Synthesis of an α-Amylase Inhibitor: Highly Stereoselective

Nov 14, 2014 - Here, we describe the efficient synthesis of α-amylase inhibitor 1. To introduce the most expensive C ring unit at a late stage in the...
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Synthesis of an α‑Amylase Inhibitor: Highly Stereoselective Glycosidation and Regioselective Manipulations of Hydroxyl Groups in Carbohydrate Derivatives Tsuyoshi Ueda,* Masaki Hayashi, Yutaka Ikeuchi, Takumi Nakajima, Eiji Numagami, and Satoshi Kobayashi Process Technology Research Laboratories, Pharmaceutical Technology Division, Daiichi Sankyo Co., Ltd., 1-12-1 Shinomiya, Hiratsuka, Kanagawa 254-0014, Japan S Supporting Information *

ABSTRACT: Here, we describe the efficient synthesis of α-amylase inhibitor 1. To introduce the most expensive C ring unit at a late stage in the synthesis, we developed 1,2-cis-O-glycosidation of AB and C ring intermediates. Taking advantage of the effect of non-neighboring protecting groups, reaction solvents, and temperature for the glycosidation led to high stereoselectivity and high yield of the 1,2-cis-glycoside product bearing the API skeleton. We also explored protection and deprotection methods for regioselective manipulation of hydroxyl groups in A and B ring intermediates. One-pot benzylation of the 2,3-hydroxyl groups of D-glucose under phase-transfer conditions and regioselective anomeric deacetylation with N-methylpiperazine were developed for the syntheses of A, B, and AB ring intermediates. Thus, the efficiency of the process was dramatically improved. The raw material cost of API was reduced to approximately one-third that of the original route, and the total process was decreased by six steps.

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INTRODUCTION (2R,3R,4R)-4-Hydroxy-2-(hydroxymethyl)pyrrolidine-3-yl 4-O(6-deoxy-α-D-glucopyranosyl)-α-D-glucopyranoside (1) is an αamylase inhibitor designed by Daiichi Sankyo to suppress postprandial hyperglycemia and excessive insulin secretion for type II diabetes (Figure 1).1,2 In the same therapeutic category,

ful general method for 1,2-cis glycosidation has been developed despite considerable progress and extensive efforts toward glycosidation techniques.5−7 Second, methods for the regioselective manipulation of hydroxyl groups in carbohydrate derivatives are required for the efficient synthesis of API.8 Differentially and/or partially protected intermediates are key feedstocks for the glycosidation reaction. The synthesis of these compounds is complicated by the several steps required conventional synthetic strategies. In addition, neighboring and even non-neighboring protecting groups often play important roles in modulating the reactivity of glycosyl donors and acceptors as well as directing the stereochemistry of glycosidation reactions. Therefore, selecting suitable protecting groups is critical for success.5−7 First-Generation Method. A practical method applicable to the multikilogram synthesis of 1 is required to support preclinical and early clinical studies. At this stage, a medicinal chemistry route is expected to be sufficiently reliable for the bulk supply. Therefore, we initially focused on modification of the original method. The first-generation method for 1 based on a medicinal chemistry route is shown in Scheme 1. 1,2,3,4Tetrabenzoyl-6-deoxy-β-D-glucopyranose (2)9−11 and 2,3-Odibenzyl-4,6-O-benzylidene-D-glucopyranose (3)12 as A and B units were prepared from D-glucose (5) over five and six steps, respectively, by modifying reported procedures. C ring units, N,5-O-carbonyl-2-O-benzoyl-1,4-deoxyimino-D-arabinitol (4), were synthesized from diacetone-D-glucose (6) over 15 steps as described previously.13 Schmidt glycosidation,14 which is a key step of this synthesis, was performed by using trichloroacetimidate 7 and C unit 4 in

Figure 1. Structure of α-amylase inhibitor 1.

α-glycosidase inhibitors3 such as voglibose and acarbose are already widely used in clinical practice. However, they often induce gastrointestinal tract-related symptoms such as diarrhea and flatulence as side effects.4 It is preferable to inhibit αamylase specifically at an early step of sugar metabolism. Accordingly, there is considerable interest in developing αamylase inhibitors that do not cause side effects in order to enhance patient satisfaction. To support the development and potential commercial production of α-amylase inhibitor 1, a scalable cost-effective synthesis that allows timely and cost-effective large-scale production is required. This represents a significant challenge because of the complexity of the trisaccharide structure comprising 6-deoxy-D-glucose (A ring), D-glucose (B ring), and 1,4-deoxyimino-D-arabinitol (C ring). The stereoselective construction of the 1,2-cis-O-glycoside bond is expected to be especially challenging. In contrast to a reliable methodology for 1,2-trans-O-glycoside formation based on neighboring-group participation in 2-O-acyl-protected glycosyl donor, no success© XXXX American Chemical Society

Received: September 21, 2014

A

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Scheme 1. First-generation method for synthesizing 1

Scheme 2. New synthesis strategy for α-amylase inhibitor 1

the presence of a catalytic amount of trimethylsilyl trifluoromethanesulfonate (TMSOTf) to afford 8 with moderate selectivity (α/β = 92:8). Deprotection of benzylidene acetal and the following toluoylation gave a BC unit 9 for which crystallization was found to be effective for removing the undesired β-isomer. 1,2-trans-Glycosidation of A unit 2 and BC unit 9 with a stoichiometric amount of TMSOTf proceeded selectively because of the neighboring-group participation by benzoyl group. Hydrolysis of benzoyl groups and cyclic carbamate afforded the final intermediate 11 with a yield of 71% from 9. Finally, deprotection of benzyl ether in hydrogenolysis conditions afforded 1 with a yield of 93%. Although this first-generation process proved suitable for the manufacturing of medium-sized lots of 1 (up to 45 kg), several drawbacks would be encountered when running this process at a commercial production scale, including (1) the large number of steps (total 33 steps), (2) linear route with low overall yield (22%) in the synthesis of C unit 4, (3) moderate selectivity of 1,2-cis-glycosidation, (4) introduction of the most expensive C unit 4 at an early stage of the process, resulting in poor costefficiency, (5) the use of a stoichiometric amount of expensive TMSOTf in the formation of the 1,2-trans-glycoside bond, and (6) tedious protection and deprotection steps in the syntheses of each units. These issues more than tripled the manufacturing cost of API compared to the target cost. Herein, we will detail the development of a stereoselective synthesis of 1 to overcome above issues.

trichloroacetimidate, which is easily activated by a catalytic amount of Lewis acid such as TMSOTf, is the most popular and reliable.5−7 Therefore, we applied this methodology to the 1,2-cis-glycoside-bond formation as well as the first-generation method. The ether protecting group at the 2-O position of the donor is well-known to favor the formation of 1,2-cis-glycoside by virtue of the anomeric effect. Therefore, benzyl ether was selected as the protecting groups at the 2 and 3 positions of the B unit. In this approach, the particularly high stereoselectivity in the reaction is especially desirable, because the undesired 1,2-transglycoside product formed at a late stage in the synthesis would complicate the purification and quality control of API. 1,2-cis-Glycosidation of AB and C Units: Selection of the Protecting Groups. Non-neighboring protecting groups

