Total Synthesis of the Trisaccharide Antigen of the Campylobacter

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Total Synthesis of the Trisaccharide Antigen of the Campylobacter jejuni RM1221 Capsular Polysaccharide via De Novo Synthesis of the 6-Deoxy-D-manno-heptose Building Blocks Xiaoman Wang, Yan Chen, Junchang Wang, and You Yang J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02394 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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The Journal of Organic Chemistry

Total Synthesis of the Trisaccharide Antigen of the Campylobacter jejuni RM1221 Capsular Polysaccharide via De Novo Synthesis of the 6-Deoxy-D-manno-heptose Building Blocks Xiaoman Wang,† Yan Chen,† Junchang Wang, and You Yang* Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China. Supporting Information Placeholder

ABSTRACT: A de novo approach utilizing the D-proline-catalyzed and LDA-promoted aldol reactions as key steps for the preparation of differentiated-protected 6-deoxy-D-manno-heptose building blocks was developed. PPh 3AuBAr4F-catalyzed glycosylation with the 6-deoxy-D-manno-heptosyl ortho-hexynylbenzoate as donor was demonstrated as a direct and practical method for the stereoselective synthesis of the -linked 6-deoxy-D-manno-heptoside as the major product. Coupling of the 6-deoxy--D-mannoheptosyl H-phosphonate with the 3-hydroxyl disaccharide acceptor based on H-phosphonate chemistry was described for the construction of the trisaccharide skeleton with the acid-labile phosphodiester linkage. Finally, first total synthesis of the unique trisaccharide antigen of the capsular polysaccharide of C. jejuni RM1221 that belongs to HS:53 serotype complex was accomplished for further evaluation as vaccine candidate against C. jejuni RM1221 infection.

INTRODUCTION Campylobacter jejuni is a spiral-shaped Gram-negative bacterium that is one of the most common causes of human gastroenteritis and diarrhoea throughout the world.1-4 As food-borne disease, the infection of C. jejuni is frequently found and sometimes fatal in young children resulting from undercooked poultry, contaminated water, and raw milk.5-7 The clinical symptoms of C. jejuni infection mainly include diarrhea, abdominal cramps, fever, headache, and vomiting.4 As for immunocompromised patients, C. jejuni illness may induce severe post-infection complications such as Guillain-Barrésyndrome (GBS) and reactive arthritis.8-10 Although antibiotics are usually available to combat against C. jejuni, the emergence of antibiotic-resistant bacteria poses a serious threat to human health.11 Thus, development of an effective vaccine could be a significant step in controlling C. jejuni infection and protecting people from gastroenteritis and diarrhoea.12,13 On the cell surface of C. jejuni, the capsular polysaccharide (CPS) is considered as one of the virulence factors that mediate the interactions of bacteria with the host.12,14 As such, the

Figure 1. The structure of capsular polysaccharide of C. jejuni RM1221. CPS serves as an attractive antigen for developing CPS-based vaccine against C. jejuni infection.15,16 In 2007, Vinogradov et al. reported the CPS structure of C. jejuni RM1221 of the Penner serotype HS:53, which consists of unique (1→3)-linked trisaccharide repeating units and is very relevant to clinicallyimportant C. jejuni strains especially in the developing countries (Figure 1).14,17 Each trisaccharide repeating unit contains one -, and one -6-deoxy-D-manno-heptose residues, as well as one -6-deoxy-D-manno-heptosyl phosphate moiety. Due to partial expression of acid-labile xylulose residues at the O-2 and O-4 positions of the sugar ring B of the trisaccharide repeating units, the glycans are quite heterogeneous and thus

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difficult to be separated for production of well-defined oligosaccharides aiming at biological studies. In contrast, synthesis of the CPS repeating unit of C. jejuni RM1221 can provide a sufficient amount of homogeneous and well-defined sugar antigens for vaccine development. The structural features of the CPS of C. jejuni RM1221 renders the synthesis of the trisaccharide repeating unit quite challenging. First, traditional C-6 one-carbon homologation strategy for the preparation of 6-deoxy-D-manno-heptose building blocks requires multiple selective protectiondeprotection procedures and often employs toxic reagents such as potassium cyanide and mercury salt.18,19 Second, stereoselective construction of the -glycosidic linkages of the 6deoxy-D-manno-heptosides is notoriously difficult and usually associated with indirect and lengthy procedures due to the anomeric effect and the steric repulsion of the C-2 substituent.20-23 Third, stereocontrolled assembly of the -linked phosphodiester bridge between the 6-deoxy-D-manno-heptose residues and global deprotection of phosphoglycans are complicated in terms of the correct configuration and the inherent instability of the anomeric phosphodiester linkage.24,25 Although synthesis of the CPS repeating unit of C. jejuni RM1221 have been pursued for around ten years, only partial structures of the trisaccharide repeating unit were obtained. 26 Here, we disclose the first total synthesis of the unique trisaccharide antigen 1 of the C. jejuni RM1221 CPS, featuring de novo synthesis27 of the 6-deoxy-D-manno-heptose building blocks, PPh3AuBAr4F-catalyzed -selective glycosylation with the 6-deoxy-D-manno-heptosyl ortho-hexynylbenzoates as donors, and stereocontrolled assembly of the 6-deoxy--Dmanno-heptosyl phosphodiester linkage.

RESULTS AND DISCUSSION As depicted in Scheme 1, the trisaccharide antigen 1 representing one repeating unit of the C. jejuni RM1221 CPS, is equipped with a 5-amino-1-pentyl linker28 at the reducing end for subsequent conjugation to a carrier protein. In view of the different types of glycosidic linkages between the 6-deoxy-Dmanno-heptose residues, target trisaccharide 1 is divided into three key 6-deoxy-D-manno-heptose building blocks: the Hphosphonate 2, the ortho-hexynylbenzoate 3, and the 3-OH acceptor 4 bearing an -linked spacer. The building blocks 2-4 are designed to be obtained by de novo synthesis via the two types of aldol reactions commencing from simple materials 57. Glycosylation of a mixture of the 4-O-benzoyl-directed 6deoxy--D-manno-heptosyl ortho-hexynylbenzoate 3 with the 3-OH acceptor 4 under the catalysis of PPh3AuBAr4F (BAr4F = tetrakis[3,5-bis(trifluoromethyl)phenyl]borate) is expected to give the -6-deoxy-D-manno-heptosyl disaccharide in a more practical way compared to the pioneering work regarding the direct glycosylation of 4,6-di-O-benzoyldirected -D-mannosyl ortho-hexynylbenzoate as the only anomer for the synthesis of -mannosides by Yu and coworkers.29 The allyl group in the resulting -disaccharide is selectively removed to afford the 3-OH disaccharide acceptor, which will be coupled with the H-phosphonate 2 via Hphosphonate chemistry24,25 to give access to target trisaccharide 1 after global deprotection.

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Scheme 1. Retrosynthetic analysis of the trisaccharide antigen 1 of C. jejuni RM1221 capsular polysaccharide.

De novo synthesis of the key 6-deoxy-D-manno-heptose building block 16 started from the first aldol reaction (Scheme 2). D-Proline (0.5 equiv)-catalyzed aldol reaction of ketone 6 with 2,2-dimethoxyacetaldehyde 7 in DMSO afforded 2,3anti--hydroxyl ketone 8 in 67% yield (de = 89%, ee = 78%) in multi-gram scale.30 Notably, replacement of DMSO with DMF or reducing the amount of D-proline led to decreased yield of the aldol product. The resulting hydroxyl group in 8 was masked with the TBS group using TBSCl and imidazole to give ketone 9 in 93% yield for the second aldol reaction. Unexpectedly, LDA (3.3 equiv)-promoted aldol reaction of ketone 9 with benzyloxyacetaldehyde 5 in THF at –78 °C provided ,-unsaturated ketone 10 as a single anomer in 84% yield without the formation of the expected -hydroxyl ketone,31 However, the stereochemistry of the newly-forming double bond could not be proven by the NMR spectra due to the complexity of the trisubstituted double bond system. Reduction of ketone 10 with L-selectride was accompanied by migration of the 2-TBS group to the 4-OH position probably due to the formation of the thermodynamically more stable product,32 affording alcohol 11 in 83% yield with excellent 2,3-anti-3,4-syn-selectivity. Migration of the silyl group from 10 to 11 was supported by the subsequent benzylation at the 2OH position, which was fully elucidated by analysis of the NMR spectra of the derived compound 16. Only trace amount of the non-migrated product was found in this reaction. Benzylation of the remaining 2-OH group in 11 with benzyl bromide in the presence of NaH and TBAI gave compound 12 in high yield (85%). A range of acids including Lewis acids and Brønsted acids such as CSA, para-toluenesulfonic acid, acetic acid and SnCl2, were then screened for selective cleavage of the isopropylidene acetal group in 12. It turned out that the biphasic system that contained 3M aqueous HCl in CH2Cl2 resulted in a very clean reaction for the removal of the isopropylidene acetal group, without affecting the dimethyl acetal and the TBS groups, affording -hydroxyl ketone 13 in a good 84% yield. Silylation of the 3-hydroxyl in 13 with TBSCl in the presence of imidazole in DMF at 60 °C was carried out to


