The organic layers were dried by Na2SO4, and concentrated under reduced pressure. The residue was purified by a silica gel column with EtOAc/petroleum ether (1:20) to afford compound 13g-3 (4.40 g, 58 %) as colorless oil. [a]25D – 2.2 (c 0.99, CDCl3). 1H NMR (CDCl3, 400 MHz) δ 8.07 (d, J = 8.7 Hz, 2 H), 6.97 (d, J = 8.6 Hz, 2 H), 5.57 (d, J = 6.2 Hz, 1 H), 4.64 (dt, J = 5.9, 2.8 Hz, 1 H), 4.13 (dd, J = 11.7, 2.7 Hz, 1 H), 3.99 (dd, J = 11.6, 2.9 Hz, 1 H), 3.90 (s, 3 H), 2.42 (s, 1 H), 1.75 (s, 3 H), 1.09 (d, J = 1.6 Hz, 11 H), 1.07 (s, 7 H); 13C{1H} NMR (CDCl3, 75 MHz) δ173.0, 165.2, 164.0, 132.0, 121.4, 113.8, 81.4, 78.4, 74.8, 74.7, 61.6, 55.5, 44.6, 23.2, 17.8, 11.9 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C25H36O6Si 461.2359; found: 461.2376. (2R,3S,4R)-4-ethynyl-5-hydroxy-4-methyl-2(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-3-yl 4-

methoxybenzoate (13g-4). 13g-4 was prepared by a general LiAlH(OtBu)3-mediated reduction procedure as colorless oil (821 mg, 46% yield). [a]25D + 1.3 (c 0.96, CDCl3). 1H NMR (CDCl3, 300 MHz) δ 8.11 - 7.97 (m, 2 H), 6.91 (dq, J = 8.0, 3.0 Hz, 2 H), 5.40 (dd, J = 37.9, 5.7 Hz, 1 H), 5.16 (dd, J = 21.6, 8.3 Hz, 1 H), 4.40 - 4.27 (m, 1 H), 4.02 - 3.83 (m, 2 H), 3.84 (d, J = 1.3 Hz, 3 H), 2.34 (d, J = 17.3 Hz, 1 H), 1.44 (d, J = 13.4 Hz, 3 H), 1.23 (s, 1 H), 1.23 - 0.91 (m, 20 H), 0.84 (s, 1 H); 13C{1H} NMR (CDCl3, 75 MHz) δ165.7, 165.5, 163.7, 163.6, 132.0, 131.9, 122.2, 122.0, 113.7, 113.7, 103.1, 102.5, 84.2, 83.1, 82.6, 81.3, 75.4, 72.0, 63.1, 62.9, 55.5, 49.1, 49.1, 29.7, 22.9, 19.8, 17.94, 17.9, 17.8, 11.9 ppm. HRMS (ESITOF) m/z: [M + H - H2O]+ Calcd for C25H37O5Si 445.2405; found: 445.2409. (2R,3S,4R)-5-((diphenoxyphosphoryl)oxy)-4-ethynyl-4methyl-2-(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-3-yl 4-methoxybenzoate (13g). 13g was prepared by the general phosphorylation procedure and in one-pot for the next step. (2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)-4-ethynyl-4-methyl-2(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-3-yl 4methoxybenzoate (14g-β) and (2R,3S,4R,5S)-5-(2,4-dioxo-3,4dihydropyrimidin-1(2H)-yl)-4-ethynyl-4-methyl-2(((triisopropylsilyl)oxy)methyl)tetrahydrofuran-3-yl 4methoxybenzoate (14g-α). Based on the general procedure for the glycosylation of phosphates, 13g was converted to products 14g-a and 14g-b. Compound 14g-b was obtained as colorless oil (24.4 , 16% yield). [a]25D + 3.3 (c 1.1, CDCl3). 1H NMR (CDCl3, 300 MHz) δ 8.47 (s, 1 H), 8.09 - 7.99 (m, 2 H), 7.75 (d, J = 8.2 Hz, 1 H), 7.00 - 6.90 (m, 2 H), 6.18 (s, 1 H), 5.74 (dd, J = 8.2, 2.2 Hz, 1 H), 5.62 (d, J = 6.3 Hz, 1 H), 4.44 (dt, J = 6.4, 3.2 Hz, 1 H), 4.07 - 3.89 (m, 2 H), 3.88 (s, 3 H), 2.44 (s, 1 H), 1.23 – 0.90 (m, 20 H); 13C{1H} NMR (CDCl3, 100 MHz) δ 165.1, 163.9, 162.8, 150.2, 140.7, 131.9, 121.6, 113.8, 100.9, 90.2, 83.3, 80.3, 63.4, 55.5, 48.9, 23.2, 17.9, 17.5, 11.9 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C29H41N2O7Si 557.2683; found: 557.2677. Compound 14g-a was obtained as colorless oil (13.7 mg, 9% yield). [a]25D – 2.9 (c 1.1, CDCl3). 1H NMR (CDCl3, 300 MHz) δ 9.65 (s, 1 H), 8.15 - 7.98 (m, 3 H), 6.98 - 6.88 (m, 2 H), 6.42 (s, 1 H), 5.73 (d, J = 8.2 Hz, 1 H), 5.51 (d, J = 8.3 Hz, 1 H), 4.40 - 4.29 (m, 1 H), 4.16 (dd, J = 11.9, 2.1 Hz, 1 H), 3.86 (s, 3 H), 2.45 (s, 1 H), 1.33 (s, 3 H), 1.22 – 0.96 (m, 21 H); 13C{1H} NMR (CDCl3, 100 MHz) δ 165.0, 163.8, 163.3, 150.5, 140.2, 131.9, 121.5, 113.8, 102.1, 90.1, 82.4, 81.6, 74.3, 73.4, 60.6, 55.4, 47.1, 29.6, 20.0, 17.9, 17.8, 11.8 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C29H41N2O7Si 557.2683; found: 