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RESULTS AND DISCUSSION Our synthetic strategy is shown in Scheme 2. In this strategy, the most expensive C ring unit is introduced at a late stage in the synthesis, in which 1,2-cis-glycosidation of AB and C units are a key step. Among the various glycoside-bond formation developed to date, Schmidt glycosidation based on O-glycosyl B

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Table 1. Effects of protecting groups on the selectivity of cis-glycosidation

a

entry

donor

acceptor

temp. (°C)

α/β ratioa

conv.b (%)

1 2 3 4 5 6 7 8 9

12a 12b 12c 12d 12c 12c 12c 12c 12c

4 4 4 4 4 13a 13b 14a 14b

−20 −20 −20 −20 −40 −40 −40 −40 −40

84:16 84:16 89:11 88:12 94:6 93:7 89:11 90:10 93:7

83 64 86 87 97 (76)c 76 85 92 99 (84)c

Determined by HPLC. bRatio of the product to the remaining donor in HPLC analysis. cHPLC yield.

Table 2. Solvent study of cis-glycosidation

a

entry

solvent

α/β ratioa

yielda (%)

1 2 3 4 5 6 7 8 9 10 11

CH2Cl2/i-Pr2O (3:2) toluene xylene chlorobenzene anisole CPME TBME DME toluene/Bu2O (3:2) toluene/dioxane (9:1) toluene/DME (9:1)

93:7 94:6 96:4 93:7 91:9 96:4 97:3 96:4 94:6 89:11 96:4

84 82 70 85 75 75 63 76 34 71 86

Determined by HPLC.

of intermediates are crucial, because they could affect not only the reactivity and selectivity of the glycosidation reaction, but also the synthetic difficulty of each unit. Therefore, we initially tested the glycosidations of acceptor 4 and donors 12a−d with different protecting groups in order to determine the best structure for each unit (Table 1). Interestingly, the acetyl group at the 6-position of the B unit (entries 3 and 4) significantly enhanced the selectivity compared to benzoyl and toluoyl groups (entries 1 and 2). It can be hypothesized that an acetyl group at the 6-position of glucosyl donor shields the β face of

the sugar ring, thus promoting high selectivity in glycosidation reaction; however, this long-range assistance is not wellestablished in glycosidation chemistry.5−7 Reducing the reaction temperature to −40 °C dramatically improved conversion and selectivity to afford the corresponding 1,2-cisglycoside product with an α/β ratio of 94:6 (entry 5). On the basis of these results, 12c was selected as a suitable donor. Next, acceptors 13a,b and 14a,b were tested for the glycosidation with 12c (entries 6−9). Unexpectedly, the protecting groups of the acceptor also significantly affected the reaction. Only NC

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Scheme 3. Retrosynthesis of AB and C units

Scheme 4. Synthesis of B unit 17aa

a

Compounds in brackets were used for the next step without isolation.

comprising toluene and an ether-type solvent (entries 9−11). The optimal solvent was toluene/DME (9:1 v/v); the yield increased significantly to 86% as the α/β ratio remained high (96:4) (entry 11). Development of the Synthesis of AB and C Units. We subsequently explored the efficient synthesis of the AB and C units (12c and14b) in which the protecting groups must be manipulated such that each can be selectively introduced or removed during the synthetic route. Unlike the first-generation method using a stoichiometric amount of expensive TMSOTf, catalytic Schmidt glycosidation was utilized to form the 1,2trans-glycoside bond in order to reduce the required amount of expensive TMSOTf. AB ring unit 12c was synthesized from the corresponding A unit 16 and B unit 17a, both of which can be prepared from inexpensive methyl-α-glucopyranose (18) by modifying the precedent procedures. The C unit 14b can prepared from 19, the synthesis of which from diacetone-glucose (6) has been reported and applied in the first-generation method.13 The synthesis of 19 from D-xylose (20) was attempted in pursuit of a more efficient method for the C unit (Scheme 3).

methoxylcarbonyl-2,5-O-dibenzoyl-1,4-deoxyimino-D-arabinitol (14b) demonstrated excellent reactivity comparable to 4. Meanwhile, 14b was easier to prepare than 4 bearing an oxazolidinone ring, which allows 1 step to be saved in the preparation of the C unit. Therefore, it was selected as a suitable C ring intermediate. 1,2-cis-Glycosidation of 12c and 14b: Solvent Study. The reaction solvent in 1,2-cis-glycosidation is well-known to be one of the most important factors affecting anomeric stereoselectivity. In particular, ether-type solvents generally participate in the glycosidation processes; as a result, the equatorial carbocation is preferentially formed, leading to axial glycosidic bond formation.7 Therefore, we subsequently examined the effect of solvent on the glycosidation of 12c and 14b at −40 °C (Table 2). As expected, ether-type solvents such as CPME, TBME, and DME demonstrated high stereoselectivity (α/β ratio = 96:4 to 97:3). However, a significant amount of unreacted acceptor 14b was observed after the reactions, and the yields were moderate (entries 6−8); this might be because 12c or its carbocation would be less stable in those solvents than in nonpolar solvents such as toluene. Therefore, we subsequently tried using mixed solvents D

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Synthesis of B Unit 17a. Field and Mukhopadhyay15 report the synthesis of 17a via the formation of cyclic orthoesters and subsequent benzylation of the other alcohols. However, their method suffers from low efficiency because of the requirement of excess amounts of BnBr and NaH, which is a safety issue owing to the rapid hydrogen evolution. Therefore, we improved upon this method by applying the phase-transfer conditions using BnCl, tetrabutylammonium bromide (TBAB), and NaOH (aq) to afford a mixture of 17a and 17b with good yield (HPLC assay yield: 85%). Fortunately, the acetyl group at the 4-position in 17b could be transferred to the 6-position in the presence of NEt3 in i-PrOH, which gave B unit 17a (57%) from 18 (Scheme 4, Method I).16 As an another high-yield access, the mixture of 17a and 17b was hydrolyzed to give 22 (82%), which was subsequently acetylated regioselectively to afford 17a (95%) (Scheme 4, Method II). Synthesis of A Unit 25. 23 was prepared via selective iodination and the subsequent one-pot acetylation using Garegg and Samuelsson’s procedure.17 The crystallization of 23 from i-PrOH was effective for removal of triphenylphosphine oxide to afford 23 with a yield of 85%. Deiodination under hydrogenolysis conditions and subsequent acetolysis using Ac2O and H2SO4 in AcOH gave the desired A ring intermediate 25 (91%) (Scheme 5).