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produce 14 in 93% yield. Selective reduction of ketone 14 with L-selectride followed by cleavage of the TBS groups and concomitant cyclization with aqueous acetic acid at 60 °C generated the corresponding hemiacetal 15, which was then acetylated to provide 6-deoxy-D-manno-heptose building block 16 in satisfactory yield with the -anomer as the predominant product. The structure of compound 16 was unambiguously verified by analyzing the 1H, 13C, and 2D NMR spectra. From the COSY and HSQC spectra, the sugar ring protons of 16 were assigned according to the 1H–1H and 13C– 1 H correlations (H-1: 6.10 ppm, d, 3J = 2.0 Hz; H-2: 3.82 ppm, dd, 3J = 2.0, 3.2 Hz; H-3: 5.17 ppm, dd, 3J = 3.6, 10.0 Hz; H-4: 5.30 ppm, t, 3J = 10.0 Hz; H-5: 4.02 ppm, dt, 3J = 2.4, 10.0 Hz). Collectively, these coupling constants of the ring protons of 16 are consistent with a D-manno-configured pyranose ring. Compared to the traditional approach for the synthesis of the free 6-deoxy-D-manno-heptose starting from methyl -D mannopyranoside (7 steps, 45% overall yield), 33 the de novo approach based on the simple aldol reactions provided the advanced intermediate 16 in 10 steps with 14% overall yield, which is more convenient for the stereo- and regioselective construction of the glycosidic linkages in the fina product 1. Scheme 2. De novo synthesis of the 6-deoxy-D-mannoheptose building block 16.

With the key intermediate 16 available in multi-gram quantity, we turned to the synthesis of building blocks 2-4. Selective removal of the anomeric acetate in 16 with benzylamine gave the corresponding hemiacetal in 70% yield (Scheme 3). Subjection of the above hemiacetal to salicylchlorophosphite34 in acetonitrile and pyridine led to the exclusive formation of 6dexoy--D-manno-heptosyl H-phosphonate 2 in 75% yield, as determined by analysis of the 1H, 31P, and coupled 13C NMR spectra of 2 [ 5.65 (d, 3JH1,P = 7.6 Hz, 1 H, H-1), 6.88 (d, 1 JH,P = 647.2 Hz, 1 H, PH); P 0.31 (dd, 1JH,P =647.4 Hz, 3 JH1,P = 7.9 Hz); C 93.2 (d, 2JC1,P = 4.0 Hz, 1JC1,H1 = 173.0 Hz)].35

The conversion of the anomeric acetate 16 to the thioglycoside36 using 5-tert-butyl-2-methylbenzenethiol37 under the promotion of BF3·Et2O, removal of the acetyl groups with sodium methoxide, and tin-mediated selective installation of the allyl group at the 3-OH position allowed for the synthesis of thioglycoside 17 in 60% yield over three steps (Scheme 4).38 Benzoylation of the 4-OH group in 17 with benzoyl chloride in the presence of DMAP in pyridine at 0 to 90 °C gave 18 in 80% yield. The benzoyl group at the 4-OH position and the allyl group at the 3-OH position in 18 were elucidated by analysis of the 1H NMR and COSY spectra of 18. Cleavage of thioglycoside 18 using a NBS/DTBP/AgOTf system in acetonitrile and water followed by condensation with orthohexynylbenzoic acid under the assistance of EDCI and DMAP proceeded smoothly to afford glycosyl orthohexynylbenzoate39 3 as a mixture of / anomers in 79% yield (: = 1:2.5), which was easily separated by silica gel chromatography. Scheme 4. Synthesis of the 6-deoxy-D-mannoheptopyranosyl ortho-hexynylbenzoate 3.

On the basis of the anomeric effect,40 BF3·Et2O-promoted glycosylation of heptosyl acetate 16 with linker 19 underwent a clean reaction, providing -linked 6-deoxy-D-mannoheptoside 20 as the only product in 87% yield (1JC1,H1 = 168.1 Hz; Scheme 5). After this, deacetylation of 20 with sodium methoxide followed by tin-mediated selective allylation of the 3-hydroxyl group using allyl bromide in the presence of TBAI and CsF in toluene at 80 °C gave alcohol 21 in 57% yield over two steps. Acetylation of the 4-hydroxyl group in 21 and subsequent removal of the allyl group with PdCl2 in methanol afforded 3-OH acceptor 4 in 90% yield over two steps.41 Scheme 5. Synthesis of -linked heptopyranosyl acceptor 4.

Scheme 3. Synthesis of the 6-deoxy--D-mannoheptopyranosyl hydrogenphosphonate 2.


6-deoxy-D-manno-


With the building blocks 2-4 available in hand, we set out to assemble them into target trisaccharide 1 of C. jejuni RM1221 capsular polysaccharide. Compared to the mannosylation,20,29,42 -selective glycosylation with the 6deoxy-D-manno-heptosyl donors was less explored and more challenging due to the absence of the 6-hydroxyl group and the formation of 1,2-cis--glycosidic linkages.21-23 Gratifyingly, PPh3AuBAr4F (0.35 equiv)-catalyzed glycosylation of 6deoxy--D-manno-heptosyl ortho-hexynylbenzoate 3 with acceptor 4 in PhCl at –42 °C provided -disaccharide 22 as the major product in an excellent 97% yield (: = 5:1; -anomer: 1 JC1,H1 = 166.0 Hz, 1JC1′,H1′ = 154.0 Hz; -anomer: 1JC1,H1 = 1 167.4 Hz, JC1′,H1′ = 171.5 Hz; Scheme 6). As described in the previous literature,29 the good -selectivity in the glycosylation reactions with 3 might be attributed to an SN2 substitution of the 1--mannosyloxy-isochromenylium-4-gold(I) complex or an SN1 reaction via a contact ion pair. Under the similar conditions, glycosylation of 6-deoxy--D-manno-heptosyl ortho-hexynylbenzoate 3 with 4 promoted by PPh3AuBAr4F (0.35 equiv) also produced disaccharide 22 in 94% yield albeit with slightly decreased -selectivity (: = 2.8:1). Since the both provided the diglycosylation with 3 or 3 saccharide 22 as the major product, a mixture of orthohexynylbenzoate 3 (: = 1:2.5) was employed as donor for the coupling with 4 to provide disaccharide 22 in excellent yield and good -selectivity (95%, : = 3.5:1) in a practical manner.

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three 6-deoxy-D-manno-heptose residues (H1 = 5.50, 4.91 and 4.88 ppm;H6 = 2.18 and 1.85–1.71 ppm), were found to be in good agreement with those reported for isolated CPS of C. jejuni RM1221 (Figure 2 and the Supporting Information for details).17 Scheme 7. Completion of trisaccharide antigen 1 of C. jejuni capsular polysaccharide.

Scheme 6. Glycosylation of 6-deoxy-D-mannoheptopyranosyl ortho-hexynylbenzoate 3 with-linked 6deoxy-D-manno-heptopyranosyl acceptor 4.

Figure 2. The 1H NMR spectrum of the final product 1.

CONCLUSION Removal of the allyl group in disaccharide 22with PdCl2 afforded disaccharide acceptor 23 in 90% yield (Scheme 7). Coupling of disaccharide 23 with H-phosphonate 2 in the presence of PivCl in pyridine followed by oxidation with iodine in pyridine and water effected the formation of the acidlabile phosphodiester linkage,24,25,43 affording trisaccharide 24 in 74% yield over two steps. The anomeric configuration of 24 was unambiguously determined by analysis of the coupling constants between the anomeric carbons and protons (sugar A: JC1,H1 = 168.0 Hz; sugar B: JC1′,H1′ = 155.0 Hz; sugar C: JC1′′,H1′′ = 172.0 Hz). Finally, global deprotection of trisaccharide 24 involving saponification of the acetyl and benzoyl groups with sodium methoxide in methanol, and hydrogenolysis of the benzyl and benzyloxycarbonyl groups over Pd/C in methanol and water, furnished target trisaccharide 1 in 86% yield over two steps after purification by Sephadex LH-20 column. The structure of 1 was confirmed by a combination of spectroscopic analysis (1H, 13C{1H}, and 2D NMR spectra, ESI-HRMS). The NMR spectra of trisaccharide antigen 1, especially the chemical shifts of the anomeric protons and the H-6 of the

In conclusion, we have developed a de novo approach for the synthesis of the orthogonally-protected 6-deoxy-D-mannoheptopyranose building blocks, enabling the first total synthesis of the unique trisaccharide antigen of C. jejuni RM1221 CPS. De novo synthesis of the 6-deoxy-D-mannoheptopyranose building blocks was effectively achieved based on the D-proline-catalyzed aldol reaction and the LDApromoted aldol reaction. Employing PPh3AuBAr4F as catalyst, glycosylation with the 6-deoxy-D-manno-heptopyranosyl ortho-hexynylbenzoate as donor provided the 6-deoxy-D-mannoheptopyranoside in excellent yield with good -selectivity, serving as a direct and practical method for the construction of 6-deoxy--D-manno-heptose-containing oligosaccharides. The acid-labile phosphodiester linkage embedded in the 6-deoxyD-manno-heptose-containing phosphosugar was obtained by coupling of the -glycosyl H-phosphonate with the 3-hydroxyl disaccharide acceptor via the H-phosphonate method. The present total synthesis provides a well-defined trisaccharide antigen of C. jejuni RM1221 CPS for subsequent immunological evaluation as vaccine candidate against gastroenteritis and diarrhoea.