557.2683. (2R,3S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4methyl-5-oxo-4-((triisopropylsilyl)ethynyl)tetrahydrofuran-3yl methyl terephthalate (13h-1). To a 100 mL round bottom flask was charged compound 13a-1 (4.40 g, 10.0 mmol, 1 equiv) and DCM (50 mL). The resulting solution was cooled to 10 °C. DMAP (0.12 g, 1.0 mmol, 0.1 equiv) and triethylamine (3.03 g, 30.0 mmol, 3 equiv) were added respectively. Then, methyl 4-(chlorocarbonyl)benzoate (3.00 g, 15.0 mmol, 1.5 equiv) was added while maintaining the temperature at 10 °C. The resulting solution was stirred for about 16 hours at room temperature. The solution was quenched with saturated NH4Cl (40 mL), and extracted by DCM (40 mL x 2). The organic layers were dried by Na2SO4, and concentrated under reduced pressure. The residue was purified by a silica gel column with EtOAc/petroleum ether (1:20) to afford compound 13h-1 (4.50 g, 75 %) as a white solid, m.p. 111.5-113.0 °C.



[a]25D +3.2 (c 1.1, CDCl3). 1H NMR (CDCl3, 300 MHz) δ 8.16 - 8.00 (m, 4 H), 5.46 (d, J = 6.6 Hz, 1 H), 4.59 (dt, J = 6.2, 2.8 Hz, 1 H), 3.97 (dd, J = 11.9, 2.8 Hz, 1 H), 3.92 (s, 3 H), 3.83 (dd, J = 11.9, 2.9 Hz, 1 H), 1.69 (s, 3 H), 0.91 (s, 20 H), 0.83 (s, 9 H), 0.061 (s, 3 H), 0.050 (s, 3 H); 13C{1H} NMR (CDCl3, 75 MHz) δ 172.6, 166.0, 164.6, 134.5, 132.7, 13.0, 129.5, 101.0, 88.4, 80.5, 75.6, 61.2, 52.5, 45.5, 25.8, 23.4, 18.4, 18.3, 11.0, 1.8, -5.4 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C32H51O7Si2 603.3173; found: 603.3189. (2R,3S,4R)-4-ethynyl-2-(hydroxymethyl)-4-methyl-5oxotetrahydrofuran-3-yl methyl terephthalate (13h-2). To a 100 ml round bottom flask was charged 13h-1 (4.00 g, 6.60 mmol, 1 equiv) and THF (50 mL). The resulting solution was cooled to 5 °C and acetic acid (1.8 g, 30 mmol, 4.5 equiv) was added followed by 1 M TBAF in THF solution (30.0 ml, 30.0 mmol, 4.5 equiv) over 30 minutes. The resulting solution was stirred for about 20 hours at room temperature. The mixture was quenched with saturated NH4Cl (100 mL), then extracted by EtOAc (50 mL x 2). The organic layers were dried by Na2SO4, and concentrated under reduced pressure. The residue was purified by a silica gel column with EtOAc/petroleum ether (1:5) to afford 13h-2 (1.90 g, 86% yield) as colorless oil. [a]25D + 7.7 (c 0.99, CDCl3). 1H NMR (CDCl3, 300 MHz) δ 8.16 - 8.01 (m, 4 H), 5.46 (d, J = 6.6 Hz, 1 H), 4.64 (dt, J = 6.3, 2.8 Hz, 1 H), 4.06 (dd, J = 13.0, 2.5 Hz, 1 H), 3.92 (s, 3 H), 3.83 (dd, J = 13.0, 3.2 Hz, 1 H), 2.80 (s, 1 H), 2.43 (s, 1 H), 1.69 (s, 3 H); 13C{1H} NMR (CDCl3, 75 MHz) δ172.6, 166.1, 165.0, 134.7, 132.5, 129.9, 129.8, 81.1, 75.4, 75.2, 60.4, 52.6, 44.5, 22.7 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C17H17O7 333.0974; found: 333.0977. (2R,3S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4ethynyl-4-methyl-5-oxotetrahydrofuran-3-yl methyl terephthalate (13h-3). To a 100 mL round bottom flask was charged 13h-2 (1.70 g, 5.10 mmol, 1 equiv), imidazole (0.830 g, 12.2 mmol,2.4 equiv) and DMF (50 mL). Then, TBDMSCl (1.00 g, 6.63 mmol, 1.3 equiv) was charged to the solution. The resulting solution was stirred for about 16 hours at room temperature. The solution was quenched with saturated NH4Cl (40 mL), and extracted by EtOAc (40 mL x 2). The organic layers were dried by Na2SO4, and concentrated under reduced pressure. The residue was purified by a silica gel column with EtOAc/petroleum ether (1:20) to afford 13h-3 (1.60 g, 70 %) as a white solid, m.p. 100.5-100.9 °C. [a]25D – 1.3 (c 1.2, CDCl3). 