Table 3. Regioselective anomeric deacetylation of 25

a

amine

conversiona (%)

yielda (%)

1 2 3 4 5 6 7 8 9 10

BnNH2 n-PrNH2 i-PrNH2 Me2NHb Et2NH n-Pr2NH MeBnNH morpholine Me2N−C2H4−NH2 N-Me-piperazine

100 100 72 100 1 2 72 100 100 100

68 75 76 85 trace trace 78 96 73 87

Determined by HPLC. bTHF solution.

than the basicity of amine significantly affects the reaction. In addition, secondary amines, especially cyclic amines, exhibited excellent reactivities while suppressing the overreaction of the product. Next, N-Me-piperazine and N,N-dimethylethylenediamine were tested, because the corresponding acetamide can be removed by simple extraction using acidic aqueous solution. Consistent with the above-mentioned findings, the reaction using N-methylpiperazine, which is a cyclic secondary amine, resulted in the complete conversion of 25 and a good yield of 26. Furthermore, as expected, the corresponding acetamide was easily removed from the reaction mixture by washing with diluted aqueous HCl (entry 10). Synthesis of AB Unit 29. Having obtained required intermediates, 1,2-trans-glycosidation of trichloroacetimidate 16, which was prepared from 25 over 2 steps without isolation, and B unit 17a in the presence of a catalytic amount of TMSOTf was attempted. The reaction proceeded selectively, and the subsequent acetolysis using Ac2O and H2SO4 in AcOH afforded the desired product 28 with an isolated yield of 80%. Regioselective deacetylation with N-methylpiperazine, which was developed for the synthesis of 26, was also effective for the reaction of 28 to give AB unit 29 in excellent yield (Scheme 6). Synthesis of C Unit 14b from 20. Fleet and Witty report the synthesis of 19 from diacetone-glucose (6) over nine steps,13 which was successfully applied to the synthesis of C unit 4 in the first-generation method. However, this route requires one carbon degradation by Malaprade glycol oxidative cleavage with NaIO4 and the subsequent reduction to afford the protected D-xylose derivative 35. To reduce the number of steps, we subsequently explored the synthesis of 14b from Dxylose (20) itself (Scheme 7). Regioselective protection of 20 and the following tosylation afforded 30 with a yield of 64%.29 The introduction of the azide function at C-5 and the following benzylation gave 31. Treatment with HCl in MeOH and mesylation at the 3-O position gave 32 as an anomeric mixture. Hydrogenolysis caused the reduction of azide to give the corresponding amine, which underwent cyclization to afford bicyclic 19; 19 was then protected by methylcarbamate and hydrolyzed with HCl to afford 33. Reduction with NaBH4 gave the diol 34 with a yield of 59% over nine steps. Protection by BzCl at the 2,5-O positions and the deprotection of benzyl ether afforded the desired 14b with a yield of 86% (33% overall yield from 20). Compared to the first-generation method, this

Scheme 5. Synthesis of A unit 25a

a

entry

Compounds in brackets were used for the next step without isolation,

Regioselective Anomeric Deacetylation of 25. Selective deacylation on C-1 of the carbohydrate residue often plays an important role in anomeric functional group transformations. Various existing methods for regioselective anomeric deacetylation are used for oligosaccharide synthesis.18−27 Among them, aminolysis is the most effective owing to the low cost and ease of handling of amine. Benzylamine is frequently used for this conversion,28 but an excess amount of amine is sometimes required, and the removal of benzylacetamide, a byproduct of the reaction, poses a barrier to scaling-up. The reactivity of other amines for this conversion is not well-understood. Therefore, we examined the regioselective deacetylation of 25 with various amines (Table 3). Complete conversions of 25 were observed when using primary amines such as BnNH2 and n-PrNH2. However, the yields were moderate, and several unknown impurities that were assumed to arise from the overreaction of 26 were detected (entries 1 and 2). While secondary amines such as Et2NH and n-Pr2NH afforded only trace amounts of 26 (entries 5 and 6), Me2NH exhibited the highest reactivity, achieving complete conversion and good yield of 26 (entries 4). Surprisingly, morpholine, which has weak basicity (pKa = 8.36), afforded the highest product yield and suppressed the overreaction of 26 (entry 8), although it was difficult to remove the corresponding acetamide from the reaction mixture. These results suggest the steric effect rather E

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Scheme 6. Synthesis of AB unit 29a

a

Compounds in brackets were used for the next step without isolation.

Scheme 7. Synthesis of C unit 14b from 20a

a

Compounds in brackets were used for the next step without isolation.

route has three fewer steps and increased the total yield from

and 14b (Scheme 8). The donor 12c was prepared from 29 in the presence of 1.5 mol % DBU. 12c could be utilized for the next glycosidation without any purification. Slowly adding the reaction mixture of 12c to a solution containing C unit 14b and

22% to 33%. End-Game Synthesis. Finally, we examined the synthesis of α-amylase inhibitor 1 via the glycosidation reaction of 12c F

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Scheme 8. Synthesis of α-amylase inhibitor 1 via glycosidation of AB and C unitsa

a

Compounds in brackets were used for the next step without isolation.

Scheme 9. Outline of second-generation method

Aminolysis with N-methylpiperazine was effective for regioselective anomeric deacetylation and was successfully applied to the reaction of 25 and 28 to afford the corresponding A and AB units with good yields.

TMSOTf in toluene-DME allowed the reaction to proceed well to afford 15 with an α/β ratio of 96/4. The hydrolysis of the Oacetyl, benzoyl, and N-methoxycarbonyl groups afforded 11, which is the same final intermediate as that in the firstgeneration method. Fortunately, the undesired β-isomer was removed effectively by crystallization of 11. Finally, deprotection of benzyl ether in hydrogenolysis conditions afforded the final product with a yield of 93%. Compared to the firstgeneration method, this route dramatically reduced the consumption rate of the expensive C unit in the synthesis of API.