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EXPERIMENTAL SECTION General Information. All reactions were performed with anhydrous solvents in oven-dried glassware with magnetic stirring under argon or nitrogen unless otherwise stated. The chemicals were used as supplied except where noted. Analytical thin layer chromatography (TLC) was conducted on precoated plates of silica gel (0.25-0.3 mm, Shanghai, China). The TLC plates were visualized by exposure to UV light or by staining with a sulfuric acid-ethanol solution. Silica gel column chromatography was performed on silica gel AR (100200 mesh, Shanghai, China). Optical rotations (OR) were measured with a Rudolph Research Analytical Autopol I automatic polarimeter. NMR spectra were recorded with a Bruker Avance III 400 or Bruker Avance III 500 spectrometer. The 1H and 13C{1H} NMR spectra were calibrated against the residual proton and carbon signals of the solvents as internal references (CDCl3 H = 7.26 ppm and C = 77.2 ppm; D2O: H = 4.79) while the 31P NMR spectra were referenced to external 85% H3PO4. Multiplicities are quoted as singlet (s), broad singlet (br s), doublet (d), triplet (t), quartet (q), doublet of doublets (dd), doublet of triplets (dt) or multiplet (m). Highresolution mass spectra were recorded on ESI-TOF spectrometers. (R)-4-((S)-1-Hydroxy-2,2-dimethoxyethyl)-2,2-dimethyl1,3-dioxan-5-one 8. To a solution of 2,2-dimethyl-1,3-dioxan5-one 6 (23.39 g, 179.73 mmol) in DMSO (45 mL) at room temperature was added D-Proline (10.35 g , 89.86 mmol). After stirring at room temperature for 30 min, 2,2dimethoxyacetaldehyde 7 (62.37 g, 359.46 mmol, 60% aq.) was added and the solution was stirred for an additional 24 h. The mixture was quenched with sat. aq. NH4Cl and extracted with CH2Cl2 (4 × 200 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 4/1) to afford 8 (28.21 g, 67%, de = 89%, ee = 78%) as a colorless syrup: The ee value of the product was measured by HPLC using a chiral stationary phase (Chiralpak AD-3, n-hexane/iso-propanol 8:2) relative to the racemic sample: major isomer 7.38 min, minor isomer 8.13 min; []D25 = +86.5 (c 0.4, CHCl3); 1H NMR (400 MHz, CDCl3) δ 4.67 (d, J = 6.8 Hz, 1 H, H-1), 4.46 (dd, J = 1.2, 6.8 Hz, 1 H), 4.27 (dd, J = 1.6, 16.4 Hz, 1 H), 4.10 (m, 1 H), 4.00 (d-like, J = 16.4 Hz, 1 H), 3.45 (s, 3 H), 3.40 (s, 3 H), 2.45 (d, J = 3.6 Hz, 1 H, OH), 1.48 (s, 6 H); 13C{1H} NMR (100 MHz, CDCl3) δ 206.6, 103.3, 100.6, 76.2, 71.2, 67.1, 55.4, 54.4, 25.0, 23.0; HRMS (ESI) m/z calcd for C 10H18O6Na [M + Na]+ 257.1001, found 257.0997. (R)-4-((S)-1-((tert-Butyldimethylsilyl)oxy)-2,2-dimethoxy ethyl)-2,2-dimethyl-1,3-dioxan-5-one 9. To a solution of ketone 8 (28.21 g, 120.43 mmol) in anhydrous CH2Cl2 (230 mL) at room temperature was added imidazole (36.89 g, 541.92 mmol) and TBSCl (72.60 g, 481.71 mmol) under argon. The resulting mixture was stirred at room temperature overnight until TLC indicated complete conversion of starting material. The reaction was quenched with sat. aq. NaHCO3 and extracted with CH2Cl2 (4 × 400 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 10/1) to afford 9 (39.0 g, 93%) as a yellow syrup: [] D25 = +64.4 (c 0.6,

CHCl3); 1H NMR (400 MHz, CDCl3) δ 4.59 (d, J = 7.6 Hz, 1 H, H-1), 4.34 (t-like, J = 1.2 Hz, 1 H), 4.21 (dd, J = 1.6, 16.0 Hz, 1 H), 4.05 (dd, J = 2.0, 7.6 Hz, 1 H), 3.89 (d-like, J = 15.6 Hz, 1 H), 3.45 (s, 3 H), 3.39 (s, 3 H), 1.47 (s, 3 H), 1.44 (s, 3 H), 0.87 (s, 9 H), 0.10 (s, 3 H), 0.09 (s, 3 H); 13C{1H} NMR (100 MHz, CDCl3) δ 206.3, 105.6, 100.4, 77.9, 73.7, 67.3, 56.2, 55.8, 25.9, 25.1, 23.1, 18.3, -4.4, -4.7; HRMS (ESI) m/z calcd for C16H32O6SiNa [M + Na]+ 371.1866, found 371.1863. (R,Z)-4-(2-(Benzyloxy)ethylidene)-6-((S)-1-((tert-butyl dimethylsilyl)oxy)-2,2-dimethoxyethyl)-2,2-dimethyl-1,3dioxan-5-one 10. To a solution of compound 9 (39.00 g, 112.00 mmol) in anhydrous THF (160 mL) at –78 °C was added LDA (185 mL, 370.00 mmol, 2 M in THF) under argon. After stirring at –78 °C for 2 h, benzyloxyacetaldehyde 5 (67.28 g, 448.02 mmol) was added and the mixture was stirred at –78 °C for an additional 3 h. The reaction was quenched with concentrated phosphate buffer (pH 7.5; 550 mL) and extracted with Et2O (3 × 400 mL). The combined organic layers were washed with sat. aq. NaHCO3 and brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 25/1) to afford 10 (45.18 g, 84%) as a colorless syrup: []25D = +1.1 (c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.34–7.28 (m, 5 H), 6.00 (t, J = 6.4 Hz, 1 H), 4.56 (d, J = 7.6 Hz, 1 H, H-1), 4.50 (s, 2 H), 4.47 (d, J = 1.6 Hz, 1 H), 4.21 (dd, J = 6.8, 14.4 Hz, 1 H), 4.14 (dd, J = 6.4, 14.4 Hz, 1 H), 4.00 (dd, J = 1.6, 7.2 Hz, 1 H), 3.44 (s, 3 H), 3.39 (s, 3 H), 1.57 (s, 3 H), 1.50 (s, 3 H), 0.86 (s, 9 H), 0.11 (s, 3 H), 0.08 (s, 3 H); 13 C{1H} NMR (100 MHz, CDCl3) δ 190.4, 147.4, 138.3, 128.5, 127.9, 127.8, 112.8, 105.5, 100.2, 78.8, 75.3, 72.6, 63.9, 56.3, 55.9, 28.2, 25.9, 23.2, 18.3, -4.3, -4.6; HRMS (ESI) m/z calcd for C25H40O7SiNa [M + Na]+ 503.2441, found 503.2440. (S)-1-((4R,5S,Z)-6-(2-(Benzyloxy)ethylidene)-5-((tert-butyl dimethylsilyl)oxy)-2,2-dimethyl-1,3-dioxan-4-yl)-2,2dimethoxy ethan-1-ol 11. To a solution of compound 10 (45.18 g, 94.08 mmol) in anhydrous THF (200 mL) at –78 °C was added dropwise 1 M solution of L-selectride in THF (280 mL, 280 mmol) under argon. After the reaction was stirred for 2 h, the mixture was warmed to room temperature, and then diethyl ether (200 mL) and sat. aq. NH4Cl (200 mL) were added and stirred for an additional 10 min. The mixture was extracted with ethyl acetate (3 × 300 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 20/1) to afford 11 (37.66 g, 83%) as a colorless syrup: []20D = +3.1 (c 0.3, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.34–7.27 (m, 5 H), 5.16 (dd, J = 5.6, 8.0 Hz, 1 H, H-6), 4.53–4.46 (m, 2 H), 4.38 (d, J = 5.6 Hz, 1 H, H-1), 4.29 (d, J = 3.2 Hz, 1 H), 4.22 (dd, J = 8.0, 11.6 Hz, 1 H), 4.07 (d, J = 3.2 Hz, 1 H), 4.01 (m, 2 H), 3.94 (t, J = 4.4 Hz, 1 H), 3.47 (s, 3 H), 3.43 (s, 3 H), 1.54 (s, 3 H), 1.39 (s, 3 H), 0.92 (s, 9 H), 0.16 (s, 3 H), 0.12 (s, 3 H); 13 C{1H} NMR (125 MHz, CDCl3) δ 150.9, 138.6, 128.5, 128.0, 127.7, 110.3, 105.1, 102.0, 75.0, 72.5, 70.3, 67.2, 63.9, 56.6, 55.6, 28.6, 26.0, 21.7, 18.4, -4.5, -4.8; HRMS (ESI) m/z calcd for C25H42O7SiNa [M + Na]+ 505.2597, found 505.2599. (((4R,5S,Z)-4-((S)-1-(Benzyloxy)-2,2-dimethoxyethyl)-6-(2(benzyloxy)ethylidene)-2,2-dimethyl-1,3-dioxan-5-yl)oxy) (tert-butyl)dimethylsilane 12. A solution of compound 11 (37.66 g, 78.09 mmol) in anhydrous THF (50 mL) was added to a stirred suspension of NaH (16.87 g, 702.80 mmol, 60% in