1H NMR (CDCl3, 300 MHz) δ 8.17-8.10 (m, 4 H), 5.51 (d, J = 5.7 Hz, 1 H), 4.63 (m, 1 H), 4.03-3.86 (m, 5 H), 2.40 (s, 1 H), 1.74 (s, 3 H), 0.88 (s, 9 H), 0.085 (s, 3 H), 0.073 (s, 3 H); 13C{1H} NMR (CDCl3, 75 MHz) δ 173.0, 165.2, 164.0, 132.0, 121.4, 113.8, 81.4, 78.4, 74.8, 74.7, 61.6, 55.5, 44.6, 23.2, 17.8, 11.9 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C23H31O7Si 447.1839; found: 447.1837. (2R,3S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4ethynyl-5-hydroxy-4-methyltetrahydrofuran-3-yl methyl terephthalate (13h-4). 13h-4 was prepared by a general LiAlH(OtBu)3-mediated reduction procedure as colorless oil (897 mg, 50% yield). [a]25D + 1.9 (c 1.2, CDCl3). 1H NMR (CDCl3, 300 MHz) δ 8.12 (qd, J = 8.4, 4.8 Hz, 4 H), 5.47 5.05 (m, 2 H), 4.42 - 3.28 (m, 4 H), 2.36 (d, J = 6.4 Hz, 1 H), 1.45 (d, J = 13.6 Hz, 3 H), 0.90 (d, J = 17.1 Hz, 9 H), 0.29 0.02 (m, 6 H); 13C{1H} NMR (CDCl3, 75 MHz) δ172.9, 166.0, 164.8, 134.6, 132.8, 129.8, 129.7, 81.1, 75.7, 75.1, 61.4, 52.5, 44.6, 25.8, 23.2, 18.3, -5.4, -5.5 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C23H33O7Si 449.1995; found: 449.1995. (2R,3S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-5((diphenoxyphosphoryl)oxy)-4-ethynyl-4-

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methyltetrahydrofuran-3-yl methyl terephthalate (13h). 13h was prepared by the standard phosphorylation procedure and in one-pot for the next step. (2R,3S,4R,5R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-5(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-ethynyl-4methyltetrahydrofuran-3-yl methyl terephthalate (14h-β) and (2R,3S,4R,5S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-5-(2,4dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-ethynyl-4methyltetrahydrofuran-3-yl methyl terephthalate (14h-α). Based on the general procedure for the glycosylation of phosphates, 13h was converted to products 14h-a and 14h-b. Compound 14h-b was obtained as a white solid (10.4 mg, 7% yield), m.p. 135.0-136.0 °C. [a]25D + 5.6 (c 1.1, CDCl3). 1H NMR (CDCl3, 300 MHz) δ 9.03 (s, 1 H), 8.11 (s, 4 H), 7.71 (d, J = 8.2 Hz, 1 H), 6.16 (s, 1 H), 5.73 (d, J = 8.2 Hz, 1 H), 5.57 (d, J = 6.3 Hz, 1 H), 4.43 (dt, J = 6.3, 3.1 Hz, 1 H), 3.93 (s, 3 H), 3.91 - 3.72 (m, 2 H), 2.44 (s, 1 H), 1.59 (s, 3 H), 1.23 (s, 1 H), 0.88 (s, 9 H), 0.06 (d, J = 3.1 Hz, 6 H); 13C{1H} NMR (CDCl3, 100 MHz) δ 166.1, 164.7, 163.2, 150.4, 140.6, 134.5, 132.9, 129.8, 129.7, 101.1, 90.1, 82.7, 80.1, 63.0, 52.6, 48.9, 25.8, 23.1, 18.3, -5.4, -5.5 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C27H35N2O8Si 543.2162; found: 543.2164. Compound 14h-a was obtained as a white solid (8.9 mg, 6% yield), m.p. 141.0-142.0 °C. [a]25D + 4.7 (c 0.79, CDCl3). 1 H NMR (CDCl3, 300 MHz) δ 8.85 (s, 1 H), 8.18 – 8.02 (m, 5 H), 6.40 (s, 1 H), 5.72 (d, J = 8.2 Hz, 1 H), 5.44 (d, J = 8.1 Hz, 1 H), 4.34 (dd, J = 8.1, 2.0 Hz, 1 H), 4.08 (dd, J = 12.0, 2.0 Hz, 1 H), 3.93 (s, 3 H), 3.77 (dd, J = 12.1, 1.9 Hz, 1 H), 3.46 (s, 1 H), 2.45 (s, 1 H), 1.31 (s, 3 H), 0.90 (s, 8 H), 0.08 (s, 6 H); 13C{1H} NMR (CDCl3, 100 MHz) δ 166.1, 164.6, 162.8, 150.3, 140.1, 134.6, 132.8, 129.8, 129.7, 102.3, 90.1, 82.1, 81.4, 77.4, 77.2, 77.0, 76.6, 74.6, 74.2, 60.5, 52.6, 47.1, 25.8, 20.0, 18.4, -5.6 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C27H35N2O8Si 543.2162; found: 543.2164. 