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EXPERIMENTAL SECTION

General. Commercially available chemicals including solvents were purchased from commercial supplier and used as received. NMR spectra were measured on a JEOL JNMECA500 NMR spectrometer at 500 MHz for 1H spectra and 125 MHz for 13C spectra. Reactions were monitored by HPLC analysis that was performed using a Agilent 1100 or Shimazu LC 10 series. HPLC methods are described below. Gradient condition of each methods was set in consideration of retention time of a measuring compound. HPLC Method A. Column: SPELCO Ascentis RP-amide 4.6 × 250 mm (5 μm), eluents: MeCN/2−10 mM aq. NH4OAc (25/75−90/10), detector: UV (210 or 220 nm), or Corona CAD, flow rate: 1.0 mL/min, column temp.: 40 °C, injection: 10 μL. HPLC Method B. Column: Imtakt Unison UK-C8 4.6 × 150 mm (3 μm), eluents: MeCN/2−10 mM aq. NH4OAc (25/75− 80/20), detector: UV (210 or 220 nm), flow rate: 1.0 mL/min, column temp.: 40 °C, injection: 10 μL. HPLC Method C. Column: L-column 4.6 × 250 mm (5 μm), eluents: MeCN/2 mM aq. NH4OAc (25/75−85/15), detector:

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CONCLUSION The outline of the second-generation synthetic method for αamylase inhibitor 1 is shown in Scheme 9. This method has dramatically better efficiency than the medicinal chemistry route (i.e., the first-generation method). The raw material costs are around one-third those of the original route, and the total number of steps was reduced from 33 to 27. In this work, the key to success was the development of highly stereoselective 1,2-cis-glycosidation of the AB and C ring intermediates, which made it possible to introduce the most expensive C unit at a late stage in the synthesis. Furthermore, the efficient synthesis of AB units was achieved by manipulating hydroxyl groups, which also reduced costs. In the synthesis of the AB units, onepot regioselective benzylation of 18 in phase-transfer conditions was developed, and the B unit 17a was obtained in three steps. G

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UV (210 or 220 nm), flow rate: 1.0 mL/min, column temp.: 40 °C, injection: 10 μL. HPLC Method D. Column: SUPELCO Discovery HSF5 4.6 × 250 mm (5 μm), eluents: MeCN/0.01% aq. TFA (40/60, isocratic), detector: ELSD, flow rate: 1.0 mL/min, column temp.: 40 °C, injection: 10 μL. Synthesis of A Unit 25 (Scheme 5). Methyl-2,3,4-tri-Oacetyl-6-deoxy-6-iodo-α- D -glucopyranoside (23). PPh 3 (89.15 g, 339.89 mmol) and imidazole (29.45 g, 432.39 mmol) were added to a mixture of methyl-α-D-glucopyranoside (18) (60.00 g, 308.99 mmol) in THF (300 mL) at rt. The reaction mixture was warmed to 70 °C, and then a solution of I2 (86.26 g, 339.89 mmol) in THF (150 mL) was added dropwise over 3 h. After completion of the reaction (confirmed by TLC), the mixture was cooled to room temperature, and then pyridine (146.65 g, 1853.94 mmol) and Ac2O (157.72g, 1544.95 mmol) were added to the mixture. The mixture was warmed to 35 °C and stirred for 17 h. After completion of the reaction (confirmed by TLC), EtOAc (720 mL) and 10% aq. NaCl (480 mL) were added, and the organic layer was separated. The organic layer was washed with 10% aq. NaCl (360 mL) containing c-HCl (150 mL) and 10% aq. NaCl (480 mL) containing Na2S2O3 (24 g). The obtained organic layer was concentrated to 420 mL, i-PrOH (900 mL) was added, and the mixture was concentrated to 600 mL. The precipitation of 23 was observed during the concentration. After i-PrOH (120 mL) was added to the slurry, the mixture was cooled to 0 °C, stirred for 1 h, and filtered. The product was washed with chilled iPrOH (180 mL) and dried in vacuo at 40 °C to afford 23 (111.41 g, 83.8%) as a white solid. 1 H NMR (500 Hz, CDCl3) δ: 5.47 (t, 1H, J = 10.0 Hz), 4.96 (d, 1H, J = 3.5 Hz), 4.90−4.85 (m, 2H), 3.82−3.78 (m, 1H), 3.48 (s, 3H), 3.30 (dd, 1H, J = 11.0, 2.5 Hz), 3.14 (dd, 1H, J = 8.5, 11.0 Hz), 2.08 (s, 3H), 2.06 (s, 3H), 2.01 (s, 3H); 13C NMR (125 Hz, CDCl3) δ: 170.1, 170.0, 169.6, 96.6, 72.4, 70.8, 69.6, 68.6, 55.7, 20.7, 20.6, and 3.6; IR (KBr pellet) 1740, 1264, 1233, and 1036 cm−1; HRMS (ESI+) [M + Na]+ calcd for C13H19INaO8: 453.0022; found 453.0007; [α]D20 = +117.20 (c = 1.0, CH2Cl2). 1,2,3,4-Tetra-O-acetyl-6-deoxy-D-glucopyranose (α,β-Mixture) (25). To a mixture of 23 (50.00 g, 116.23 mmol) and AcONa (19.07g, 232.46 mmol) in EtOAc (500 mL) and H2O (100 mL), 5% Pd/C (moisture content: 54.5%) (16.48 g) was added. The mixture was stirred for 5.5 h at 35 °C under H2 pressure (3 bar). After completion of the reaction (confirmed by HPLC Method A), the mixture was filtered and washed with EtOAc (250 mL). The filtrate was washed with 10% aq. NaCl (400 mL) containing Na2S2O3 (22.05 g, 139.48 mmol) and 10% aq. NaCl (250 mL). The organic layer was concentrated to 50 mL, and AcOH (100 mL) was added. The mixture was concentrated to 50 mL of Ac2O (15.77g, 232.46 mmol); H2SO4 (5.70 g, 58.12 mmol) was added, and the mixture was stirred at 25 °C for 5 h. After completion of the reaction (confirmed by HPLC Method A), AcONa (9.53 g, 116.23 mmol) was added, and the mixture was stirred for 15 min. EtOAc (500 mL) and 5% aq. NaHCO3 (500 mL) were added. The organic layer was separated, washed with 5% aq. NaHCO3 (500 mL) 2 times and concentrated to 100 mL. i-PrOH (400 mL) was added, and the mixture was concentrated to 175 mL. The precipitation of 25 was observed, and then H2O (800 mL) was added dropwise. The slurry was stirred at 0 °C, filterd, washed with chilled iPrOH/H2O (16/84 mL), and dried in vacuo to afford 25 (34.80 g, 90.1%) as a white solid.