oil) in anhydrous THF (200 mL) at 0 °C under argon. After the resulting mixture was stirred at 0 °C for 15 min, benzyl bromide (28 mL, 235.71 mmol) and TBAI (1.30 g, 3.52 mmol) were then added. The reaction was stirred at room temperature for 12 overnight. After TLC showed no starting material, the reaction was quenched with sat. aq. NaHCO3 and extracted with EtOAc (3 × 500 mL). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo to yield the crude product which was purified by silica gel column chromatography (petroleum ether/EtOAc: 20/1) to afford 12 (37.99 g, 85%) as a colorless syrup: []D20 = +42.9 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.35–7.25 (m, 10 H), 5.12 (dd, J = 5.2, 7.2 Hz, 1 H), 5.02 (d-like, J = 11.2 Hz, 1 H), 4.56 (d-like, J = 11.2 Hz, 1 H), 4.51 (d, J = 1.2 Hz, 1 H), 4.49 (s, 2 H), 4.17–4.13 (m, 2 H), 4.04 (dd, J = 5.6, 12.4 Hz, 1 H), 3.96 (dd, J = 0.8, 9.2 Hz, 1 H), 3.92 (dd, J = 1.2, 9.2 Hz, 1 H), 3.52 (s, 3 H), 3.50 (s, 3 H), 1.50 (s, 3 H), 1.35 (s, 3 H), 0.90 (s, 9 H), 0.06 (s, 3 H), 0.03 (s, 3 H); 13C{1H} NMR (100 MHz, CDCl3) δ 151.0, 139.2, 138.6, 128.5, 128.3, 127.8, 127.7, 127.6, 127.4, 111.0, 105.3, 101.9, 77.3, 74.9, 72.8, 72.4, 65.7, 63.9, 57.0, 56.4, 28.7, 26.1, 21.7, 18.5, -4.2, -4.4; HRMS (ESI) m/z calcd for C32H48O7SiNa [M + Na]+ 595.3067, found 595.3069. (4S,5R,6S)-1,6-Bis(benzyloxy)-4-((tert-butyldimethylsilyl) oxy)-5-hydroxy-7,7-dimethoxyheptan-3-one 13. To a solution of compound 12 (37.99 g, 66.38 mmol) in CH2Cl2 (300 mL) at room temperature was added 3 M solution of HCl in H 2O (400 mL). The reaction was stirred vigorously at room temperature for 5.5 h. After TLC showed no starting material, the reaction was quenched with sat. aq. NaHCO3 and extracted with CH2Cl2 (3 × 200 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 10/1) to afford 13 (29.68 g, 84%) as a yellow syrup: []D20 = –3.8 (c 0.87, CHCl3); 1 H NMR (400 MHz, CDCl3) δ 7.34–7.26 (m, 10 H), 4.89 (dlike, J = 11.2 Hz, 1 H), 4.59 (d, J = 3.6 Hz, 1 H), 4.50 (d-like, J = 11.2 Hz, 1 H), 4.42 (s, 2 H), 4.40 (d, J = 3.2 Hz, 1 H), 3.98 (dt, J = 3.2, 7.6 Hz, 1 H), 3.70–3.64 (m, 1 H), 3.62–3.56 (m, 2 H), 3.50 (s, 3 H), 3.45 (s, 3 H), 3.01 (d, J = 7.6 Hz, 1 H), 2.89– 2.72 (m, 2 H), 0.92 (s, 9 H), 0.07 (s, 3 H), 0.00 (s, 3 H); 13 C{1H} NMR (100 MHz, CDCl3) δ 209.5, 138.5, 138.3, 128.5, 128.4, 127.9, 127.8, 127.7, 127.6, 105.9, 79.3, 78.6, 73.9, 73.3, 72.0, 65.4, 56.7, 56.0, 38.9, 26.1, 18.5, -4.3, -4.8; HRMS (ESI) m/z calcd for C29H44O7SiNa [M + Na]+ 555.2754, found 555.2755. (4S,5R,6S)-1,6-Bis(benzyloxy)-4,5-bis((tert-butyldimethyl silyl)oxy)-7,7-dimethoxyheptan-3-one 14. To a solution of compound 13 (29.68 g, 55.76 mmol) in anhydrous DMF (300 mL) at room temperature was added imidazole (39.86 g, 585.48 mmol) and TBSCl (84.04 mg, 557.60 mmol) under argon. The resulting mixture was stirred at 60 °C in the oil bath overnight until TLC indicated complete conversion of starting material. The reaction was quenched with sat. aq. NaHCO3 and extracted with CH2Cl2 (4 × 300 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 20/1) to afford 14 (33.52 g, 93%) as a colorless syrup: []25D = +13.0 (c 0.3, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.27– 7.22 (m, 10 H), 4.70 (d-like, J = 11.2 Hz, 1 H), 4.63–4.59 (m,

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2 H), 4.27 (m, 2 H), 4.21 (m, 2 H), 3.46 (m, 2 H), 3.44 (s, 3 H), 3.34 (s, 3 H), 3.31 (m, 1 H), 2.86 (m, 1 H), 2.68 (m, 1 H), 0.95 (s, 9 H), 0.89 (s, 9 H), 0.13 (s, 6 H), 0.10 (s, 3 H), -0.04 (s, 3 H); 13C{1H} NMR (125 MHz, CDCl3) δ 207.9, 138.8, 138.6, 128.4, 128.3, 127.8, 127.7, 127.5, 127.4, 104.9, 84.2, 80.7, 76.5, 75.8, 73.1, 66.2, 57.3, 54.9, 41.0, 26.2, 26.1, 18.7, 18.2, 4.4, -4.6, -4.7, -5.3; HRMS (ESI) m/z calcd for C35H58O7Si2Na [M + Na]+ 669.3619, found 669.3623. 2,7-Di-O-benzyl-3,4-di-O-acetyl-6-deoxy-D-manno-heptopy ranosyl acetate 16. To a solution of compound 14 (8.0 g, 12.38 mmol) in anhydrous THF (120 mL) at –78 °C was added dropwise 1 M solution of L-selectride in THF (123.80 mL, 123.80 mmol) under argon. After the reaction was stirred for 12 h, the reaction mixture was warmed to room temperature, and then diethyl ether (90 mL) and sat. aq. NH 4Cl (90 mL) were added and stirred for an additional 10 min. The mixture was extracted with ethyl acetate (4 × 300 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 30/1) to afford the corresponding alcohol (6.02 g, 75%) as a colorless syrup. HRMS (ESI) m/z calcd for C35H60O7Si2Na [M + Na]+ 671.3775, found 671.3770. A solution of the above alcohol (6.02 g, 9.28 mmol) in acetic acid/water (7/3, v/v, 120 mL) was stirred at 60 °C in the oil bath for 12 h. The mixture was concentrated in vacuo and purified by silica gel column chromatography (CH2Cl2/MeOH: 60/1) to afford 15 (1.08 g) as a white solid and the corresponding intermediates. The above intermediates were added to a solution of acetic acid/water (7/3, v/v, 50 mL) and stirred at 60 °C in the oil bath for 12 h. The mixture was concentrated in vacuo and purified by silica gel column chromatography (CH2Cl2/MeOH: 60/1) to afford 15 (1.36 g). Collectively, the cyclization reaction (repeat 2 times) provided 15 (2.44 g, 70%) as white solid: HRMS (ESI) m/z calcd for C21H26O6Na [M + Na]+ 397.1627, found 397.1628. To a solution of 15 (2.44 g, 6.52 mmol) in anhydrous pyridine (200 mL) at room temperature was added dropwise acetic anhydride (62 mL, 65.20 mmol) under argon. After stirring at room temperature for 8 h, the reaction was quenched with sat. aq. NaHCO3 and extracted with CH2Cl2 (3 × 200 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 12/1) to afford 16 (3.03 g, 93%) as a -anomer: 1H NMR (400 MHz, CDCl3) δ 7.35–7.27 (m, 10 H), 6.10 (d, J = 2.0 Hz, 1 H, H-1), 5.30 (t, J = 10.0 Hz, 1 H, H-4), 5.17 (dd, J = 3.6, 10.0 Hz, 1 H, H-3), 4.72 (d-like, J = 12.4 Hz, 1 H, OCH2Ph), 4.60 (d-like, J = 12.0 Hz, 1 H, OCH2Ph), 4.47 (m, 2 H, OCH2Ph), 4.02 (dt, J = 2.4, 10.0 Hz, 1 H, H-5), 3.82 (dd, J = 2.0, 3.2 Hz, 1 H, H-2), 3.63– 3.54 (m, 2 H, H-7), 2.04 (s, 3 H), 2.03 (s, 3 H), 1.99 (s, 3 H), 1.94–1.86 (m, 1 H, H-6a), 1.81–1.72 (m, 1 H, H-6b); 13C NMR (125 MHz, CDCl3) δ 170.5, 170.0, 169.1, 138.6, 137.4, 128.6, 128.5, 128.2, 127.7, 127.6, 91.3 (C-1, 1JC1,H1 = 175.5 Hz), 74.5, 73.3, 73.2, 71.1, 69.7, 69.1, 65.8, 31.8, 21.1, 21.0, 20.9; -anomer: 1H NMR (400 MHz, CDCl3) δ 7.38–7.27 (m, 10 H), 5.69 (s, 1 H, H-1), 5.25 (t, J = 10.0 Hz, 1 H, H-4), 4.90 (dd, J = 3.2, 10.0 Hz, 1 H, H-3), 4.81 (d-like, J = 12.4 Hz, 1 H, OCH2Ph), 4.69 (d, J = 12.4 Hz, 1 H, OCH2Ph), 4.48 (m, 2 H, OCH2Ph), 4.00 (d, J = 2.8 Hz, 1 H, H-2), 3.70 (dt, J = 2.8, 10.0 Hz, 1 H, H-5), 3.65–3.56 (m, 2 H, H-7), 2.11 (s, 3 H),


Page 7 of 12 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


2.02 (s, 3 H), 1.93 (s, 3 H), 1.90–1.80 (m, 2 H, H-6); HRMS (ESI) m/z calcd for C27H32O9Na [M + Na]+ 523.1944, found 523.1945. 2,7-Di-O-benzyl-3,4-di-O-acetyl-6-deoxy--D-manno-hepto pyranosyl hydrogenphosphonate, triethylammonium salt 2. To a solution of compound 16 (69 mg, 0.14 mmol) in THF/MeOH (7/2, v/v, 1.8 mL) at room temperature was added slowly a solution of BnNH2 (165 mg, 1.54 mmol) in THF (0.3 mL). After stirred at room temperature overnight, the reaction was quenched with aq. HCl (0.5 mL, 1 N) and sat. aq. NH 4Cl, and extracted with CH2Cl2 (3 × 8.0 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 10/1→3/1) to afford the corresponding hemiacetal (45 mg, 70%) as a colorless syrup. HRMS (ESI) m/z calcd for C25H30O8Na [M + Na]+ 481.1838, found 481.1837. A solution of the above hemiacetal (14 mg, 0.03 mmol) in acetonitrile/pyridine (3/4, v/v, 0.7 mL) was added dropwise to a stirred solution of salicylchlorophosphite (10 mg, 0.05 mmol) in acetonitrile (0.3 mL) at 0 °C under argon. The mixture was stirred at room temperature for 1 h until TLC showed complete conversion of starting material. The reaction was quenched with pyridine (0.5 mL) and H2O (0.5 mL) and stirred for an additional 15 min. The mixture was extracted with CH2Cl2, and the organic layers were washed with 0.1 M triethylammonium bicarbonate (TEAB) solution, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (CH2Cl2/MeOH/Et3N: 10/1/0.1) to afford 2 (14 mg, 75%) as a white solid: []20D = +3.6 (c 0.5, CHCl3); 1H NMR (400 MHz, CDCl3) δ 12.0 (br s, 1 H), 7.29–7.16 (m, 10 H), 6.88 (d, 1JH,P = 647.2 Hz, 1 H, PH), 5.65 (d, 3JH1,P = 7.6 Hz, 1 H, H-1), 5.23–5.15 (m, 2 H), 4.65 (d-like, J = 12.0 Hz, 1 H), 4.50 (d-like, J = 12.4 Hz, 1 H), 4.40 (m, 2 H), 4.05 (dt, J = 2.4, 9.2 Hz, 1 H), 3.81 (br s, 1 H), 3.58–3.51 (m, 2 H), 2.99 (m, 6 H), 1.94 (s, 3 H), 1.88 (s, 3 H), 1.82–1.70 (m, 2 H), 1.27 (t, J = 7.2 Hz, 9 H); 13C NMR (100 MHz, CDCl3) δ 170.4, 170.1, 138.6, 137.9, 128.5, 128.1, 127.9, 127.6, 93.2 (d, 2JC1,P = 4.0 Hz, 1JC1,H1 = 173.0 Hz), 76.1 (d, J = 6.5 Hz), 73.2, 73.0, 71.3, 70.1, 68.4, 66.4, 45.8, 31.9, 21.0, 8.7; 31P NMR (162 MHz, CDCl3) δ 0.31 (dd, 1JH,P =647.4 Hz, 3JH1,P = 7.9 Hz). HRMS (ESI) m/z calcd for C25H30O10P [M – Et3N – H]– 521.1582, found 521.1578. 5-tert-Butyl-2-methylphenyl 2,7-di-O-benzyl-3-O-allyl-6deoxy-1-thio-D-manno-heptopyranoside 17. To a solution of compound 16 (900 mg, 1.80 mmol) in anhydrous CH2Cl2 (80 mL) at room temperature was added 5-tert-butyl-2methylbenzenethiol (487 mg, 2.70 mmol) and BF3·OEt2 (766 mg, 5.40 mmol) under argon. After stirring at room temperature overnight, the reaction mixture was quenched with sat. aq. NaHCO3 and extracted with CH2Cl2 (3 × 150 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was dissolved in anhydrous MeOH (30 mL) and then added NaOMe (59 mg, 1.10 mmol). After stirring at room temperature for 4 h, the reaction was neutralized with Amberlite IR120 H+ resin. The mixture was filtered, and the filtrate was concentrated in vacuo. The residue was dissolved in anhydrous toluene (18 mL) and then (Bu3Sn)2O (1.07 g, 1.80 mmol) was added. After stirring at 110 °C in the oil bath for 4 h, the temperature was cooled to 80 °C and then CsF (410 mg, 2.70

mmol,) TBAI (776 mg, 2.10 mmol), and AllBr (774 mg, 6.40 mmol) were successively added. After stirring at 80 °C in the oil bath for 24 h, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 6/1) to afford 17 (622 mg, 60% over three steps) as a colorless syrup: []20D = +20.0 (c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.51 (d, J = 2.0 Hz, 1 H), 7.36–7.23 (m, 10 H), 7.18 (dd, J = 2.0, 8.0 Hz, 1 H), 7.10 (d-like, J = 8.0 Hz, 1 H), 5.91 (m, 1 H), 5.55 (d, J = 1.2 Hz, 1 H, H-1), 5.30 (dd, J = 1.6, 17.2 Hz, 1 H), 5.21 (dd, J = 1.6, 10.4 Hz, 1 H), 4.75 (d-like, J = 12.4 Hz, 1 H), 4.64 (d-like, J = 12.8 Hz, 1 H), 4.40 (m, 2 H), 4.12 (m, 1 H), 4.06–3.96 (m, 3 H), 3.89 (dt, J = 2.0, 9.6 Hz, 1 H), 3.64–3.59 (m, 2 H), 3.55– 3.50 (m, 1 H), 2.87 (d, J = 2.0 Hz, 1 H), 2.30 (s, 3 H), 2.25– 2.18 (m, 1 H), 1.96–1.87 (m, 1 H), 1.29 (s, 9 H); 13C{1H} NMR (125 MHz, CDCl3) δ 150.0, 138.4, 138.0, 135.8, 134.6, 133.8, 130.1, 128.6, 128.5, 128.2, 128.1, 128.0, 127.8, 127.6, 124.5, 117.8, 85.4, 79.6, 75.8, 73.1, 72.4, 71.6, 70.8, 70.6, 67.1, 34.7, 32.7, 31.6, 20.3; HRMS (ESI) m/z calcd for C35H44O5SNa [M + Na]+ 599.2807, found 599.2805. 5-tert-Butyl-2-methylphenyl 2,7-di-O-benzyl-3-O-allyl-4-Obenzoyl-6-deoxy-1-thio-D-manno-heptopyranoside 18. To a solution of compound 17 (212 mg, 0.37 mmol) in anhydrous pyridine (1.3 mL) at 0 °C was added DMAP (13 mg, 0.11 mmol) and benzoyl chloride (155 mg, 1.10 mmol). The mixture was stirred at 90 °C in the oil bath for 12 h until TLC indicated complete conversion of starting material. The reaction was quenched with sat. aq. NaHCO3 and extracted with CH2Cl2 (4 × 10 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 12/1) to afford 18 (202 mg, 80%) as a white syrup: []20D = +45.3 (c 0.1, CHCl3); 1 H NMR (400 MHz, CDCl3) δ 8.09–7.11 (m, 18 H), 5.78–5.69 (m, 1 H), 5.60 (s, 1 H, H-1), 5.58 (t, J = 10.0 Hz, 1 H, H-4), 5.20 (dd, J = 1.6, 17.2 Hz, 1 H), 5.09 (dd, J = 1.2, 10.4 Hz, 1 H), 4.80 (s, 2 H), 4.41 (dt, J = 4.0, 9.6 Hz, 1 H, H-5), 4.25 (m, 2 H), 4.10 (dd, J = 2.4, 2.8 Hz, 1 H, H-2), 4.05 (dd, J = 5.6, 12.8 Hz, 1 H), 3.94 (dd, J = 5.6, 12.8 Hz, 1 H), 3.90 (dd, J = 2.8, 9.6 Hz, 1 H, H-3), 3.58–3.48 (m, 2 H, H-7), 2.32 (s, 3 H), 1.98–1.93 (m, 2 H, H-6), 1.31 (s, 9 H); 13C{1H} NMR (125 MHz, CDCl3) δ 165.8, 150.0, 138.6, 138.1, 135.5, 134.5, 133.7, 133.2, 130.2, 130.0, 128.6, 128.5, 128.4, 128.2, 128.0, 127.6, 127.5, 127.4, 124.4, 117.5, 85.6, 77.7, 76.7, 73.0, 72.9, 72.4, 71.4, 69.4, 66.7, 34.8, 32.0, 31.6, 20.3; HRMS (ESI) m/z calcd for C42H48O6SNa [M + Na]+ 703.3069, found 703.3068. 2,7-Di-O-benzyl-3-O-allyl-4-O-benzoyl-6-deoxy-D-mannoheptopyranosyl ortho-hexynylbenzoate 3. To a solution of thioglycoside 18 (202 mg, 0.30 mmol) in acetonitrile (10 mL) at room temperature was successively added DTBP (220 mg, 1.15 mmol), AgOTf (229 mg, 0.89 mmol), H 2O (0.175 mL) and NBS (158 mg, 0.89 mmol). After stirring at room temperature for 1.5 h, the reaction was quenched with sat. aq. Na2S2O3 and extracted with EtOAc (4 × 30 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude residue was dissolved in anhydrous CH2Cl2 (5.0 mL), and then DMAP (49 mg, 0.40 mmol), EDCI (96 mg, 0.50 mmol) and orthohexynylbenzoic acid (61 mg, 0.30 mmol) were added. After stirring at room temperature for 1 h under argon, the reaction was quenched with sat. aq. NaHCO3 and extracted with



CH2Cl2 (4 × 10 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 40/1→30/1) to afford 3 (167 mg, 79%, α:β = 1:2.5) as a colorless syrup. 3： []20D = 11.8 (c 0.4, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.06–7.17 (m, 19 H), 6.45 (d, J = 2.0 Hz, 1 H, H-1), 5.77–5.67 (m, 1 H), 5.62 (t, J = 9.6 Hz, 1 H, H-4), 5.15 (dd, J = 1.6, 17.2 Hz, 1 H), 5.05 (dd, J = 1.2, 10.4 Hz, 1 H), 4.86 (m, 2 H), 4.38 (s, 2 H), 4.28 (dt, J = 2.8, 10.0 Hz, 1 H, H-5), 4.04–4.00 (m, 3 H), 3.93 (dd, J = 5.6, 12.8 Hz, 1 H), 3.63 (m, 1 H, H-7a), 3.55 (m, 1 H, H-7b), 2.50 (t, J = 6.8 Hz, 2 H), 2.02–1.94 (m, 1 H, H-6a), 1.92–1.84 (m, 1 H, H-6b), 1.63–1.57 (m, 4 H), 1.52– 1.43 (m, 2 H), 0.94 (t, J = 7.2 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 165.8, 164.4, 138.5, 137.9, 135.0, 134.5, 133.2, 132.2, 130.8, 130.6, 130.1, 129.9, 128.5, 128.3, 128.1, 127.9, 127.6, 127.4, 125.1, 117.3, 96.8, 93.0 ( 1JC1,H1 = 174.5 Hz), 79.8, 76.9, 74.1, 73.2, 73.1, 71.8, 71.3, 70.2, 65.9, 32.0, 30.9, 22.2, 19.8, 13.8; HRMS (ESI) m/z calcd for C44H46O8Na [M + Na] + 725.3090, found 725.3089. 3β：[] D20 = –17.1 (c 0.4, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.06–7.20 (m, 19 H), 5.96 (d, J = 0.8 Hz, 1 H, H-1), 5.76–5.66 (m, 1 H), 5.55 (t, J = 9.6 Hz, 1 H, H-4), 5.18 (dd, J = 1.6, 17.2 Hz, 1 H), 5.09 (dd, J = 1.6, 10.4 Hz, 1 H), 4.91 (m, 2 H), 4.45 (m, 2 H), 4.11 (d-like, J = 1.6 Hz, 1 H, H-2), 4.04 (dd, J = 5.6, 13.2 Hz, 1 H), 3.95– 3.85 (m, 2 H), 3.72 (dd, J = 2.8, 9.6 Hz, 1 H, H-3), 3.66 (m, 1 H, H-7a), 3.57 (m, 1 H, H-7b), 2.46 (t, J = 7.2 Hz, 2 H), 2.00– 1.94 (m, 2 H, H-6), 1.65–1.58 (m, 4 H), 1.52–1.43 (m, 2 H), 0.93 (d, J = 7.2 Hz, 3 H); 13C NMR (125 MHz, CDCl3) δ 165.8, 163.8, 138.7, 138.2, 134.7, 134.4, 133.3, 132.3, 130.8, 130.5, 130.0, 129.9, 128.5, 128.4, 128.3, 127.8, 127.7, 127.5, 127.2, 125.7, 117.6, 97.1, 93.6 (1JC1,H1 = 160.1 Hz), 79.4, 79.2, 74.4, 73.9, 73.2, 72.4, 71.9, 71.3, 66.0, 32.0, 30.8, 22.2, 19.7, 13.8; HRMS (ESI) m/z calcd for C44H46O8Na [M + Na]+ 725.3090, found 725.3089. N-Benzyl-benzyloxycarbonyl-5-aminopentyl 2,7-di-O-benzyl -3,4-di-O-acetyl-6-deoxy--D-manno-heptopyranoside 20. To a solution of compound 16 (1.24 g, 2.48 mmol) and linker 19 (1.05 g, 3.22 mmol) in anhydrous CH2Cl2 (35 mL) at 0 °C was added BF3·OEt2 (0.92 mL, 7.44 mmol) under argon. After stirring at room temperature overnight, the reaction mixture was quenched with sat. aq. NaHCO3 and extracted with CH2Cl2 (3 × 100 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 12/1) to afford 20 (1.66 g, 87%) as a colorless syrup: [] 20D = +12.7 (c 0.8, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.36–7.17 (m, 20 H), 5.26–5.15 (m, 4 H), 4.72 (d-like, J = 6.4 Hz, 1 H), 4.63 (m, 2 H), 4.46 (m, 4 H), 3.89 (t-like, J = 9.6 Hz, 1 H), 3.79 (br s, 1 H), 3.65 (m, 1 H), 3.58 (m, 2 H), 3.25–3.13 (m, 3 H), 2.02 (s, 3 H), 1.99 (s, 3 H), 1.93–1.86 (m, 1 H), 1.81–1.72 (m, 1 H), 1.49–1.43 (m, 4 H), 1.22–1.14 (m, 2 H); 13C NMR (125 MHz, CDCl3) δ 170.4, 170.2, 156.8, 156.3, 138.5, 138.1, 137.9, 137.1, 136.9, 128.7, 128.5, 128.4, 128.0, 127.9, 127.7, 127.6, 127.4, 127.3, 97.7 (1JC1,H1 = 168.1 Hz), 75.9, 73.4, 73.1, 71.9, 70.5, 67.5, 67.3, 66.7, 66.2, 50.6, 50.3, 47.2, 46.2, 31.7, 29.2, 28.1, 27.6, 23.5, 21.1, 21.0; HRMS (ESI) m/z calcd for C45H53O10NNa [M + Na]+ 790.3567, found 790.3568. N-Benzyl-benzyloxycarbonyl-5-aminopentyl 2,7-di-Obenzyl-3-O-allyl-6-deoxy--D-manno-heptopyranoside 21. To

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a solution of compound 20 (1.66 g, 2.163 mmol) in anhydrous MeOH (50 mL) at room temperature was added NaOMe (82 mg, 1.43 mmol) under argon. After stirring at room temperature for 4 h until TLC indicated complete conversion of starting material, the reaction was neutralized with Amberlite IR120 H+ resin. The mixture was filtered, and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 6/1) to afford the corresponding diol as a colorless syrup: []20D = +9.1 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.27–7.08 (m, 20 H), 5.09 (d-like, J = 14.0 Hz, 2 H), 4.70 (br s, 1 H), 4.64 (dlike, J = 11.6 Hz, 1 H), 4.51 (d-like, J = 11.6 Hz, 1 H), 4.45– 4.38 (m, 4 H), 3.68 (br s, 1 H), 3.62–3.44 (m, 6 H), 3.19–3.08 (m, 3 H), 2.31 (brs, 1H), 2.12 (m, 1 H), 1.75 (m, 1 H), 1.42– 1.35 (m, 4 H), 1.11 (m, 2 H); HRMS (ESI) m/z calcd for C41H49O8NNa [M + Na]+ 706.3356, found 706.3358. To a solution of the above diol in anhydrous toluene (25 mL) at room temperature was added (Bu3Sn)2O (1.26 g, 2.12 mmol). After the mixture was stirred at 110 °C in the oil bath for 4 h, the temperature was cooled to 80 °C and then CsF (556 mg, 3.66 mmol,) TBAI (942 mg, 2.55 mmol), and AllBr (769 mg, 6.36 mmol) were successively added. After stirring at 80 °C in the oil bath for 24 h, the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 6/1) to afford 21 (890 mg, 57%) as a colorless syrup: [] 20D = +2.5 (c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.35–7.17 (m, 20 H), 5.95–5.86 (m, 1 H), 5.27 (d-like, J = 17.2 Hz, 1 H), 5.17 (dlike, J = 10.8 Hz, 3 H), 4.75–4.70 (m, 3 H), 4.52–4.46 (m, 4 H), 4.04 (dd, J = 5.2, 12.4 Hz, 1 H), 3.98 (m, 1 H), 3.78 (t, J = 9.6 Hz, 1 H), 3.72 (br s, 1 H), 3.64 (m, 3 H), 3.55 (m, 2 H), 3.26–3.14 (m, 3 H), 2.23 (m, 1 H), 1.81 (m, 1 H), 1.45 (m, 4 H), 1.20 (m, 2 H); 13C{1H} NMR (125 MHz, CDCl3) δ 138.6, 138.5, 138.1, 134.8, 128.7, 128.6, 128.5, 128.4, 128.1, 128.0, 127.9, 127.8, 127.7, 127.5, 127.3, 117.3, 98.1, 79.8, 74.1, 73.0, 72.9, 70.8, 70.6, 69.6, 67.4, 67.2, 66.9, 50.6, 50.4, 47.3, 46.2, 32.4, 29.2, 28.1, 27.7, 23.7; HRMS (ESI) m/z calcd for C44H53O8NNa [M + Na]+ 746.3669, found 746.3668. N-Benzyl-benzyloxycarbonyl-5-aminopentyl 2,7-di-Obenzyl-4-O-acetyl-6-deoxy--D-manno-heptopyranoside 4. To a solution of compound 21 (177 mg, 0.24 mmol) in anhydrous pyridine (5.0 mL) under argon was added dropwise acetic anhydride (0.23 mL, 2.40 mmol). After stirring at room temperature for 8 h, the reaction was quenched with sat. aq. NaHCO3 and extracted with CH2Cl2 (3 × 10 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was dissolved in anhydrous MeOH (11 mL) and then PdCl2 (28 mg, 0.16 mmol) was added. After stirring at room temperature for 3 h at room temperature, the mixture was diluted with CH2Cl2 (30 mL) and filtered. The filtrate was concentrated in vacuo, and the residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 4/1) to afford 4 (156 mg, 90% over two steps) as a colorless syrup: []D25 = +5.4 (c 0.1, CHCl3); 1H NMR (500 MHz, CDCl3) δ 7.35–7.17 (m, 20 H), 5.17 (d-like, J = 16.5 Hz, 2 H), 4.91 (t, J = 9.5 Hz, 1 H, H-4), 4.77 (d-like, J = 7.0 Hz, 1 H), 4.72 (d-like, J = 11.5 Hz, 1 H), 4.57 (d-like, J = 11.5 Hz, 1 H), 4.46 (m, 4 H, H-1 and OCH2Ph), 3.84–3.78 (m, 2 H, H-3 and H-5), 3.70 (br s, 1 H, H-2), 3.65–3.61 (m, 1 H, H-7a), 3.59–3.55 (m, 2 H), 3.29–3.14 (m, 3 H), 2.26 (d, J = 11.0 Hz, 1 H, OH), 2.08 (s, 3 H), 1.94–1.88 (m, 1 H, H-6a),


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1.74–1.69 (m, 1 H, H-6b), 1.45 (m, 4 H), 1.18 (m, 2 H); C{1H} NMR (100 MHz, CDCl3) δ 171.3, 156.8, 156.3, 138.4, 138.0, 137.7, 137.0, 136.9, 128.7, 128.6, 128.5, 128.2, 128.0, 127.9, 127.7, 127.4, 127.3, 96.7, 78.6, 73.7, 73.3, 73.2, 70.0, 67.3, 66.3, 50.6, 50.3, 47.1, 46.2, 31.7, 29.2, 28.1, 27.6, 23.5, 21.2; HRMS (ESI) m/z calcd for C43H51O9NNa [M + Na]+ 748.3462, found 748.3461. N-Benzyl-benzyloxycarbonyl-5-aminopentyl (2,7-di-Obenzyl-3-O-allyl-4-O-benzoyl-6-deoxy-D-manno-heptopyra nosyl)-(1→3)-2,7-di-O-benzyl-4-O-acetyl-6-deoxy--Dmanno-heptopyranoside 22. A mixture of donor 3(52 mg, 0.072 mmol), acceptor 4 (43 mg, 0.06 mmol), 4Å MS (95 mg) and Ph3PAuCl (10 mg, 0.021 mmol) in anhydrous PhCl (1.8 mL) was stirred at –42 °C for 30 min under argon. A solution of AgBAr4F (91 μL, 0.23 M in Et2O) was added. The mixture was stirred at the same temperature for 12 h followed by addition of Ph3P (15 mg) to quench the reaction. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 22/1→8/1) to provide 22 (12 mg, 16%) as a colorless syrup and 22 (60 mg, 81%) as a colorless syrup. A mixture of donor 3 (43 mg, 0.06 mmol), acceptor 4 (35 mg, 0.05 mmol), 4Å MS (78 mg) and Ph3PAuCl (8 mg, 0.017 mmol) in anhydrous PhCl (1.5 mL) was stirred at -42 °C for 30 min under argon. A solution of AgBAr4F (74 μL, 0.23 M in Et2O) was added. The mixture was stirred at the same temperature for 12 h followed by addition of Ph3P (12 mg) to quench the reaction. The mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 22/1→8/1) to provide 22 (15 mg, 25%) as a colorless syrup and 22 (42 mg, 69%) as a colorless syrup. 22: []20D = +1.4 (c 0.1, CHCl3); 1H NMR (500 MHz, CDCl3) δ 8.07–7.22 (m, 35 H), 5.74–5.67 (m, 1 H), 5.47 (t, J = 9.5 Hz, 1 H), 5.22–5.12 (m, 4 H), 5.02 (m, 2 H), 4.83 (d-like, J = 12.5 Hz, 1 H), 4.72 (d, J = 12.5 Hz, 1 H), 4.64 (m, 2 H), 4.45 (m, 5 H), 4.32 (m, 2 H), 4.10–4.02 (m, 3 H), 3.93–3.87 (m, 3 H), 3.83–3.76 (m, 2 H), 3.62 (m, 2 H), 3.55 (m, 2 H), 3.45 (br s, 1 H), 3.24–3.12 (m, 3 H), 1.92 (m, 1 H), 1.85–1.79 (m, 5 H), 1.75–1.69 (m, 1 H), 1.45 (m, 4 H), 1.20 (m, 2 H); 13 C NMR (125 MHz, CDCl3) δ 170.0, 165.8, 156.8, 156.3, 138.8, 138.6, 138.5, 138.4, 138.0, 137.0, 136.9, 134.5, 133.2, 130.2, 129.9, 128.7, 128.6, 128.5, 128.4, 128.1, 128.0, 127.9, 127.7, 127.6, 127.5, 127.4, 127.3, 117.5, 100.9 ( 1JC1′,H1′ = 171.5 Hz), 97.7 (1JC1,H1 = 167.4 Hz), 78.2, 77.3, 76.5, 74.8, 73.2, 73.1, 73.0, 72.7, 72.6, 72.5, 71.5, 67.9, 67.3, 67.0, 66.3, 65.9, 50.6, 50.3, 47.2, 46.3, 32.2, 31.7, 29.5, 28.2, 27.7, 23.6, 20.9; HRMS (ESI) m/z calcd for C74H83O15NNa [M + Na]+ 1248.5660, found 1248.5658. 22: [] D20 = –19.2 (c 0.1, CHCl3); 1H NMR (500 MHz, CDCl3) δ 8.04–7.15 (m, 35 H), 5.67–5.60 (m, 1 H), 5.36 (t, J = 9.5 Hz, 1 H), 5.31 (t, J = 9.5 Hz, 1 H), 5.17 (d-like, J = 13.0 Hz, 2 H), 5.10 (d-like, J = 17.0 Hz, 1 H), 5.03 (d-like, J = 10.5 Hz, 1 H), 4.90 (m, 2 H), 4.70 (t, J = 10.0 Hz, 2 H), 4.49–4.35 (m, 7 H), 4.21 (m, 2 H), 3.85 (m, 2 H), 3.77 (br s, 1 H), 3.69 (m, 4 H), 3.59 (m, 3 H), 3.44 (m, 1 H), 3.28 (m, 2 H), 3.14 (m, 2 H), 2.01–1.93 (m, 5 H), 1.85– 1.80 (m, 2 H), 1.45 (m, 4 H), 1.17 (m, 2 H); 13C NMR (125 MHz, CDCl3) δ 170.6, 165.8, 156.8, 156.3, 139.0, 138.7, 138.5, 138.0, 137.0, 136.9, 134.8, 133.2, 130.3, 129.9, 128.7, 128.6, 128.5, 128.2, 128.1, 128.0, 127.9, 127.7, 127.6, 127.5, 127.4, 127.3, 117.0, 97.6 (1JC1′,H1′ = 154.0 Hz), 97.0 (1JC1,H1 = 13

166.0 Hz), 79.0, 73.9, 73.8, 73.7, 73.2, 73.0, 72.2, 71.0, 70.7, 70.5, 67.3, 67.1, 66.3, 66.0, 50.6, 50.3, 47.1, 46.2, 31.8, 31.7, 29.3, 28.1, 27.6, 23.5, 21.3; HRMS (ESI) m/z calcd for C74H83O15NNa [M + Na]+ 1248.5660, found 1248.5662. N-Benzyl-benzyloxycarbonyl-5-aminopentyl (2,7-di-Obenzyl-4-O-benzoyl-6-deoxy--D-manno-heptopyranosyl)(1→3)-2,7-di-O-benzyl-4-O-acetyl-6-deoxy--D-mannoheptopyranoside 23. To a solution of disaccharide 22 (40 mg, 0.03 mmol) in anhydrous MeOH (2 mL) was added PdCl 2 (4 mg, 0.02 mmol). After stirred at room temperature for 3 h under argon, the mixture was diluted with CH2Cl2 (5.0 mL) and filtered. The filtrate was concentrated in vacuo, and the residue was purified by silica gel column chromatography (petroleum ether/EtOAc: 4/1→2/1) to afford 23 (32 mg, 90%) as a colorless syrup: []20D = –11.2 (c 0.1, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.02–7.14 (m, 35 H), 5.27 (t, J = 10.0 Hz, 1 H), 5.15 (d-like, J = 9.2 Hz, 2 H), 5.06 (t, J = 9.6 Hz, 1 H), 5.00 (d-like, J = 12.0 Hz, 1 H), 4.87 (s, 1 H), 4.71 (d-like, J = 12.4 Hz, 1 H), 4.52–4.38 (m, 7 H), 4.27 (s, 1 H), 4.16 (dd, J = 3.2, 10.0 Hz, 1 H), 3.86 (t, J = 9.6 Hz, 1 H), 3.77 (br s, 1 H), 3.70–3.64 (m, 3 H), 3.59 (m, 3 H), 3.45 (m, 2 H), 3.30–3.09 (m, 4 H), 2.39 (d, J = 10.4 Hz, 1 H), 1.97–1.93 (m, 5 H), 1.82– 1.75 (m, 2 H), 1.42 (m, 4 H), 1.16 (m, 2 H); 13C{1H} NMR (125 MHz, CDCl3) δ 170.5, 166.5, 156.8, 156.3, 138.6, 138.5, 138.4, 138.0, 137.9, 137.0, 136.9, 133.3, 130.0, 128.7, 128.6, 128.5, 128.3, 128.2, 128.0, 127.9, 127.8, 127.7, 127.5, 127.3, 97.9, 97.1, 77.6, 74.8, 74.2, 74.1, 73.6, 73.2, 73.1, 72.5, 72.2, 70.7, 70.6, 67.4, 67.3, 67.0, 66.3, 66.0, 50.6, 50.3, 47.2, 46.2, 31.8, 31.7, 29.3, 28.1, 27.7, 23.5, 21.2; HRMS (ESI) m/z calcd for C71H79O15NNa [M + Na]+ 1208.5347, found 1208.5345. N-Benzyl-benzyloxycarbonyl-5-aminopentyl (2,7-di-Obenzyl-3,4-di-O-acetyl-6-deoxy--D-manno-heptopyranosyl phosphonate)-(1→3)-(2,7-di-O-benzyl-4-O-benzoyl-6-deoxy-D-manno-heptopyranosyl)-(1→3)-2,7-di-O-benzyl-4-Oacetyl-6-deoxy--D-manno-heptopyranoside triethylammonium salt 24. To a mixture of hydrogenphosphate 2 (31 mg, 0.05 mmol) and disaccharide acceptor 23 (49 mg, 0.04 mmol) in anhydrous pyridine (2.5 mL) was added pivaloyl chloride (25 mg, 0.20 mmol). The mixture was stirred at room temperature for 1 h under argon until TLC indicated complete conversion of starting material. The reaction was cooled to 0 °C and a solution of iodine (48 mg, 0.20 mmol) in pyridine/H 2O (19/1, v/v, 0.5 mL) was added slowly. After stirring at 0 °C for 0.5 h, the mixture was diluted with CH2Cl2, washed with sat. aq. Na2S2O3 and 0.1 M TEAB solution. The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (CH2Cl2/MeOH/Et3N: 20/1/0.1) to afford 24 (53 mg, 74%) as a colorless syrup: []D20 = –13.7 (c 0.8, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.12–7.13 (m, 45 H), 5.52 (dd, J = 1.6, 6.8 Hz, 1 H, H-1′′), 5.42 (t, J = 9.6 Hz, 1 H), 5.26–5.20 (m, 3 H), 5.14 (d-like, J = 7.2 Hz, 2 H), 5.03 (d-like, J = 12.0 Hz, 1 H), 4.76 (br s, 1 H, H-1′), 4.71 (dd, J = 4.4, 12.0 Hz, 2 H), 4.58 (d-like, J = 11.6 Hz, 1 H), 4.57 (br s, 1 H, H-1), 4.51– 4.33 (m, 11 H), 4.13 (dd, J = 2.8, 9.6 Hz, 1 H), 3.99 (br s, 2 H), 3.83–3.75 (m, 2 H), 3.63–3.46 (m, 8 H), 3.30–3.07 (m, 4 H), 2.70 (m, 6 H), 1.98 (s, 3 H), 1.87 (s, 3 H), 1.81 (s, 3 H), 1.80– 1.72 (m, 6 H), 1.39 (m, 4 H), 1.14 (m, 2 H), 1.00 (t, J = 7.2 Hz, 9 H); 13C NMR (125 MHz, CDCl3) δ 170.4, 170.2, 170.0, 166.5, 156.8, 156.3, 140.1, 138.8, 138.6, 138.4, 138.2, 138.0, 137.0, 136.9, 133.2, 130.2, 128.7, 128.6, 128.5, 128.4, 128.3,



128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.3, 127.1, 126.8, 98.2 (1JC1′,H1′ = 155.0 Hz, C-1′), 97.6 (1JC1,H1 = 168.0 Hz, C-1), 93.7 (1JC1′′,H1′′ = 172.0 Hz, C-1′′), 77.9, 76.7, 75.5, 75.0, 74.6, 73.2, 73.1, 72.9, 72.7, 72.3, 72.2, 71.2, 70.8, 70.7, 70.0, 68.8, 67.3, 66.7, 66.4, 66.2, 50.6, 50.3, 47.2, 46.2, 45.6, 32.0, 31.8, 31.7, 29.2, 28.1, 27.6, 23.4, 21.0, 20.9, 8.5; 31P NMR (162 MHz, CDCl3) δ -4.1. HRMS (ESI) m/z calcd for C96H107O25NP [M – Et3N – H]– 1704.6875, found 1704.6893. 5-Aminopentyl (6-deoxy--D-manno-heptopyranosyl phosphonate)-(1→3)-(6-deoxy--D-manno-heptopyranosyl)(1→3)-6-deoxy--D-manno-heptopyranoside triethylammonium salt 1. Compound 24 (18 mg, 0.01 mmol) was dissolved in a solution of sodium methoxide in methanol (1.2 M, 1 mL). The reaction mixture was stirred at room temperature overnight. Concentration in vacuo and purification by silica gel column chromatography (CH2Cl2/MeOH/Et3N: 20/1/0.01) gave the corresponding tetraol as a colorless syrup. A mixture of the resulting tetraol and Pd/C (100 mg, 10%) in the methanol/water (5/1, v/v, 5.4 mL) was stirred under an atmosphere of H2 at room temperature for 48 h. Filtration, concentration in vacuo and elution through Sephadex LH-20 column (H2O + 0.2% Et3N) provided 1 (7 mg, 86% over two steps) as a white solid: []D20 = 4.4 (c 0.25, H2O); 1H NMR (500 MHz, D2O) δ 5.50 (d, J = 6.5 Hz, 1 H, H-1′′), 4.91 (s, 1 H, H-1), 4.88 (s, 1 H, H-1′), 4.29 (d, J = 2.5 Hz, 1 H, H-2′), 4.19 (m, 2 H, H-2 and H-3′), 4.07 (br s, 1 H, H-2′′), 4.03 (dd, J = 3.0, 9.0 Hz, 1 H, H3), 3.96–3.91 (m, 2 H), 3.86–3.76 (m, 8 H), 3.74–3.66 (m, 2 H), 3.60 (m, 2 H), 3.52 (m, 1 H), 3.16–3.06 (m, 2 H), 2.18 (m, 3 H), 1.85–1.71 (m, 7 H), 1.55–1.46 (m, 2 H); 13C{1H} NMR (125 MHz, D2O) δ 99.2 (C-1), 96.3 (C-1′), 95.9 (2JC1,P = 4.9 Hz, C-1′′), 77.9 (2JC3′,P = 5.8 Hz), 77.1, 72.6, 70.3, 70.2, 70.1, 70.0, 69.8, 69.1, 69.0, 68.5, 67.2, 66.7, 58.1, 58.0, 46.6, 39.3, 33.4, 33.2, 33.1, 27.9, 26.5, 22.5, 8.1; 31P NMR (202 MHz, D2O) δ -2.6. HRMS (ESI) m/z calcd for C26H49O19NP [M – Et3N – H]– 710.2642, found 710.2635.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ NMR spectra for new compounds (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

ORCID You Yang: 0000-0003-4438-2162

Author Contributions †

X.W., and Y.C. contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Financial support from the National Thousand Young Talents Program (Y100-4Q-1701, YC0140103), the National Natural Science Foundation of China (21871081) and the Fundamental Research Funds for the Central Universities (22221818014) is gratefully acknowledged.

REFERENCES

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Total Synthesis of the Trisaccharide Antigen of the Campylobacter

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