1-((2R,4aR,6R,7R,7aS)-7-ethynyl-2-isopropoxy-7-methyl-2oxidotetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-6yl)pyrimidine-2,4(1H,3H)-dione (1). Approach 1: To a 3 L three-neck round bottom, equipped with an overhead stirrer, a thermocouple, and a nitrogen inlet, was charged ester 19 (123.7 g, 0.3225 mol), methanol (403 mL). The reaction mixture was stirred at room temperature to become homogenous solution. To the resulting solution was slowly charged 22 wt% sodium methoxide in methanol solution (87.09 g, 0.3548 mol) while maintaining the batch temperature at 20-30 °C. After addition, the reaction mixture was stirred at 20-30 °C until the ester 19 was completely consumed. The reaction mixture was adjusted to PH = 5-6 by 5 M solution of phosphoric acid in methanol. Methyl ethyl ketone (MEK) (495 mL) was charged to the reaction mixture and was stirred at room temperature for 0.5 h. The solid (inorganic salts) was then filtered off and the solid was washed with MEK (258 mL). The combined organic solution was concentrated to around 371 mL (total volume) at 50-55 °C. A fresh MEK (371 mL) was charged to the batch. The resulting mixture was concentrated to around 371 mL. The process was repeated until the content of methanol in the batch was < 0.7 mol% against to product 1-((2R,3R,4S,5R)-3ethynyl-4-hydroxy-5-(hydroxymethyl)-3methyltetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione (19a) (when R = H for compound 19) (by NMR). The reaction mixture was seeded and afforded a thick slurry. Then, toluene (672 mL) was charged. The resulting slurry was stirred at 0 °C overnight. The crystalline solid was collected by filtration (good filtration rate) and washed with 5:1 toluene: methanol (495 mL). The crystalline solid was transferred to a tray-drier


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and dried overnight at 40°C to give desired product 19a as a white crystalline solid (78.1 g corrected pure product, 95% yield), m.p. 155.1-155.9 °C. [a]25D + 52.4 (c 0.68, MeOH). 1H NMR (CDCl3, 500 MHz) δ 8.27 (d, J = 8.1 Hz, 1 H), 6.26 (s, 1 H), 5.70 (d, J = 8.1 Hz, 1 H), 4.86 (s, 2 H), 4.02 (m, 2 H), 3.94 (d, J = 9.0 Hz, 1 H), 3.84 (dd, J = 13.1, 2.9 Hz, 1 H), 2.84 (s, 1 H), 1.25 (s, 3 H); 13C{1H} NMR (CDCl3, 125 MHz) δ 166.2, 152.7, 142.7, 102.5, 92.2, 84.9, 84.8, 75.7, 74.7, 60.2, 20.1 ppm. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C12H14N2O5 267.0981; found 267.0986. To a 3 L round bottom was charged 19a (75.65 g, 0.2840 mol) and 4,5-dicyanomidazole (DCI) (86.50 g, 0.7100 mol) and anhydrous acetonile (2.1 L). The resulting solution was azotropically distilled between 35 and 45 °C until the water content was less than 3 mol% at a final total volume ~ 600 mL. The resulting solution was then degassed by 3 cycles of vacuum/N2-refill to remove dissolved oxygen. This solution is referred to below as “Solution A”. In a separate 1 L round bottom was charged anhydrous THF (503 mL) and was degassed by 3 cycles of vacuum/N2-refill. Phosphordiamidite 6 (86.00 g, 0.2840 mol) was charged. This solution is referred to below as “Solution B”. To a 3 L three-neck round bottom, equipped with overhead stirrer, a thermocouple, and nitrogen inlet was charged anhydrous THF (830 mL) and was degassed by 3 cycles of vacuum/N2-refill, then warmed to an internal temperature of 55 °C. Solutions A and B were transferred into the warm THF in the 5 L round bottom from the containers on tared balances using metered pumps through separate vessel inlets. Based on the known mass of each solution, the pumping rate was adjusted (by measuring grams added per minute) such that the total mass of both Solutions A and B were added over approximately 6 h. It is extremely critical that the relative rates of addition of Solutions A and B be identical to maintain the proper stoichiometry in the reaction mixture. Once the addition were finished, the vessel and container that contained Solution A was rinsed with MeCN (50 mL) and this was added into the vessel through the pump used for Solution A with no particular regard for the time of addition. A similar rinse with THF (50 mL) was used for the vessel, container, and pump used for