H NMR (500 Hz, CDCl3) β-anomer: δ: 5.69 (d, 1H, J = 8.5 Hz), 5.21 (t, 1H, J = 9.5 Hz), 5.11 (dd, 1H, J = 8.5, 9.5 Hz), 4.86 (t, 1H, J = 10.0 Hz), 3.75−3.69 (m, 1H), 2.11 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 1.25 (d, 3H, J = 6.0 Hz); α-anomer: δ: 6.27 (d, 1H, J = 4.0 Hz), 5.43 (t, 1H, J = 10.0 Hz), 5.07 (dd, 1H, J = 6.0, 10.5 Hz), 4.86 (t, 1H, J = 10.0 Hz), 4.04−4.00 (m, 1H), 2.11 (s, 3H), 2.06 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.21 (d, 3H, J = 6.0 Hz); 13C NMR (125 Hz, CDCl3) δ: 170.2, 170.1, 169.6, 169.3, 169.1, 169.0, 91.6, 73.2, 72.9, 72.7, 70.9, 70.6, 69.7, 69.5, 20.8, 20.6, 20.54, 20.52, 20.4, 17.3, and 17.2; IR (KBr pellet) 1755, 1377, 1228, 1150, 1076, and 1039 cm−1; HRMS (ESI+) [M + Na]+ calcd for C14H20NaO9: 355.1005; found 355.1013; [α]D20 = +119.70 (c = 1.0, CH2Cl2). Synthesis of B Unit 17a (Scheme 4, Method I). Methyl 2,3-di-O-benzyl-6-O-acetyl-α-D-glucopyranoside (17a). To a mixture of methyl-α-D-glucopyranoside (18) (15.00 g, 77.24 mmol) in MeCN (75 mL), CH3C(OMe)3 (13.92 g, 115.86 mmol) and TsOH·H2O (0.294 g, 1.54 mmol) were added. The mixture was warmed to 50 °C and stirred while MeOH was removed at 50 °C in vacuo and MeCN was added to maintain the volume of the mixture. After completion of the reaction (confirmed by HPLC Method A), NEt3 (0.391 g, 3.62 mmol) was added, and the mixture was concentrated to 37.5 mL. Tetrabutylammonium bromide (1.25 g, 3.86 mmol), BnCl (39.12 g, 308.96 mmol), and 48% aq. NaOH (30 mL) were added, and the mixture was stirred at 40 °C for 4 h. After completion of the reaction (confirmed by HPLC Method A), toluene (150 mL) and H2O (75 mL) were added, and the organic layer was separated, washed with 1 M HCl aq. (81 mL), and concentrated to 48 mL. i-PrOH (75 mL) and NEt3 (15.63 g, 154.48 mmol) were added, and the mixture was stirred at rt for 55 h. After completion of the reaction (confirmed by HPLC Method A), toluene (117 mL) and 1 M aq. HCl (170 mL, 169.93 mmol) were added, and then the pH of the mixture was adjusted to ca. 7.0 with 48% aq. NaOH. The organic layer was separated and concentrated to 45 mL. i-PrOH (22 mL) and ethylcyclohexane (113 mL) was added dropwise. The precipitation of 17a was observed, and the slurry was stirred at 0 °C and filtered. The product was washed with chilled ethylcyclohexane (69 mL) and dried in vacuo to afford 17a (18.46 g, 57.4%) as a white solid. 1 H NMR (500 Hz, CDCl3) δ: 7.38−7.29 (m, 10H), 5.00 (d, 1H, J = 11.5 Hz), 4.77 (d, 1H, J = 11.5 Hz), 4.74 (d, 1H, J = 11.5 Hz), 4.66 (d, 1H, J = 12.0 Hz), 4.62 (d, 1H, J = 3.5 Hz), 4.40 (dd, 1H, J = 11.5, 4.0 Hz), 4.21 (dd, 1H, J = 12.0, 2.5 Hz), 3.79 (t, 1H, J = 9.5 Hz), 3.75−3.72 (m, 1H), 3.51 (dd, 1H, J = 3.5, 9.5 Hz), 3.42 (t, 1H, J = 9.0 Hz), 3.38 (s, 3H), 2.51 (d, 1H, J = 2.5 Hz), 2.07 (s, 3H); 13C NMR (125 Hz, CDCl3) δ: 171.3, 138.6, 137.9, 128.6, 128.5, 128.1, 128.0, 127.96, 127.88, 98.2, 81.1, 79.4, 75.5, 73.2, 69.8, 69.2, 63.2, 55.2, and 20.8.; IR (KBr pellet) 3524, 2917, 1727, 1264, 1116, 1061, 1035, and 744 cm−1; HRMS (ESI+) [M + Na]+ calcd for C23H28NaO7: 439.1733; found 439.1745; [α]D20 = +41.50 (c = 1.0, CH2Cl2). Synthesis of 22 (Scheme 4, Method II). Methyl-2,3-diO-benzyl-α-D-glucopyranoside (22). To a mixture of methylα-D-glucopyranoside (18) (100.00 g, 514.99 mmol) in MeCN (500 mL), CH3C(OEt)3 (125.32 g, 772.49 mmol), and TsOH· H2O (1.96 g, 10.30 mmol) was added. The mixture was stirred at rt for 5 h. After completion of the reaction (confirmed by HPLC Method A), NEt3 (2.60 g, 25.75 mmol) was added and the mixture was concentrated to 250 mL. Tetrabutylammonium bromide (8.55 g, 25.75 mmol), BnCl (260.77 g, 2059.96 1

H

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(7.0 mL, 39.12 mmol) in toluene (1950 mL) at −30 °C over 2.5 h and stirred for 1 h. After the reaction (confirmed by HPLC Method A), NEt3 (8.24 mL, 58.68 mmol) was added, and the mixture was concentrated to 1300 mL. The residue was washed with 25% aq. MeOH (1300 mL) three times, and the organic layer was concentrated to 260 mL. AcOH (650 mL) was added, and the mixture was concentrated to 260 mL. After addition of AcOH (390 mL), H2SO4 (9.1 mL, 170.71 mmol) and Ac2O (91 mL, 962.68 mmol) were added at 20 °C, and the mixture was stirred at 20 °C for 4 h. After completion of the reaction (confirmed by HPLC Method A), AcONa (45.5 g, 554.67 mmol) was added, and the mixture was stirred for 30 min. H2O (520 mL) and i-PrOH (910 mL) was added, and the precipitation of 28 was observed. After stirring for 17 h at rt, H2O (1300 mL) was added dropwise. The slurry was cooled to 0 °C, stirred for 2 h, and filtered. The product was washed with chilled 40% aq. i-PrOH (650 mL) and dried in vacuo to afford 28 (156.12 g, 79.5%, α/β = 4:1) as a white solid. 1 H NMR (500 Hz, CDCl3) α-anomer: δ: 7.37−7.21 (m, 10H), 6.27 (d, 1H, J = 3.5 Hz), 5.07 (d, 1H, J = 9.5 Hz), 4.98− 4.90 (m, 3H), 4.81−4.75 (m, 1H), 4.68−4.64 (m, 2H), 4.61− 4.57 (m, 1H), 4.40 (dd, 1H, J = 2.5, 12.5 Hz), 4.10 (dd, 1H, J = 4.5, 12.0 Hz), 3.91−3.86 (m, 2H), 3.78−3.75 (m, 1H), 3.62 (dd, 1H, J = 3.5, 9.5 Hz), 3.37−3.32 (m, 1H), 2.17 (s, 3H), 2.08 (s, 3H), 2.05 (s, 3H), 2.00 (s, 3H), 1.99 (s, 3H), 1.01 (d, 3H, J = 6.0 Hz); β-anomer: δ: 7.37−7.21 (m, 10H), 5.58 (d, 1H, J = 8.0 Hz), 5.10−5.07 (m, 1H), 4.98−4.90 (m, 3H), 4.81−4.75 (m, 2H), 4.72−4.70 (m, 1H), 4.61−4.57 (m, 1H), 4.42 (dd, 1H, J = 2.0, 12.0 Hz), 4.12 (dd, 1H, J = 5.5, 12.5 Hz), 3.80 (t, 1H, J = 8.5 Hz), 3.69 (t, 1H, J = 8.5 Hz), 3.64−3.62 (m, 1H), 3.54 (t, 1H, J = 8.5 Hz), 3.42−3.36 (m, 1H), 2.10 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H), 1.04 (d, 3H, J = 6.0 Hz); 13C NMR (125 Hz, CDCl3) δ: 170.4, 170.2, 169.6, 169.4, 169.2, 138.8, 137.3, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.75, 127.70, 127.2, 126.9, 100.7, 93.5, 89.3, 79.3, 78.3, 77.7, 75.0, 73.2, 72.98, 72.97, 72.4, 70.5, 70.0, 62.1, 21.0, 20.8, 20.6, 20.5, and 17.1; IR (KBr pellet) 1745, 1374, 1247, 1220, 1072, and 1039 cm−1; HRMS (ESI+) [M + Na]+ calcd for C36H44NaO15: 739.2578; found 739.2558; [α]D20 = +44.20 (c = 1.0, CH2Cl2). Synthesis of 29 (Scheme 6). 6-O-Acetyl-2,3-di-O-benzyl4-O-(2,3,4-tri-O-acetyl-6-deoxy-β-D-glucopyranosyl)-D-glucopyranose (29). To a solution of 1,6-di-O-acetyl-2,3-di-Obenzyl-4-O-(2,3,4-tri-O-acetyl-6-deoxy-β-D-glucopyranosyl)-Dglucopyranose (28) (60.00 g, 83.71 mmol) in THF (120 mL), N-methylpiperazine (33.54 g, 334.85 mmol) was added at 15 °C. The mixture was stirred at 15 °C for 48 h. After completion of the reaction (confirmed by HPLC Method A), the mixture was cooled to 0 °C. EtOAc (600 mL) and 1 M aq. HCl (600 mL) were added, and the organic layer was separated, washed with H2O (600 mL) and 1% aq. NaHCO3 (600 mL), and concentrated to 180 mL. i-PrOH (360 mL) was added, and the mixture was concentrated to 180 mL. i-PrOH (240 mL) was added, and then H2O (1260 mL) was added dropwise over 2 h. During the addtion, the precipitation of 29 was observed. The slurry was stirred at rt for 1 h and filtered. The product was washed with 25% aq. i-PrOH (300 mL) and dried in vacuo to afford 29 (52.68 g, 93.3%) as a white solid. 1 H NMR (500 Hz, CDCl3) α/β-mixture: δ: 7.38−7.24 (m, 20H), 5.15−5.14 (m, 1H), 5.11−5.06 (m, 2H), 4.98−4.92 (m, 4H), 4.89−4.84 (m, 3H), 4.81−4.75 (m, 2H), 4.73−4.69 (m, 3H), 4.65−4.61 (m, 3H), 4.49−4.43 (m, 2H), 4.13−4.03 (m, 3H), 3.90 (t, 1H, J = 9.0 Hz), 3.76−3.61 (m, 4H), 3.55−3.51

mmol), and 48% aq. NaOH (200 mL) were added and the mixture was stirred at 40 °C for 4 h. After completion of the reaction (confirmed by HPLC Method A), toluene (1000 mL) and H2O (500 mL) were added, and the organic layer was separated and washed with 2 M HCl aq. (500 mL). 2 M aq. NaOH (450 mL) and MeOH (50 mL) were added, and the mixture was stirred at 55 °C for 3 h. The organic layer was separated, washed with H2O (300 mL), and concentrated to 500 mL. Toluene (400 mL) was added, and then heptane (700 mL) was added dropwise at 45 °C. The mixture was stirred for 1 h, and the precipitation of 22 was observed. Heptane (700 mL) was added dropwise at 45 °C, and the slurry was stirred at 0 °C and filtered. The product was washed with chilled mixed solution of toluene (100 mL) and heptane (200 mL) and dried in vacuo to afford 22 (157.21 g, 81.5%) as a white solid. 1 H NMR (500 Hz, CDCl3) δ: 7.38−7.28 (m, 10H), 5.01 (d, 1H, J = 11.0 Hz), 4.76 (d, 1H, J = 12.0 Hz), 4.71 (d, 1H, J = 12.0 Hz), 4.65 (d, 1H, J = 11.5 Hz), 4.59 (d, 1H, J = 3.5 Hz), 3.81−3.71 (m, 3H), 3.62−3.58 (m, 1H), 3.52 (d, 1H, J = 9.0 Hz), 3.49 (dd, 1H, J = 3.5, 9.5 Hz), 3.37 (s, 3H), 2.52 (brs, 1H), 2.09 (brs, 1H); 13C NMR (125 Hz, CDCl3) δ: 138.7, 137.9, 128.6, 128.5, 128.1, 128.0, 127.9, 127.88, 98.1, 81.3, 79.7, 75.4, 73.1, 70.7, 70.3, 62.3, and 55.2; IR (KBr pellet) 3286, 2922, 1739, 1453, 1120, 1061, 1028, 756, 737, and 698 cm−1; HRMS (ESI+) [M + Na]+ calcd for C21H26NaO6: 397.1627; found 397.1613; [α]D20 = +49.25 (c = 1.0, CH2Cl2). Synthesis of B Unit 17a (Scheme 4, Method II). Methyl2,3-di-O-benzyl-6-O-acetyl-α-D-glucopyranoside (17a). To a solution of methyl 2,3-di-O-benzyl-α-D-glucopyranoside (22) (150.00 g, 400.61 mmol) in EtOAc (450 mL), NEt3 (46.62 g, 460.70 mmol), and Ac2O (47.03 g, 460.70 mmol) was added at 20 °C. The mixture was stirred for 16 h. After completion of the reaction (confirmed by HPLC Method A), H2O (450 mL) was added, and the organic layer was separated. i-PrOH (900 mL) was added, and the mixture was concentrated to 450 mL. After addition of i-PrOH (300 mL), the mixture was stirred for 1 h, and the precipitation of 17a was observed. H2O (1200 mL) was added dropwise, and the slurry was cooled to 0 °C and filtered. The product was washed with chilled mixed solution of i-PrOH (150 mL) and H2O (300 mL) and dried in vacuo to afford 17a (156.21 g, 93.6%) as a white solid. Synthesis of 28 (Scheme 6). 1,6-Di-O-acetyl-2,3-di-Obenzyl-4-O-(2,3,4-tri-O-acetyl-6-deoxy-β-D-glucopyranosyl)D-glucopyranose (28). To a mixture of 1,2,3,4-tetra-O-acetyl-6deoxy-D-glucopyranose (α,β-mixture) (25) (130.00 g, 391.21 mmol) in THF (195 mL), N-methylpiperazine (58.77 g, 586.82 mmol) was added dropwise at 15 °C. The mixture was stirred at 15 °C for 14 h. After completion of the reaction (confirmed by HPLC Method A), the mixture was cooled to 0 °C, and cHCl (65.21 g, 625.94 mmol) was added dropwise. EtOAc (1300 mL) and H2O (390 mL) were added, and the organic layer was separated and washed with H2O (390 mL) three times. The organic layer was concentrated to 260 mL, and then toluene (1300 mL) was added. The solution was concentrated to 520 mL. The addtion of toluene (1300 mL) and the following concentration to 520 mL were repeated to control the water content less than 100 ppm. To the solution, Cl3CCN (73.43 g, 508.57 mmol) and DBU (0.89 g, 5.87 mmol) was added. The mixture was stirred at 25 °C for 14 h. After completion of the reaction (confirmed by HPLC Method B), toluene (900 mL) was added. The solution was added dropwise to the mixture of methyl 2,3-di-O-benzyl-6-O-acetyl-α-Dglucopyranoside (17a) (114.05 g, 273.85 mmol) and TMSOTf I