Solution B. After the end of all additions, the reaction mixture was stirred at 55 °C for 13 h to allow full isomerization of the P(III) stereocentre of 20. The reaction mixture was cooled to ~ -15 °C. To a 5 L round bottom, equipped with overhead stirrer, a thermocouple, and nitrogen inlet was charged with iodine (86.50 g, 0.3410 mol) and THF (335 mL) and the suspension stirred until all iodine had dissolved. To this mixture was added pyridine (114.5 mL, 1.420 mol) and water (26.5 mL). The solution was cooled to ~ -20 °C. The above intermediate 20 solution in the 3 L round bottom was transferred into the iodine mixture while maintaining the internal temperature of the iodine mixture between -20 and -15 °C. Post end of addition, the solution was stirred at an internal temperature near -20 °C for 0.5 h. The reaction was sampled and checked by HPLC for full consumption of the intermediate 20. While the solution was kept below 10 °C, residual iodine was quenched by addition of aq. Na2SO3 (52.3 g dissolved in 419 mL of water). The reaction mixture changes color from dark red to bright yellow, and white precipitate forms. The solution should be checked for residual oxidant with test strips. While the solution was warmed to 20 °C, it was diluted with 15% brine (550 mL). This dissolves precipitated solids and leads to a phase separation. The top organic layer was concentrated at < 25 °C under

reduced pressure to around 1 L. The resulting concentrated solution was extracted with dichloromethane (1.6 L) and the aqueous layer back extracted with dichloromethane (150 mL). The combined organic layers were washed twice with 2 N HCl (1.1 L x 1 and 540 mL x 1). The HCl wash layers were combined and back extracted with dichloromethane (530 mL). The dichloromethane phases were combined and washed twice with 4% sodium bicarbonate solution (2.1 L x 1 and 1.1 L x 1). The bicarbonate wash layers were combined and back extracted with dichloromethane (530 mL). The dichloromethane phases were combined and washed with 5% brine (1.1 L). The brine wash layer was back extracted with dichloromethane (530 mL) and the organic phases combined. Silica gel (222 g) was charged to a pressure filter and wetted with dichloromethane (380 mL). The above organic solution was filtered through the silica and then the silica washed with dichloromethane/acetone (85:15 by volume, 3.15 L). The filtrates were combined and concentrated under reduced pressure to around 500 mL. Acetone (700 mL) was charged and the volume reduced to around 500 mL. Product crystallized as a dense white crystalline solid during the distillation with acetone. 2-Propanol (1.0 L) was charged and the volume reduced to around 500 mL. The slurry was stirred at 20°C for 1 h. The solid was collected by filtration, washed with 2-propanol (200 mL) and dried in vacuum at 45°C for 2 days to give desired product 1 as a white crystalline solid (66.87 g, 64% yield overall), m.p. 208.6 °C (decompose). [a]25D + 9.0 (c 1.4, MeOH).1H NMR (DMSO-d6, 500 MHz) δ 11.61 (s, H), 7.83 (d, J = 8.1 Hz, 1 H), 6.31 (s, 1 H), 5.72 (d, J = 8.1 Hz, 1H), 4.67 (m, 3 H), 4.22 (d, J = 9.5 Hz, 1 H), 4.16 (, td, J = 9.5, 5.6 Hz, 1 H), 3.64 (s, 1 H), 1.39 (d, J = 6.2 Hz, 3 H), 1.37 (d, J = 6.2 Hz, 3 H), 1.21 (s, 3 H); 13C{1H} NMR (DMSO-d6, 125 MHz) δ 163.1, 150.8, 140.8, 102.7, 92.3, 82.9, 78.5, 82.3, 73.7, 71.7, 69.0, 44.6, 23.8, 23.5, 19.7 ppm. HRMS (ESITOF) m/z: [M + H]+ Calcd for C15H20N2O7P 371.1008; found 371.1015. 1-((2R,4aR,6R,7R,7aS)-7-ethynyl-2-isopropoxy-7-methyl-2oxidotetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-6yl)pyrimidine-2,4(1H,3H)-dione (1). Approach 2: To a 1 L round bottom, equipped with overhead stirrer, a thermocouple, and nitrogen inlet was charged with benzoate 19 (28.82 g, 0.07200 mol) and MeCN (180 ml). 1H-Imidazole-4,5dicarbonitrile, (DCI, 2.146 g, 0.01817 mol) was added followed by 1-isopropoxy-N,N,N',N'tetraisopropylphosphinediamine 6 (27.42 g, 0.09400 mol) over 10 minutes. The resulting reaction mixture was heated at 55 °C and the slurry became homogenous solution after 15 minutes. It indicated that the reaction was completed. 30 wt% NaOMe in methanol (31.40 g, 0.1740 mol) was added and the reaction mixture was stirred at room temperature for 0.5 h. The mixture was diluted with 10 wt% sodium chloride aqueous (150 ml) and EtOAc (300 mL). The layers were separated. The organics were then concentrated to ~ 100 mL under reduced pressure. The residue was diluted with EtOAc (300 mL) and concentrated by the removal of 300 ml of solvent. MeCN (600 mL) were added and the stream was concentrated to ~ 100 mL by removal of ~ 600 mL of MeCN. The stream was diluted again with MeCN (600 mL) and concentrated to ~ 100 mL solution by removal of ~ 600 ml of MeCN. The final stream was diluted with THF (KF = 17 ppm, 300 mL) to give a final stream of phosphoramidite 21 in around 3 : 10 MeCN : THF in 100% HPLC assay yield. This solution was filtered into a 1 L round bottom, which was equipped with overhead stirrer, a thermocouple, and nitrogen inlet for the next reaction. The resulting filtrate was heated to 55 °C.



1H-Imidazole-4,5-dicarbonitrile (17.01 g, 0.1440 mol) was dissolved in THF : MeCN (6:1; 200 mL, KF = 55 ppm). The resulting solution was added to above the heated solution of over ~ 3 h while maintaining the temperature at ~ 55 °C. After completely addition, the reaction mixture was stirred at ~ 55 °C for 16 h. The reaction mixture was cooled to room temperature, and then filtered. The flask and pad were washed with THF (60 mL). The combined organic filtrates, which contained the intermediate 20 in a typical 95:5 dr were added to a 1L dropping funnel - Solution A. A solution of iodine (21.93 g, 0.08640 mol), pyridine (28.48 g, 0.3600 mol), water (6.485, 0.3600 mol) in THF (120 mL) was prepared in a 2 L Radley's reactor-ready vessel. This solution was cooled to < -15 °C - Solution B. Solution A was added into Solution B while maintaining the internal temperature of Solution B < -15 °C. Addition required ~ 65 minutes. The reaction mixture was stirred at -15 °C for 0.5 h after the end of addition. HPLC showed that the reaction was complete. The reaction mixture was warmed to -10 °C, and then added ~ 1 M Na2SO3 (9.16 g dissolved in 72 mL water). 15 wt% Sodium chloride solution (132 mL) was added, then warmed contents to > 10 °C. Drained from vessel into separatory funnel, then removed bottom aqueous layer. Top organic layer was assayed by HPLC against standard to give desired product 1 (20.39 g, 76% HPLC yield from 21). The organic solution was charged to a 500 mL round bottom, which equipped with overhead stirrer, a thermocouple, and nitrogen inlet. The solution was concentrated under reduced pressure to a final volume of ~ 82 mL. The solution was seeded and stirred at 35 °C for 0.5 h while thin slurry developed. IPA (347 mL) was slowly added over 1.5 h. The resulting slurry was distilled under reduced pressure to a final volume ~ 204 mL. The resulting slurry was stirred at 45 °C for 1 h, and then cooled to 18 °C overnight. The crystalline solid was collected by filtration, rinsed with IPA (41 mL), dried in oven under vacuum at 50 °C overnight to give API (1) (18.60 g, 69% isolated yield overall from 19). Both 1H NMR and 13 C NMR data matched the first approach.

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X-ray data collections and experimentation, Fugui Zhang, Fujun Di, Kai Hou, Haitao Guo, Xianghui Wen, Yong Wang (WuXi AppTec, Inc.) and Shiping Ye, Zhengou Liu (Pharmaron, Inc.) for experimentation, Kevin Maloney, David Tschaen, and Steven Oliver (Merck & Co., Inc., Rahway, NJ, USA) for helpful discussion and/or proofreading.

REFERENCES

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Copies of 1H and 13C NMR spectra of compounds tert-2iodoacrylate, 2a, 8, 7b-a, 7a-b, 7b-b, 9, 4, 15, 16, 17, 19, 1, 13a1 to 4, 13c1 to 2, 13d1 to 3, 13e1 to 4, 13g1 to 4, 13h1 to 4, 14a-a and b, 14c-a and b, 14d-a and b, 14e-a and b, 14g-a and b, 14ha and b; Schemes for the preparation of 13a-e and 13g-h; Computational details of compounds 13e and 13f; X-ray crystallography data of compound 4. (PDF)

AUTHOR INFORMATION Corresponding Author * Email: [email protected]; [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We gratefully acknowledge Richard Ball, Justin Newman, Joe Lynch (Merck & Co., Inc., Rahway, NJ, USA) for single crystal


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Biomol. Chem., 2014, 12, 1184-1197. (c) Christensen, H. M.; Oscarson, S.; Jensen, H. H. Common side reactions of the glycosyl donor in chemical glycosylation. Carbohydr Res. 2015, 408, 51-95. (d) Kageyama, M.; Nagasawa, T.; Yoshida, M.; Ohrui, H.; Kuwahara, S. Enantioselective Total Synthesis of the Potent AntiHIV Nucleoside EfdA. Org. Lett. 2011, 13, 5264-5266. (e) McLaughlin, Mark; Kong, Jongrock; Belyk, Kevin M.; Chen, Billy; Gibson, Andrew W.; Keen, Stephen P.; Lieberman, David R.; Milczek, Erika M.; Moore, Jeffrey C.; Murray, David; Peng, F.; Qi, J.; Reamer, R. A.; Song, Z. J.; Tan, L.; Wang, L.; Williams, M. J. Enantioselective Synthesis of 4′-Ethynyl-2fluoro-2′-deoxyadenosine (EFdA) via Enzymatic Desymmetrization. Org. Lett. 2017, 19, 926-929. (f) Ding, F.; Ishiwate, A.; Ito Y. Stereodivergent Mannosylation Using 2- O-( ortho-Tosylamido)benzyl Group. Org. Lett. 2018, 20, 4833-4837. Selected references for the glycosylation using the diphenyl phosphate as leaving group: (a) Arihara, R.; Kakita, K.; Yamada, K.; Nakamura, S.; Hashimoto, S. Synthesis of the Tetrasaccharide Repeating Unit from Acinetobacter baumannii Serogroup O18 Capitalizing on Phosphorus-Containing Leaving Groups. J. Org. Chem. 2015, 80, 4278-4288. (b) Hashimoto, Y.; Tanikawa, S.; Saito, R.; Sasaki, K. β-Stereoselective Mannosylation Using 2,6-Lactones. J. Am. Chem. Soc. 2016, 138, 14840-14843. (c) Nakamura, S.; Tsuda, T.; Suzuki, N.; Hashimoto, S. Direct and Stereoselective Synthesis of 2-Azido-2deoxy-β-D-mannosides Using the Phosphate Method. Heterocycles 2009, 77, 843-848. (d) Tsuda, T.; Nakamura, S.; Hashimoto, S. A highly stereoselective construction of 1,2-trans-β-glycosidic linkages capitalizing on 2-azido-2-deoxy-d-glycosyl diphenyl phosphates as glycosyl donors. Tetrahedron 2004, 60, 10711-10737. (e) Plante, O. J.; Palmacci, E. R.; Seeberger, P. H. Formation of β-Glucosamine and β-Mannose Linkages Using Glycosyl Phosphates. Org. Lett. 2000, 2, 3841-3843. Selected references for the glycosylation of 2’-deoxynucleosides through 3’-directing and 5’-directing group: (a) Liu, Z.; Li, D.; Yin, B.; Zhang, J. Highly stereoselective synthesis of 2′-deoxy-β-ribonucleosides via a 3′-(N-acetyl)-glycyl-directing group. Tetrahedron Lett. 2010, 51, 240-243. (b) Sugimura, H.; Osumi, K.; Kodaka, Y.; Sujino, K. Stereoselective Synthesis of 2'-Deoxy-.beta.-D-threo-pentofuranosyl Nucleosides by the NBS-Promoted Coupling Reaction of Thioglycosides with Silylated Heterocyclic Bases. J. Org. Chem. 1994, 59, 7653-7660. (c) Huang, W.; Zhou, Y.-Y.; Pan, X.-L.; Zhou, X.-Y.; Lei, J.-C.; Liu, D.-M.; Chu, Y.; Yang, J.-S. Stereodirecting Effect of C5-Carboxylate Substituents on the Glycosylation Stereochemistry of 3-Deoxy-d-manno-oct-2-ulosonic Acid (Kdo) Thioglycoside Donors: Stereoselective Synthesis of α- and βKdo Glycosides. J. Am. Chem. Soc. 2018, 140, 3574-3582. Sherer, E. C.; Lee, C. H.; Shpungin, J.; Cuff, J. F.; Da, C.; Ball, R.; Bach, R.; Crespo, A.; Gong, X.; Welch, C. J. Systematic Approach to Conformational Sampling for Assigning Absolute Configuration Using Vibrational Circular Dichroism. J. Med. Chem. 2014, 57, 477-494. 1

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(a) Becke, A. D. Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 1993, 98, 5648-5652. (b) Lee, C. T.; Yang, W. T.; Parr, R. G. Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys. Review B 1988, 37, 785-789. (a) Marenich, A. V.; Cramer, C. J.; Truhlar, D. G. Universal solvation model based on solute electron density and on a continuum model of the solvent defined by the bulk dielectric constant and atomic surface tensions. J. Phys. Chem. B 2009, 113, 6378-6396. (b) Grimme, S.; Ehrlich, S.; Goerigk, L. Effect of the damping function in dispersion corrected density functional theory. J. Computational Chem. 2011, 32, 1456-1465. (c) Zhao, Y.; Truhlar, D. G. A new local density functional for maingroup thermochemistry, transition metal bonding, thermochemical kinetics, and noncovalent interactions. J. Chem. Phys. 2006, 125, 194101. (d) Chai, J. D.; Head-Gordon, M. Long-range corrected hybrid density functionals with damped atomatom dispersion corrections. Phys. Chem. 2008, 10, 6615-6620. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J., J.A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision D.01, Gaussian, Inc.: Wallingford, CT, 2009. The a/b ratio of compounds 17 and 18 didn’t impact the diastereoselectivity of the glycosylation. Commercial available compound; for its synthesis, see: (a) Babkov, D. A.; Chizhov, A. O.; Khandazhinskaya, A. L.; Corona, A.; Esposito, F.; Tramontano, E.; Seley-Radtke, K. L.; Novikov, M. S. An Efficient Route to Novel Uracil-Based DrugLike Molecules. Synthesis, 2015, 47, 1413-1422. (b) Battisti, U. M.; Sorbi, C.; Quotadamo, A.; Franchini, S.; Tait, A.; Schols, D.; Jeong, L. S.; Lee, S. K.; Song, J.; Brasili, L. Diastereoselective Synthesis of (1,3-Dioxan-4-yl)pyrimidine and Purin Nucleoside Analogues. Eur. J. Org. Chem. 2015, 1235–1245. (c) Li, N.-S.; Piccirilli, J. A. Efficient synthesis of 2′-Cα-aminomethyl-2′-deoxynucleosides. Chem. Comm. 2012, 48, 8754-8756. (d) Gagnieu, C. H.; Guiller, A.; Pacheco, H. Carbohydr. Chem. 1988, 180, 233-242. (a) Broeders, N. L. H. L.; Van Der Heiden, A. P.; Koole, L. H.; Kanters, J. A.; Schouten, A. 2′-O-Methyl-cis-adenosine 3′,5′-cyclic methyl monophosphate, a new model system for cAMP. Aspects of structure and reactivity. Can. J. Chem. 1993, 71, 855. (b) Broeders, N. L. H. L.; Koole, L. H.; Buck, H. M. A 400- and 600-MHz proton NMR conformational study on nucleoside cyclic 3',5' Pv-TBP systems. Conformational transmission induces diequatorial orientation of the 3',5'dioxaphosphorinane ring in a nonchair conformation. J. Am. Chem. Soc. 1990, 112, 7475. (c) Reddy, P. G.; Chun, B. –K.; Zhang, H. –R.; Rachakonda, S.; Ross, B. S.; Sofia, M. J. Stereoselective Synthesis of PSI-352938: A β-d-2′-Deoxy-2′-αfluoro-2′-β-C-methyl-3′,5′-cyclic Phosphate Nucleotide Prodrug for the Treatment of HCV. J. Org. Chem. 2011, 76, 37823790. The oxidant has been previously used in the oligonucleotide synthesis, see: Beaucage, S. L. In Current Protocols in Nucleic Acid Chemistry; Beaucage, S. L., Bergstrom, D. E., Glick, G. D., Jones, R. A., Eds.; John Wiley & Sons: New York, NY, 2001; Chapter 3.

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