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783.99 mmol), the mixture was stirred at 100 °C for 3 h. After completion of the reaction (confirmed by HPLC Method C), EtOAc (900 mL) was added. ClCO2Me (29.64 g, 313.60 mmol) was added dropwise to the mixture at 0 °C. After completion of the reaction (confirmed by HPLC Method C), 10% aq. NaCl (720 mL) was added, and the organic layer was separated (HPLC purity of the product: 69.4%). H2O (270 mL) and c-HCl (54 mL) were added, and the mixture was stirred at 30 °C for 1 h. After completion of the reaction (confirmed by HPLC Method C), the organic layer was separated and washed with 5% aq. NaHCO3 (360 mL) (HPLC purity of 33: 80.4%). To the solution, NaBH4 (7.91 g, 209.06 mmol) was added portionwise at 0 °C. The mixture was stirred at 0 °C for 20 min. After completion of the reaction (confirmed by HPLC Method C), 10% aq. NH4Cl (270 mL) was added, and the organic layer was separated and washed with 10% aq. NaCl (270 mL). Activated carbon (13.5 g) was added, and the mixture was stirred at 30 °C for 30 min, filtered, and washed with EtOAc (270 mL). The filtrate was concentrated to 360 mL and warmed to 50 °C, and heptane (450 mL) was added dropwise. During the addtion, the precipitation of 34 was observed. The slurry was stirred at rt for 1 h and filterd. The product was washed with EtOAc/heptane (1:3, 360 mL) and dried in vacuo to afford 34 (58.67 g, 56.1% from 30) as a white solid. 1 H NMR (500 MHz, CDCl3) δ 7.37−7.28 (m, 5H), 5.00 (d, 0.3H (a rotamer), J = 8.5 Hz), 4.71 (d, 0.7H (a rotamer), J = 9.0 Hz), 4.59 (d, 2H, J = 2.0 Hz), 4.20−4.15 (m, 2H), 4.05− 3.87 (m, 3H), 3.72−3.65 (m, 1H), 3.68 (s, 3H), 3.54−3.47 (m, 1H) ; 13C NMR (125 MHz, CDCl3) δ 156.4, 156.0, 137.3, 128.3, 127.8, 127.5, 127.4, 87.1, 86.0, 72.3, 71.4, 71.3, 64.9, 64.5, 61.5, 61.4, 55.0, 54.3, and 52.5 (rotamers were observed); IR (KBr pellet) 3357, 1728, 1395, 1318, 1223, 1180, 1100, and 1057 cm−1; HRMS (ESI+) [M + H]+ calcd for C14H20NO5: 282.1341; found 282.1338; [α]D20 = −28.23 (c = 1.0, DMSO). Synthesis of C Unit 14b (Scheme 7). 2,5-O-Dibenzoyl-Nmethoxycarbonyl-1,4-dideoxy-1,4-imino-D-arabinitol (14b). To a mixture of 3-O-benzyl-N-methoxycarbonyl-1,4-dideoxy1,4-imino-D-arabinitol (34) (30.00 g, 106.65 mmol) and pyridine (42.18 g, 533.25 mmol) in EtOAc (120 mL), BzCl (37.48 g, 266.63 mmol) was added dropwise at 25 °C. The mixture was warmed to 70 °C and stirred for 5 h. After that, the mixture was cooled to 25 °C and stirred for 15 h. After completion of the reaction (confirmed by HPLC Method C), EtOAc (180 mL) and 2 M aq. HCl (300 mL) were added, and the organic layer was separated and washed with 2 M aq. HCl (150 mL), 5% aq. NaHCO3 (150 mL), and 10% aq. NaCl (150 mL). The obtained organic layer was concentrated to 90 mL (HPLC purity of the product: 90.4%). To the solution, MeOH (210 mL), c-HCl (9 mL), and 5% Pd/C containing 54.5% of water (13.19 g) were added, and the mixture was stirred at 60 °C under H2 pressure (3 bar) for 1 h. After completion of the reaction (confirmed by HPLC Method C), the mixture was filtered and washed with EtOAc (120 mL). The filtrate was concentrated to 120 mL. EtOAc (180 mL) and 1 M aq. HCl (150 mL) were added, and the organic layer was separated and washed with 5% aq. NaHCO3 (150 mL) 2 times and 10% aq. NaCl (150 mL). To the obtained organic layer, activated carbon (4.5 g) was added, and the mixture was stirred for 30 min, filtered, and washed with EtOAc (120 mL). The filtrate was concentrated to 120 mL. Heptane (120 mL) was added dropwise to the solution at 50 °C, and the mixture was stirred at 50 °C for 17 h (the precipitation of 14b was observed). After

(m, 2H), 3.41−3.35 (m, 3H), 3.23 (brs, 1H), 2.10 (s, 3H), 2.09 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H), 1.99 (s, 3H), 1.98 (s, 3H), 1.71 (brs, 2H), 1.03 (d, 3H, J = 6.5 Hz), 1.01 (d, 3H, J = 6.0 Hz); 13C NMR (125 Hz, CDCl3) δ: 170.5, 170.4, 170.11, 170.08, 169.5, 169.3, 169.2, 138.7, 138.5, 137.9, 137.5, 128.2, 128.0, 127.9, 127.8, 127.7, 127.6, 127.4, 127.0, 126.9, 126.8, 100.33, 100.31, 96.9, 90.4, 82.4, 82.2, 79.12, 79.07, 76.7, 74.7, 74.5, 72.9, 72.84, 72.79, 72.3, 72.1, 69.78, 69.76, 67.9, 62.4, 62.2, 20.62, 20.60, 20.4, 20.3, 16.9, and 16.8; IR (KBr pellet) 3466, 1745, 1248, 1218, 1069, and 1039 cm−1; HRMS (ESI+) [M + Na]+ calcd for C34H42NaO14: 697.2472; found 697.2474; [α]D20 = +34.60 (c = 1.0, CH2Cl2). Synthesis of 34 (Scheme 7). 3-O-Benzyl-N-methoxycarbonyl-1,4-dideoxy-1,4-imino-D-arabinitol (34). To a solution of 5-O-tosylate-1,2-O-isopropylidene-α-D-xylofuranose (30)15 (90.00 g, 261.33 mmol) in DMSO (360 mL), NaN3 (20.39 g, 313.60 mmol) was added. The mixture was stirred at 110 °C for 5 h. After completion of the reaction (confirmed by HPLC Method C), the mixture was cooled to rt, and 20% aq. NaCl (540 mL) and toluene (450 mL) were added. The organic layer was separated, and the aqueous layer was extracted with toluene (450 mL) two times. The combined organic layer was concentrated to 360 mL (HPLC purity of the product: 96.0%). The solution was added dropwise to a solution of 60% NaH in oil (12.54 g, 313.60 mmol) in THF (495 mL) at 0 °C. BnCl (34.73 g, 274.40 mmol) and tetrabutylammonium bromide (8.42 g, 26.13 mmol) were added, and the mixture was stirred at 25 °C for 4 h. After completion of the reaction (confirmed by HPLC Method C), 20% aq. NaCl (450 mL) was added dropwise, and the organic layer was separated and concentrated to 360 mL. MeOH (900 mL) was added, and the mixture was concentrated to 360 mL. MeOH (900 mL) was added to the residue, and the mixture was concentrated to 360 mL. MeOH (270 mL) was added (HPLC purity of 31: 88.7%). To the solution of 31, 2 M HCl in MeOH (270 mL) was added, and the mixture was warmed to 55 °C and stirred for 2 h. After completion of the reaction (confirmed by HPLC Method C), the pH of the mixture was adjusted to 7−8 with 20% aq. NaOH. Then heptane (180 mL) was added, and the aqueous layer was separated and concentrated to 360 mL. EtOAc (900 mL) was added to the residue, and the mixture was concentrated to 360 mL. EtOAc (360 mL) and 20% aq. NaCl (450 mL) were added, and the organic layer was separated and concentrated to 360 mL. EtOAc (900 mL) was added to the residue, and the mixture was concentrated to 360 mL. EtOAc (270 mL) was added (HPLC purity of the product: 88.3%). To the solution, NEt3 (39.66 g, 392.00 mmol) and MsCl (32.93 g, 287.46 mmol) were added dropwise at 0 °C, and the mixture was stirred. After completion of the reaction (confirmed by HPLC Method C), 5% aq. NaHCO3 (900 mL) was added dropwise, and the mixture was stirred for 1 h. The mixture was separated, and the obtained aqueous layer was extracted by EtOAc (1350 mL). The combined organic layer was washed with 5% aq. NaHCO3 (450 mL), 1 M aq. HCl (450 mL), 5% aq. NaHCO3 (450 mL), and 20% aq. NaCl (450 mL) and concentrated to 270 mL (HPLC purity of 32: 91.0%). To the solution of 32, MeOH (450 mL) and 5% Pd/C containing 54.5% of water (9.89 g) were added, and the mixture was stirred under H2 pressure (3 bar) at 50 °C for 9 h. After completion of the reaction (confirmed by HPLC Method C), the mixture was filtered and washed with MeOH (360 mL). The filtrate was concentrated to 450 mL, and DMSO (450 mL) was added to the residue. After addition of NEt3 (79.33 g, J

dx.doi.org/10.1021/op500306p | Org. Process Res. Dev. XXXX, XXX, XXX−XXX

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that, heptane (360 mL) was added dropwise at 50 °C, and the mixture was cooled to 20 °C, stirred for 18 h, and filtered. The product was washed with EtOAc/heptane (1:4, 150 mL) and dried in vacuo to afford 14b (36.67 g, 86.1%) as a white solid. 1 H NMR (500 MHz, CDCl3) δ 8.03−8.00 (m, 4H), 7.60− 7.52 (m, 2H), 7.46−7.38 (m, 4H), 5.35−5.32 (m, 1H), 4.60 (brs, 2H), 4.53 (brs, 1H), 4.32−4.10 (m, 2H), 3.70 (brs, 3H), 3.69 (brs, 1H), 3.42−3.34 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 166.0, 165.7, 155.7, 133.3, 132.9, 132.8, 129.41, 129.36, 128.8, 128.3, 128.1, 78.1, 77.3, 75.9, 75.0, 63.7, 63.2, 63.0, 62.4, 52.6, 50.8, and 50.5 (rotamers were observed); IR (KBr pellet) 3436, 1725, 1686, 1460, 1397, 1282, 1115, and 710 cm−1; Elementary analysis. Found: C, 63.24; H, 5.32; N, 3.67%. Calcd for C21H21NO7: C, 63.15; H, 5.30; N, 3.51%; LRMS (FAB) [M + H]+ 400; [α]D20 = −23.20 (c = 1.0, DMSO). Synthesis of ABC Unit 11 (Scheme 8). (2R,3R,4R)-4Hydroxy-2-(hydroxymethyl)pyrrolidine-3-yl 2,5-O-dibenzyl-4O-(6-deoxy-α-D-glucopyranosyl)-α-D-glucopyranoside (11). To a mixture of 6-O-acetyl-2,3-di-O-benzyl-4-O-(2,3,4-tri-Oacetyl-6-deoxy-β- D -glucopyranosyl)- D -glucopyranose (29) (21.96 g, 32.55 mmol) and trichloroacetonitrile (7.05 g, 48.83 mmol) in toluene (7 mL), DBU (99 mg, 0.65 mmol) was added at 25 °C, and the mixture was stirred for 24 h. The completion of the reaction was confirmed by HPLC Method A (solution of 12c). To a solution of 2,5-O-dibenzoyl-N-methoxycarbonyl-1,4dideoxy-1,4-imino-D-arabinitol (14b) (10.00 g, 25.04 mmol) in toluene (215 mL) and DME (35 mL) at −40 °C, TMSOTf (0.68 mL, 3.76 mmol) was added dropwise. To the mixture, the solution of 12c was added dropwise at −40 °C over 10 h, and the mixture was stirred for 30 min. After completion of the reaction (confirmed by HPLC Method A), NEt3 (1.05 mL, 7.51 mmol) was added. The mixture was washed with 20% aq. NaCl (100 mL) and concentrated to 70 mL. i-PrOH (60 mL), H2O (30 mL), and 5 M aq. NaOH (120 mL) were added, and the mixture was stirred at 70 °C for 7 h. After completion of the reaction (confirmed by HPLC Method A), the mixture was cooled to 25 °C, and c-HCl (55 mL) was added (pH: