Stereoselective Synthesis of 2-Azido-2-deoxy-β-D-mannosides via Cs₂CO₃-Mediated Anomeric O-Alkylation with Primary Triflates: Synthesis of a Tetrasaccharide Fragment of Micrococcus Luteus Teichuronic Acid

Abstract

Cesium carbonate-mediated anomeric O-alkylation of various protected 2-azido-2-deoxy-D-mannoses with primary triflate electrophiles afforded corresponding 2-azido-2-deoxy-β-mannosides in good yields and excellent anomeric selectivity. In addition, 1,3-dibromo-5,5-dimethylhydantoin was found to be the optimal oxidant for preparation of those 2-azido-2-deoxy-D-mannoses from their corresponding thioglycosides. The utilization of this method was demonstrated in the synthesis of a tetrasaccharide fragment of Micrococcus luteus teichuronic acid containing N-acetyl-β-D-mannosaminuronic acid (ManNAcA).

Graphical Abstract

INTRODUCTION

N-Acetyl-β-mannosamines, i.e. β-ManNAc, exist in a number of bacterial capsular polysaccharides (CPS) and lipopolysaccharides, especially those from harmful bacteria strains.¹ Recently, chemical synthesis of structurally well-defined bacterial capsular polysaccharides (CPS) and lipopolysaccharides or their fragments has attracted wide attention due to various biological purposes. For instance, use of glycan-based antimicrobial vaccines consisting of antigenic oligosaccharides or the fragments has been proven as a viable approach for treatment of bacterial infections which also help reduce the reliance on antibiotic treatment. As a class of 1,2-cis-glycosidic linkages,^2–4 2-amino-2-deoxy-β-mannosides are difficult to construct due to the steric effect and the absence of anomeric effect. Although great success has been achieved in the chemical synthesis of β-mannosides,^5–11 stereoselective construction of 2-amino-2-deoxy-β-mannosides is rather underexplored.^1,12,13 For instance, stereoselective synthesis of 2-amino-2-deoxy-β-mannosides has been accomplished either through S_N2 inversion of the C2 triflate of β-glucopyranosides by azide anion^14–19 or involving stereoselective reduction of β-glucopyranosides-derived C2 O-acyloxime.¹ In addition, application of 4,6-O-benzylidene protecting group,^20–22 a well-established strategy for construction of β-mannosides,^23–25 or (S)-4,6-O-pyruvyl ketal,²⁶ in the synthesis 2-azido-2-deoxy-β-mannosides afforded poor to moderate anomeric selectivity. Recently, van der Marel, Codee, and co-workers reported that synthesis of 2-azido-2-deoxy-β-mannuronic acid (ManN₃A) can be achieved with good to excellent anomeric stereoselectivity by using mannosazide methyl uronate donors.^27–28 Despite all of the above-mentioned success, it is appealing to develop mild and easily operable approaches for the stereoselective synthesis of β-mannosamines and β-mannosaminuronic acids.

Early in 2016, our laboratory reported a mild and easily operable method for the stereoselective synthesis of β-mannopyranosides via Cs₂CO₃-mediated anomeric O-alkylation of mannose-derived lactols (a, Scheme 1).²⁹ We also found that this Cs₂CO₃-mediated anomeric O-alkylation was also amenable to the stereoselective construction of 2-deoxy-β-glycosides.²⁹ Based on those results, we wondered if similar strategy could be employed for stereoselective synthesis of 2-azido-2-deoxy-β-mannosides (b, Scheme 1). In addition, the utilization of this method, if successfully developed, may be applied to the stereoselective syntheses of bacterial capsular polysaccharides containing N-Acetyl-β-mannosamines (β-ManNAc) or N-acetyl-β-D-mannosaminuronic acids (ManNAcA), e.g. a tetrasaccharide fragment of Micrococcus luteus teichuronic acid (6, Figure 1).

Scheme 1.

Stereoselective synthesis of β-mannoside-type linkages via anomeric O-alkylation.

Figure 1.

Tetrasaccharide fragment of Micrococcus luteus teichuronic acid (6).

RESULTS AND DISCUSSION

Our studies commenced with the preparation of various fully or partially protected 2-azido-2-deoxy-D-mannoses. As shown in Scheme 2, borinic acid-catalyzed regioselective benzylation of the diol of the known phenyl 2-azido-3-O-benzyl-2-deoxy-1-thio-β-D-mannopyranoside 7²⁷ afforded desired product phenyl 2-azido-3,6-di-O-benzyl-2-deoxy-1-thio-β-D-mannopyranoside 8 (quantitative yield). Likewise, regioselective p-methoxybenzylation of the diol in 7 afforded 10 in 90% yield. Next, thioglycoside 8 and 10 were subjected to standard N-bromosuccinimide (NBS)-mediated oxidation³⁰ to prepare corresponding lactol 9 and 11, respectively. However, it was found that the byproduct succinimide was inseparable from lactol 9 or 11, regardless extensive efforts we carried out. Therefore, we started to investigate various oxidants for this transformation (cf. Table 1).

Scheme 2.

Synthesis of 2-azido-2-deoxy-D-mannosamines.

Conditions: a) KI, K₂CO₃, 10 mol% Ph₂BO(CH₂)₂NH₂, BnBr, CH₃CN, 40 °C; b) 1,3-dibromo-5,5-dimethylhydantoin, acetone/H₂O(15/1, v/v), 0 °C; c) KI, K₂CO₃, 10 mol% Ph₂BO(CH₂)₂NH₂, PMBCl, CH₃CN, 40 °C; d) NaH, BnBr, DMF, 0 °C to RT; e) Ac₂O, Pyridine, DMAP, CH₂Cl₂, 0 °C to RT; f) NBS, acetone/H₂O (15/1, v/v), RT.

Table 1.

Screening of various oxidants for the synthesis of 2-azido-2-deoxy-D-mannose 11.^a,b

Entry	Oxidant	Time	Yield
1	N-bromosuccinimide (NBS), 1.5 eq.	1 h	79%c
2	Br2 Solution (3.0 M in dichloromethane, 3.0 eq.)d	1 h	trace
3	2,4,4,6-tetrabromo-2,5-cyclohexadienone, 1.5 eq.	12 h	trace
4	5,5-dibromomeldrums acid, 1.5 eq.	12 h	no reaction
5	N-bromophthalimide, 1.5 eq.	12 h	58%
6	1,3-dibromo-5,5-dimethylhydantoin, 1.2 eq.	15 min	79%
7	dibromoisocyanuric acid, 1.5 eq.	15 min	60%
8	trichloroisocyanuric acid, 1.5 eq.	15 min	30%

^aGeneral condtions: thioglycoside 10 (1.0 eq.), oxidant, acetone/H₂O (15/1, v/v), 0 °C;

^bIsolated yields.

^cContaminated with succinimide.

^dReaction was carried out in CH₃CN/H₂O (10/1, v/v), 0 °C.

Thioglycoside 10 was chosen as a model substrate for screening of various bromine-based oxidants. As shown in Table 1, use of NBS as oxidant did furnish desired corresponding lactol 11, but upon purification via silica gel column chromatography lactol 11 was contaminated with succinimide which was found to be inseparable, despite various attempts (entry 1, Table 1). Use of bromine solution in dichloromethane³¹ or 2,4,4,6-tetrabromo-2,5-cyclohexadienone for oxidation of thioglycoside 10 only led to the trace amount of corresponding lactol 11 (entries 2 and 3). Among other oxidants including 5,5-dibromomeldrums acid, N-bromophthalimide, dibromoisocyanuric acid, and trichloroisocyanuric acid, 1,3-dibromo-5,5-dimethylhydantoin was discovered to be the optimal which smoothly oxidized thioglycoside 10 to afford corresponding pure lactol 11 in 79% yield. Gratifyingly, no contamination of the byproduct was detected. In addition, 1,3-dibromo-5,5-dimethylhydantoin was able to oxidize thioglycoside 8 to afford corresponding lactol 9 in 94% yield after purification.

Next, two additional 2-azido-2-deoxy-D-mannoses were prepared. As shown in Scheme 2, the C4-free alcohol of thioglycoside 10 was either benzylated or acetylated to give corresponding fully protected thioglycosides 12 (82%) or 13 (88%), respectively. While oxidation of thioglycoside 12 using 1,3-dibromo-5,5-dimethylhydantoin afforded corresponding lactol 14 in 85% yield, it was found that NBS was a better oxidant for thioglycoside 13 and pure lactol 15 was obtained in quantitative yield.

With all of the 2-azido-2-deoxy-D-mannoses in hand, we set to investigate the anomeric O-alkylation of those lactols with electrophiles (Table 2). To our delight, cesium carbonate-mediated anomeric O-alkylation of known 2-azido-2-deoxy-3,4,6-tri-O-benzyl-D-mannose (16)³² with known primary triflate 17 under standard condition (16 (1.0 eq.), 17 (2.0 eq.), Cs₂CO₃ (2.5 eq.), ClCH₂CH₂Cl, 40 °C, 24 h) furnished desired 2-azido-2-deoxy-β-D-mannoside 19 in 81% yield (β only). Under the same reaction condition, a number of partially or fully protected 2-azido-2-deoxy-β-D-mannoside disaccharides (20–24) were obtained from corresponding lactols and primary triflates (17 or 18) in good yields and excellent anomeric selectivity. It is worth noting that this β-selective anomeric O-alkylation of 2-azido-2-deoxy-D-mannoses tolerates C4-free alcohol (cf. 21) and acetate functionality (cf. 23). Although primary triflates were found to be suitable electrophiles for anomeric O-alkylation of 2-azido-2-deoxy-D-mannoses, secondary triflates did not work well for this type of reactions, due to their inferior reactivity compared to primary triflates. For instance, under the same reaction condition anomeric O-alkylation of 2-azido-2-deoxy-D-mannose 16 with D-galactose-derived C4-triflate 25 only afforded trace amount of corresponding 2-azido-2-deoxy-D-mannoside 26.

Table 2.

Synthesis of 2-azido-2-deoxy-D-mannosides via anomeric O-alkylation.^a,b

^aCondition: A: lactols (1.0 eq.), triflate (2.0 eq.), Cs₂CO₃ (2.5 eq.), ClCH₂CH₂Cl, 40 °C, 24 h;

^bIsolated yield.

This method was next applied to the synthesis of a protected tetrasaccharide fragment (30, Scheme 3) of teichuronic acid, a capsular polysaccharide from Micrococcus luteus. Structurally, Micrococcus luteus teichuronic acid (cf. 6, Figure 1) consists of alternating β-N-acetyl-D-mannosaminuronic acid (ManNAcA) and α-glucoside residues.^33–36 Biological studies suggested that M. luteus cause several diseases including recurrent bacteremia,³⁷ septic shock,³⁸ and meningitis.³⁹ In addition, it was discovered that Micrococcus luteus teichuronic acid component induces the production of inflammatory cytokines.⁴0 Additionally, it was shown that the carboxylic acid moiety is essential for the immunostimulating activity.⁴⁰ Previously, limited efforts have been reported on the synthesis of Micrococcus luteus teichuronic acid fragments involving the use of methyl 2-(benzoyloxy)iminoglycosuronate⁴¹ or mannosazide methyl uronate donors.²⁷

Scheme 3.

Synthesis of tetrasaccharide fragment of Micrococcus luteus teichuronic acid (6).

Conditions: a) 1,3-dibromo-5,5-dimethylhydantoin (DBDMH) (1.1 eq.), acetone/water (15/1, v/v), NaHCO₃ (10 eq.), 0 °C, 91%; b) Cl₃CCN, DBU (0.1 eq.), CH₂Cl₂, 0 °C, 86% yield; c) TMSOTf (10 mol%), Et₂O/CH₂Cl₂ (2/1, v/v), −60 °C, 82% (α anomer) and 8.7 % (β anomer), α/β = 9.4/1; d) 10% CF₃CO₂H, CH₂Cl₂, 78%; e) PIDA, TEMPO, CH₂Cl₂/H₂O (10/1, v/v), 2 h, 76%; f) K₂CO₃, MeI, DMF, 4 h, 94%; g) thioacetic acid/pyridine (1/1, v/v), 40 °C, 12 h, 88%; h) Na, NH3(liquid), THF, −78 °C; i) Ac₂O, THF/H₂O(10/1, v/v), NaHCO₃, RT, 40% over two steps.

Our synthesis of the tetrasaccharide fragment of Micrococcus luteus teichuronic acid (6) commenced with the traditional N-iodosuccinimide/triflic acid-mediated glycosylation of disaccharide thioglycoside donor 24 and disaccharide acceptor 21 which gave the desired tetrasaccharide 29 in 80% yield (α/β = 2.7/1). Efforts involving the use of exogenous modulator, e.g. DMF,⁴² did not improve the α-selectivity. Therefore, we decided to evaluate the corresponding trichloroacetimidate donor 28 in this glycosylation in the hope of improving the anomeric selectivity. Thus, 1,3-dibromo-5,5-dimethylhydantoin (DBDMH)-mediated oxidation of disaccharide thioglycoside 24 afforded the desired lactol 27 in 91% yield (α/β = 1.3/1) which was then subjected to reaction with trichloroacetonitrile in the presence of 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) as the base catalyst to produce corresponding disaccharide trichloroacetimidate 28 in 86% yield (α only) (Scheme 3). Next, trimethylsilyl trifluoromethanesulfonate (TMSOTf)-catalyzed glycosylation of disaccharide trichloroacetimidate 28 with disaccharide acceptor 21 furnished the desired α-linked tetrasaccharide 29 (82% yield) and its β-anomer (8.7% yield) (α/β = 9.4/1). The α-anomer 29 was subjected to the global removal of p-methoxybenzyl ether by 10% CF₃CO₂H in CH₂Cl₂ to afford the desired diol 30 in 78% yield. Next, phenyliodine(III) diacetate (PIDA)/TEMPO-mediated global oxidation of the two C6-primary alcohols afforded the corresponding di-carboxylic acid 31 (76% yield). Initially, the two carboxylic acids in 31 were methylated to give the bis-methyl ester 32 (94% yield) in which the two C2-azides were subsequently converted to the acetamides using standard protocol involving thioacetic acid/pyridine.⁴³ As a result, the protected tetrasaccharide (33) was obtained in 88% yield. Unfortunately, hydrolysis of the two methyl esters were found to be challenging, and attempts to use less basic protocol (H₂O₂, KOH)²⁷ afforded the desired bis-carboxylic acid as well as inseparable elimination products. Finally, it was found that direct subjection of di-carboxylic acid 31 to the Birch reduction at −78 °C followed by chemoselective acetylation of the diamines afforded desired tetrasaccharide fragment 6 (40% yield, two steps) of Micrococcus luteus teichuronic acid containing alternating β-ManNAcA and α-glucose residues.²⁷

CONCLUSION

In conclusion, we have developed an approach for stereoselective synthesis of 2-azido-2-deoxy-β-mannosides through cesium carbonate-mediated anomeric O-alkylation of 2-azido-2-deoxy-D-mannoses with sugar-derived primary triflates. In addition, 1,3-dibromo-5,5-dimethylhydantoin was discovered as the optimal oxidant for converting phenyl 2-azido-2-deoxy-1-thio-α-D-mannosides to their corresponding lactols. This method has demonstrated its utilization in the synthesis of a tetrasaccharide fragment of Micrococcus luteus teichuronic acid containing alternating β-ManNAcA and α-glucose residues.

EXPERIMENTAL SECTION

Materials and Methods

Proton and carbon nuclear magnetic resonance spectra (¹H NMR and ¹³C NMR) were recorded on either Bruker 600 (¹H NMR-600 MHz; ¹³C NMR 150 MHz) at ambient temperature with CDCl₃ as the solvent unless otherwise stated. Chemical shifts are reported in parts per million relative to residual protic solvent internal standard CDCl₃: ¹H NMR at δ 7.26, ¹³C NMR at δ 77.16. Data for ¹H NMR are reported as follows: chemical shift, integration, multiplicity (app = apparent, par obsc = partially obscure, ovrlp = overlapping, s = singlet, d = doublet, dd = doublet of doublet, t = triplet, q = quartet, m = multiplet) and coupling constants in Hertz. All ¹³C NMR spectra were recorded with complete proton decoupling. Structural assignments were made with additional information from gCOSY, gHSQC, and gHMBC experiments. Low resolution mass spectra (LRMS) were acquired on a Waters Acuity Premiere XE TOF LC-MS by electrospray ionization. Optical rotations were measured with Autopol-IV digital polarimeter; concentrations are expressed as g/100 mL.

All reagents and chemicals were purchased from Acros Organics, Sigma Aldrich, Fisher Scientific, Alfa Aesar, and Strem Chemicals and used without further purification. THF, dichloromethane, toluene, and diethyl ether were purified by passing through two packed columns of neutral alumina (Innovative Technology). Anhydrous DMF and benzene were purchased from Acros Organics and Sigma-Aldrich and used without further drying. All reactions were carried out in oven-dried glassware under an argon atmosphere unless otherwise noted. All reactions that require elevated temperature were carried out under traditional oil bath heating. Analytical thin layer chromatography was performed using 0.25 mm silica gel 60-F plates. Preparative thin layer chromatography was performed using 1 mm silica gel prep TLC plates. Flash column chromatography was performed using 200–400 mesh silica gel (Scientific Absorbents, Inc.). Yields refer to chromatographically and spectroscopically pure materials, unless otherwise stated.

Synthesis of 2-azido-2-deoxymannoses

Phenyl 2-azido-3,6-di-O-benzyl-2-deoxy-1-thio-β-D-mannopyranoside (8).

To a solution of known compound phenyl 2-azido-3-O-benzyl-2-deoxy-1-thio-β-D-mannopyranoside 7²⁷ (387 mg, 1.0 mmol) in CH₃CN (5 mL) were added KI (166 mg, 1.0 mmol), K₂CO₃ (152 mg, 1.1 mmol) Ph₂BO(CH₂)₂NH₂ catalyst (22.5 mg, 0.1 mmol) and BnBr (179 µL, 1.5 mmol). The mixture was stirred vigorously at 40 °C for 12 h before being filtered and the resulting filtrate was concentrated. The residue was purified by flash column chromatography (Hexanes/EtOAc = 15/1) to afford phenyl 2-azido-3,6-di-O-benzyl-2-deoxy-1-thio-β-D-mannopyranoside 8 (477 mg, quantitative) as colorless syrup.${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}=+41.6\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz, CDCl₃) δ 7.53 – 7.48 (m, 2H, H_Ar), 7.47 – 7.41 (m, 4H, H_Ar), 7.39 – 7.26 (m, 9H, H_Ar), 5.50 (d, J = 1.5 Hz, 1H, H-1), 4.80 (d, J = 11.6 Hz, 1H, -OCH₂Ar), 4.74 (d, J = 11.6 Hz, 1H, -OCH₂Ar), 4.64 (d, J = 12.0 Hz, 1H, -OCH₂Ar), 4.56 (d, J = 12.0 Hz, 1H, -OCH₂Ar), 4.33 (dt, J = 9.2, 4.4 Hz, 1H, H-5), 4.16 (dd, J = 3.6, 1.6 Hz, 1H, H-2), 4.06 (t, J = 9.4 Hz, 1H, H-4), 3.89 (dd, J = 9.2, 3.5 Hz, 1H, H-3), 3.82 – 3.79 (m, 2H, H-6a/b), 2.69 (br s, 1H, -OH). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 138.1, 137.3, 133.3, 132.0, 129.2, 128.8, 128.4, 128.4, 128.3, 128.0, 127.7, 86.5, 79.4, 73.6, 72.7, 72.2, 69.9, 68.1, 62.1. HRMS (ESI) m/z: [M + Na]⁺ Calcd for C₂₆H₂₇N₃NaO₄S 500.1620; found 500.1620.

2-Azido-3,6-di-O-benzyl-2-deoxy-D-mannopyranose (9).

To a solution of phenyl 2-azido-3,6-di-O-benzyl-2-deoxy-1-thio-β-D-mannopyranoside 8 (170 mg, 0.35 mmol) in 1.5 mL acetone and 100 μL water cooled at 0 °C was added 1,3-dibromo-5,5-dimethylhydantoin (DBDMH) (110 mg, 0.38 mmol). The resulting mixture was stirred at 0 °C for 30 minutes before being quenched with saturated NaHCO₃ and saturated Na₂S₂O₃ solution. Acetone was removed under reduced pressure, and the remaining aqueous mixture was extracted with EtOAc (3 × 10 mL). Combined extracts was sequentially washed with water (3 × 20 mL) and brine (20 mL), dried over anhydrous Na₂SO₄, filtered and concentrated. The crude residue was purified by flash column chromatography (Hexanes/EtOAc = 4/1) to furnish 127 mg (0.32 mmol, 94%) of 2-azido-3,6-di-O-benzyl-2-deoxy-D-mannopyranose 9 as a mixture of anomers (α/β = 2.9/1).${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}=-10.4\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz, CDCl3) δ 7.44 – 7.27 (m, 14H, H_Ar), 5.14 (s, 1H, H-1 α), 4.74 (d, J = 11.5 Hz, 2H, -OCH₂Ar α, -OCH₂Ar β), 4.66 – 4.61 (m, 2H, -OCH₂Ar β, H-1 β), 4.59 (d, J = 11.5 Hz, 1H, - OCH₂Ar α), 4.56 – 4.51 (m, 4H, -OCH₂Ar α, -OCH₂Ar α, -OCH₂Ar β, -OCH₂Ar β), 3.99 (m, 1H, H-5 α), 3.96 – 3.91 (m, 3H, H-2 α, H-3 α, H-2 β), 3.91 – 3.83 (m, 2H, H-4 α, H-4 β), 3.79 – 3.71 (m, 3H, H-6a α, H-6a/b β), 3.66 (dd, J = 10.3, 6.7 Hz, 1H, H-6b α), 3.58 – 3.52 (m, 2H, C1-OH β, H-3 β), 3.41 (dt, J = 9.5, 4.8 Hz, 1H, H-5 β), 3.16 (s, 1H, C1-OH α), 2.66 (s, 1H, C4-OH β), 2.47 (s, 1H, C4-OH α). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 137.7, 137.5, 128.8, 128.5, 128.3, 128.2, 128.1, 127.9, 93.0, 79.0, 73.7, 72.4, 71.1, 70.4, 67.7, 60.6. HRMS (ESI) m/z: [M + Na]⁺ Calcd for C₂₀H₂₃N₃NaO₅ 408.1535, found 408.1534.

Phenyl 2-azido-3-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-1-thio-β-D-mannopyranoside (10).

To a solution of known compound phenyl 2-azido-3-O-benzyl-2-deoxy-1-thio-β-D-mannopyranoside 7²⁷ (580 mg, 1.5 mmol) in CH₃CN (15 mL) were added KI (249 mg, 1.5 mmol), K₂CO₃ (221 mg, 1.6 mmol) Ph₂BO(CH₂)₂NH₂ catalyst (34 mg, 0.15 mmol) and PMBCl (305 µL, 2.25 mmol). The mixture was stirred vigorously at 40 °C for 12 h before being filtered and concentrated. The residue was purified by flash column chromatography (Hexanes/EtOAc = 9/1), to afford compound phenyl 2-azido-3-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-1-thio-β-D-mannopyranoside 10 (685 mg, 1.35 mmol, 90%) as colorless syrup.${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}=+43.1\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz, CDCl₃) δ 8.27 – 7.14 (m, 13H, H_Ar), 6.90 (dd, J = 8.6, 3.5 Hz, 2H, H_Ar), 5.50 (d, J = 1.6 Hz, 1H, H-1), 4.80 (d, J = 11.6 Hz, 1H, -OCH₂Ar), 4.75 (d, J = 11.6 Hz, 1H, -OCH₂Ar), 4.58 (d, J = 11.5 Hz, 1H, -OCH₂Ar), 4.49 (d, J = 11.5 Hz, 1H, -OCH₂Ar), 4.32 (dt, J = 9.3, 4.4 Hz, 1H, H-5), 4.15 (dd, J = 3.6, 1.6 Hz, 1H, H-2), 4.05 (td, J = 9.4, 2.3 Hz, 1H, H-4), 3.89 (dd, J = 9.2, 3.6 Hz, 1H, H-3), 3.84 (s, 3H, -OCH₃), 3.76 (d, J = 4.4 Hz, 2H, H-6a/b), 2.78 (br s, 1H, -OH). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 159.2, 137.3, 133.3, 131.9, 130.1, 129.3, 129.2, 128.8, 128.3, 128.2, 127.9, 113.8, 86.4, 79.4, 73.2, 72.7, 72.2, 69.6, 68.3, 62.1, 55.3. HRMS (ESI) m/z: [M + Na]⁺ Calcd for C₂₇H₂₉N₃NaO₅S, 530.1726; found 530.1733.

2-Azido-3-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-D-mannopyranose (11).

To a solution of phenyl 2-azido-3-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-1-thio-β-D-mannopyranoside 10 (2.0 g, 3.94 mmol) in 18 mL acetone and 1.2 mL water cooled at 0 °C was added 1,3-dibromo-5,5-dimethylhydantoin (DBDMH) (1.23 g, 4.33 mmol). The resulting mixture was stirred at 0 °C for 30 minutes before being quenched with saturated NaHCO₃ and saturated Na₂S₂O₃ solution (1.5 mL each). Acetone was removed under reduced pressure, and the remaining aqueous mixture was extracted with EtOAc (3×50 mL). Combined extracts were sequentially washed with water (3 ×50 mL) and brine (50 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude residue was purified by flash column chromatography (Hexanes/EtOAc = 2/1) to furnish 1.3 g (3.13 mmol, 79%) of lactol 11 as a mixture of anomers (α/β = 4/1).${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}=-5.3\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz, CDCl₃ δ 7.56 (d, J = 1.9 Hz, 1.26H, H_Ar), 7.48 – 7.16 (m, 17H, H_Ar), 6.89 (d, J = 8.7 Hz, 0.26H, H_Ar), 6.84 (d, J = 8.4 Hz, 1H, H_Ar), 5.16 (s, 1H, H-1 α), 4.81 – 4.75 (m, 2H, -OCH₂Ar α, -OCH₂Ar β), 4.69 – 4.60 (m, 3H, -OCH₂Ar α, -OCH₂Ar β, H-1 β), 4.59 – 4.48 (m, 4H, -OCH₂Ar α, -OCH₂Ar α, -OCH₂Ar β, -OCH₂Ar β), 4.28 (d, J = 8.8 Hz, 1H, C1-OH β), 4.21 (d, J = 3.6 Hz, 1H, C1-OH α), 4.06 – 4.00 (m, 1H, H-5 α), 3.93 – 3.89 (m, 1H, H-3 α), 4.24 – 4.15 (m, 12H, H-2 α, H-2 β, H-4 α, H-6a α, Ar-OCH₃ α, Ar-OCH₃ β, H-4 β, H-6a β), 3.72 – 3.62 (m, 2H, H-6b β, H-6b α), 3.49 – 3.37 (m, 2H, H-3 β, H-5 β), 3.41 (d, J = 11.6 Hz, 0.26H). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 159.4, 137.5, 129.8, 129.8, 129.7, 128.8, 128.8, 128.3, 128.9, 128.1, 113.9, 113.9, 93.8, 92.9, 79.0, 73.3, 72.4, 71.0, 70.1, 67.8, 60.7, 55.4, 55.4. HRMS (ESI) m/z: [M + Na]⁺ Calcd for C₂₁H₂₅N₃NaO₆ 438.1641; found 438.1638.

Phenyl 2-azido-3,4-di-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-1-thio-β-D-mannopyranoside (12).

To a solution of phenyl 2-azido-3-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-1-thio-β-D-mannopyranoside 10 (1.26 g, 2.48 mmol) in 8.3 mL of N,N-dimethylformamide cooled to 0 °C was added sodium hydride (60% dispersion in mineral oil, 149 mg, 3.72 mmol) portion wise. The resulting mixture was stirred at 0 °C for 30 minutes before BnBr (366 μL, 2.97 mmol) was added dropwise. The resulting mixture was warmed up and stirred at ambient temperature overnight. The reaction mixture was quenched with water and extracted with EtOAc (3×50 mL). Combined extracts were sequentially washed with water (3×50 mL) and brine (50 mL), dried over Na₂SO₄, filtered, and concentrated. The crude residue was purified by flash column chromatography (Hexanes/EtOAc = 25/1) to furnish 1.22 g (2.04 mmol, 82%) of phenyl 2-azido-3,4-di-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-1-thio-β-D-mannopyranoside 12. ${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}=+98.9\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz,CDCl₃) δ 7.67 – 7.24 (m, 18H, H_Ar), 6.95 (d, J = 8.0 Hz, 2H, H_Ar), 5.60 (d, J = 1.9 Hz, 1H, H-1), 4.95 (d, J = 10.7 Hz, 1H, -OCH₂Ar), 4.84 (m, 2H, -OCH₂Ar), 4.71 (d, J = 11.7 Hz, 1H, -OCH₂Ar), 4.60 (d, J = 10.7 Hz, 1H, -OCH₂Ar), 4.49 (d, J = 11.7 Hz, 1H, -OCH₂Ar), 4.41 – 4.34 (m, 1H, H-5), 4.21 (dd, J = 3.8, 1.6 Hz, 1H, H-2), 4.14 (dd, J = 9.0, 3.8 Hz, 1H, H-3), 4.08 (t, J = 9.3 Hz, 1H, H-4), 3.89 (dd, J = 10.8, 4.3 Hz, 1H, H-6), 3.85 (s, 3H, -OCH₃), 3.75 (d, J = 10.8 Hz, 1H, H-6). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 159.1, 138.0, 137.4, 133.4, 131.7, 130.1, 129.5, 129.1, 128.5, 128.3, 128.0, 128.0, 127.9, 127.7, 127.7, 113.6, 86.2, 79.9, 75.2, 74.5, 72.9, 72.6, 68.1, 62.7, 55.1. HRMS (ESI) m/z: [M + Na]⁺ Calcd for C₃₄H₃₅N₃NaO₅S 620.2195; found 620.2167.

2-Azido-3,4-di-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-D-mannopyranose (14).

To a solution of phenyl 2-azido-3,4-di-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-1-thio-β-D-mannopyranoside 12 (180 mg, 0.3 mmol) in 1.5 mL acetone and 100 μL water cooled at 0 °C was added 1,3-dibromo-5,5-dimethylhydantoin (DBDMH) (94.4 mg, 0.33 mmol). The resulting mixture was stirred at 0 °C for 15 minutes before being quenched with saturated NaHCO₃ and saturated Na₂S₂O₃ solution (50 μL each). Acetone was removed under reduced pressure, and the remaining aqueous mixture was extracted with EtOAc (3×10 mL). Combined extracts were sequentially washed with water (3×20 mL) and brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude residue was purified by flash column chromatography (Hexanes/EtOAc =4/1) to furnish 129 mg (0.25 mmol, 85%) of 2-azido-3,4-di-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-D-mannopyranose 14 as a mixture of anomers (α/β = 6/1).${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}=+26.2\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz, CDCl₃) δ 7.59 – 7.15 (m, 14H, HAr), 6.85 (d, J = 8.4 Hz, 1H, HAr), 5.18 (d, J = 1.8 Hz, 1H, H-1 α), 4.94 – 4.85 (m, 2H, -OCH₂Ar α, -OCH₂Ar β), 4.82 – 4.67 (m, 5H, -OCH₂Ar α, -OCH₂Ar β, -OCH₂Ar α, -OCH₂Ar β, H-1 β), 4.56 – 4.39 (m, 6H, -OCH₂Ar α, -OCH₂Ar β, -OCH₂Ar α, -OCH₂Ar β, -OCH₂Ar α, -OCH₂Ar β), 4.20 – 4.11 (m, 3H, C1-OH β, H-3 α, C1-OH α), 4.04 (m, 1H, H-5 α), 3.95 – 3.91 (m, 2H, H-2 α, H-2 β), 3.95 – 3.91 (m, 6H, -OCH₃ α, -OCH₃ β), 3.82 – 3.77 (m, 3H, H-2 β, H-6a/b β), 3.74 (t, J = 9.4 Hz, 1H, H-4 α), 3.69 – 3.63 (m, 3H, H-3 β, H-4 β, H-6a α), 3.59 (dd, J = 10.4, 6.8 Hz, 1H, H-6b α), 3.42 (m, 1H, H-5 β). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 159.2, 155.3, 137.9, 137.8, 137.7, 137.4, 133.3, 129.9, 129.9, 129.4, 128.5, 128.4, 128.3, 128.3, 128.0, 127.9, 127.9, 127.8, 127.8, 127.8, 127.7, 113.7, 113.7, 111.5, 111.3, 93.0, 92.4, 81.4, 79.27, 75.1, 75.1, 74.7, 74.6, 74.6, 74.0, 72.9, 72.8, 72.2, 72.1, 70.6, 68.8, 68.4, 62.7, 61.5, 56.0, 55.1. HRMS (ESI) m/z: [M + Na]⁺ Calcd for C₂₈H₃₁N₃NaO₆ 528.2111; found 528.2109.

Phenyl 4-O-acetyl-2-azido-3-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-1-thio-β-D-mannopyranoside (13).

To a solution of phenyl 2-azido-3-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-1-thio-β-D-mannopyranoside 10 (355 mg, 0.7 mmol) in CH₂Cl₂ (2.3 mL) was added DMAP (8.5 mg, 0.07 mmol) and pyridine (570 µL, 7.0 mmol). The resulting mixture was cooled to 0 °C and acetic anhydride (330 µL, 3.5 mmol) was added dropwise. The resulting mixture was warmed to room temperature and stirred for 2 h before being quenched with saturated NaHCO₃ solution. The mixture was extracted with CH₂Cl₂ (3×20 mL), and combined extracts were washed with water (2×50 mL) and brine (20 mL), dried over Na₂SO₄, filtered, and concentrated. The crude residue was purified by flash column chromatography (Hexanes/EtOAc = 15/1) to furnish phenyl 4-O-acetyl-2-azido-3-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-1-thio-β-D-mannopyranoside 13 (340 mg, 0.62 mmol, 88%) as a white solid.${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}=+71.3\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz,CDCl₃) δ 7.58 – 7.19 (m, 13H, H_Ar), 6.89 (d, J = 11.8 Hz, 2H, H_Ar), δ 5.47 (d, J = 1.6 Hz, 1H, H-1), 5.32 (t, J = 9.4 Hz, 1H, H-4), 4.72 (d, J = 12.0 Hz, 1H, -OCH₂Ar), 4.65 (d, J = 12.0 Hz, 1H, -OCH₂Ar), 4.46 (d, J = 11.4 Hz, 1H, -OCH₂Ar), 4.42 (d, J = 11.4 Hz, 1H, -OCH₂Ar), 4.38 (ddd, J = 9.4, 6.0, 3.1 Hz, 1H, H-5), 4.11 (dd, J = 3.1, 2.0 Hz, 1H, H-2), 3.96 (dd, J = 9.1, 3.5 Hz, 1H, H-3), 3.83 (s, 3H, -OCH₃), 3.59 (dd, J = 10.9, 6.0 Hz, 1H, H-6a), 3.55 (dd, J = 10.8, 3.1 Hz, 1H, H-6b), 1.99 (s, 3H, -C(O)CH₃). ¹³C{¹H} NMR (150 MHz, CDCl3) δ 169.7, 159.2, 137.3, 133.0, 132.2, 130.1, 129.4, 129.2, 128.7, 128.2, 128.1, 128.0, 113.7, 86.0, 73.2, 72.5, 71.4, 69.0, 68.5, 62.4, 55.3, 21.0. HRMS (ESI) m/z: [M + Na]⁺ Calcd for C₂₉H₃₁N₃NaO₆S 572.1831; found 572.1823.

4-O-Acetyl-2-azido-3-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-D-mannopyranose (15).

To a solution of phenyl 4-O-acetyl-2-azido-3-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-1-thio-β-D-mannopyranoside 13 (274 mg, 0.5 mmol) in 1.5 mL acetone and 100 μL water cooled at 0 °C was added N-bromosuccinimide (133 mg, 0.75 mmol). The resulting mixture was stirred at 0 °C for 2 h before being quenched with saturated NaHCO₃. Acetone was removed under reduced pressure, and the remaining aqueous mixture was extracted with EtOAc (3×10 mL). Combined extracts were washed sequentially with water (3 ×20 mL) and brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude residue was purified by flash column chromatography (Hexanes/EtOAc = 4/1) to furnish 231 mg (0.5 mmol, quantitative) of 4-O-acetyl-2-azido-3-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-D-mannopyranose 15 as a mixture of anomers (α/β = 5/1). ${\left[\mathit{\alpha }\right]}_{\mathbf{\text{D}}}^{\mathbf{25}}=+22.5\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz,CDCl₃) δ 7.37–7.27 (m, 6H, H_Ar), 7.23–7.17 (m, 2H, H_Ar), 6.83 (dd, J = 8.4, 3.6 Hz, 2H, H_Ar), 5.17 – 5.03 (m, 3H, H-1 α, H-4 α, H-4 β), 4.72 – 4.65 (m, 3H, H-1 β, -OCH₂Ar α, -OCH₂Ar β), 4.60 – 4.54 (m, 2H, -OCH₂Ar α, -OCH₂Ar β), 4.45 – 4.39 (m, 4H, -OCH₂Ar α, -OCH₂Ar β, -OCH₂Ar α, -OCH₂Ar β), 4.06 – 3.97 (m, 2H, overlap), 3.93 – 3.84 (m, 2H, overlap), 3.81 (s, 3H, -OCH₃), 3.70 – 3.62 (m, 2H, overlap), 3.54 – 3.40 (m, 3H, overlap), 1.95 (s, 3H, -C(O)CH₃). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 170.9, 155.6, 137.7, 133.5, 133.3, 131.9, 131.0, 130.9, 128.8, 128.7, 128.6, 128.6, 128.6, 128.2, 128.0, 128.0, 127.8, 127.8, 111.7, 111.5, 92.6, 76.2, 72.5, 72.3,69.8, 69.6, 68.5, 61.4, 56.3, 21.0, HRMS (ESI) m/z: [M + H]⁺ Calcd for C₂₃H₂₈N₃O₇ 458.1927; found 458.1915.

Synthesis of sugar-derived triflate

Phenyl 2,3,4-tri-O-benzyl-6-O-trifluoromethanesulfonyl-1-thio-α-D-glucopyranoside (18).

To a solution of phenyl 2,3,4-tri-O-benzyl-1-thio-α-D-mannopyranoside⁴⁴ (440 mg, 0.81 mmol) in 2.7 mL dichloromethane and pyridine (329 μL, 4.05 mmol) cooled at 0 °C was added triflic anhydride (163 μL, 0.97 mmol) dropwise. The resulting mixture was stirred at 0 °C for 10 minutes and then quenched with ice water. Organic layer was separated and aqueous layer was extracted with CH₂Cl₂ (3×20 mL). The combined organic extracts were washed sequentially with saturated CuSO₄ (3×20 mL) and water (3×50 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated. The residue was purified by flash column chromatography (pure CH₂Cl₂) to afford 436 mg (80%) of phenyl 2,3,4-tri-O-benzyl-6-O-trifluoromethanesulfonyl-1-thio-α-D-glucopyranoside 18.${\left[\alpha \right]}_{\mathbf{D}}^{\mathbf{25}}=+140.2\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz,CDCl₃) δ 7.53 – 7.28 (m, 20H, H_Ar), 5.63 (d, J = 5.3 Hz, 1H, H-1), 5.06 (d, J = 10.8 Hz, 1H, -OCH₂Ar), 4.97 (d, J = 11.0 Hz, 1H, -OCH₂Ar), 4.83 (d, J = 10.8 Hz, 1H, -OCH₂Ar), 4.79 (d, J = 11.6 Hz, 1H, -OCH₂Ar), 4.71 (d, J = 11.6 Hz, 1H, - OCH₂Ar), 4.61 (d, J = 11.0 Hz, 1H, -OCH₂Ar), 4.53 (m, 2H, H-6a/b), 4.48 (m, 1H, H-5), 3.95 (t, J = 9.1 Hz, 1H, H-3), 3.90 (dd, J = 9.6, 5.3 Hz, 1H, H-2), 3.51 (dd, J = 9.9, 8.6 Hz, 1H, H-4). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 138.5, 137.6, 133.8, 131.9, 129.4, 128.9, 128.8, 128.8, 128.5, 128.4, 128.4, 128.4, 128.3, 128.1, 127.8, 122.0, 119.9, 117.7, 115.6, 87.1, 82.5, 79.8, 76.4, 76.1, 75.4, 75.0, 72.9, 69.4. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₃₄H₃₄F₃O₇S₂ 675.1698; found 675.1622.

Synthesis of 2-azido-2-deoxymannosides via Cs₂CO₃-mediated O-alkylation.

General procedure.

To a mixture of lactol donor (0.1 mmol, 1.0 eq.), sugar-derived triflate acceptor 17 or 18 (2.0 eq.), and Cs₂CO₃ (2.5 eq.) was added 1,2-dichloroethane (1.0 mL). The reaction mixture was stirred at 40 °C for 24 hours. The crude reaction mixture was directly loaded onto preparative thin layer chromatography (TLC) plates and developed in various solvent system until the bands are separated. Multiple preparative TLC plates were used if needed. The silica bands containing desired product were scraped and collected in a flask. A mixed solvent containing dichloromethane/methanol (10/1, v/v) was added and this resulting heterogeneous mixture was stirred for 6 hours before being filtered, and concentrated under reduced pressure. The β-configuration of the newly formed mannosidic linkage was unambiguously assigned by measuring the ¹J_C-H coupling constants of the anomeric carbon.

Methyl 2-azido-3,4,6-tri-O-benzyl-2-deoxy-β-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranoside (19).

Disaccharide 19 was prepared from known lactol donor 16³² (47.5 mg, 0.1 mmol) and triflate acceptor 17 (119 mg, 0.2 mmol) following the general procedure. The crude reaction mixture was purified by preparative TLC (Hexanes/EtOAc = 2/1) to afford compound 19 (77.3 mg, 81%) as white solid. The J_C1′,H1′ was determined to be 159.1 Hz.${\left[\alpha \right]}_{\mathbf{D}}^{\mathbf{25}}=+4.2\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz,CDCl3) δ 7.44 – 7.21 (m, 28H, H_Ar), 7.20 – 7.14 (m, 2H, H_Ar), 4.99 (d, J = 10.8 Hz, 1H, -OCH₂Ar), 4.90 – 4.75 (m, 4H, -OCH₂Ar), 4.74 – 4.62 (m, 3H, -OCH₂Ar), 4.61 – 4.47 (m, 5H, -OCH₂Ar, H-1), 4.21 (d, J = 1.2 Hz, 1H, H-1′), 4.11 (dd, J = 10.5, 2.0 Hz, 1H, H-6′a), 4.00 (t, J = 9.2 Hz, 1H, H-3), 3.81 (ddd, J = 10.1, 5.9, 2.0 Hz, 1H, H-5), 3.75 (dd, J = 3.7, 1.1 Hz, 1H, H-2′), 3.73 – 3.60 (m, 3H, H-4′, H-6a/b), 3.58–3.44 (m, 3H, H-3′, H-2, H-6′b), 3.39 (dd, J = 9.9, 9.0 Hz, 1H, H-4), 3.34 (s, 3H, -OCH₃), 3.32 (m, 1H, H-5′). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 138.8, 138.5, 138.3, 138.2, 138.1, 137.6, 128.7, 128.6, 128.6, 128.5, 128.5, 128.4, 128.3, 128.2, 128.2, 128.2, 128.1, 128.1, 128.0, 127.9, 127.9, 127.9, 127.8, 127.8, 127.7, 99.8, 97.9, 82.2, 82.2, 80.6, 80.0, 77.7, 75.9, 75.8, 75.4, 74.8, 74.6, 73.7, 73.6, 72.3, 69.7, 69.2, 68.5, 62.1, 55.2. HRMS (ESI) m/z: [M + Na]⁺ Calcd for C₅₅H₅₉N₃NaO10 944.4098; found 944.4020.

Methyl 2-azido-3,6-di-O-benzyl-2-deoxy-β-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranoside (20).

Disaccharide 20 was prepared from lactol donor 9 (38.5 mg, 0.1 mmol) and triflate acceptor 17 (119 mg, 0.2 mmol) following the general procedure. The crude reaction mixture was purified by preparative TLC (Hexanes/EtOAc/CH₂Cl₂ = 3/1/1) to afford compound 20 (66.2 mg, 79%) as white solid. The J_C1′,H1′ was determined to be 159.6 Hz. ${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}=-29.1\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz, CDCl₃) δ 7.42 – 7.19 (m, 25H, H_Ar), 5.00 (d, J = 10.8 Hz, 1H, -OCH₂Ar), 4.87 (d, J = 11.6 Hz, 1H, -OCH₂Ar), 4.84 – 4.76 (m, 2H, -OCH₂Ar), 4.74 (d, J = 11.8 Hz, 1H, -OCH₂Ar), 4.66 (m, 2H, -OCH₂Ar), 4.61 – 4.51 (m, 4H, -OCH₂Ar (3H), H-1), 4.24 (d, J = 1.2 Hz, 1H, H-1′), 4.09 (dd, J = 10.4, 2.0 Hz, 1H, H-6′a), 4.01 (t, J = 9.2 Hz, 1H, H-3), 3.84 – 3.66 (m, 5H, H-5, H-2′, H-4′, H-6a/b), 3.57 – 3.44 (m, 2H, H-2, H-6′b), 3.40 (t, J = 9.4 Hz, 1H, H-4), 3.36–3.29 (m, 5H, -OCH₃, H-3′, H-5′), 2.75 (s, 1H, C4′-OH ). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 138.8, 138.5, 138.2, 137.9, 137.6, 128.8, 128.6, 128.5, 128.5, 128.5, 128.3, 128.1, 128.1, 128.0, 127.9, 127.9, 127.8, 127.7, 99.8, 97.9, 82.2, 79.9, 79.9, 77.6, 75.9, 74.8, 74.7, 73.9, 73.5, 72.2, 70.6, 69.7, 68.6, 68.4, 61.3, 55.2. HRMS (ESI) m/z: [M + Na]⁺ Calcd for C₄₈H₅₃N₃NaO₁₀ 854.3629; found 854.3549.

Methyl 2-azido-3-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-β-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranoside (21).

Disaccharide 21 was prepared from lactol donor 11 (41.5 mg, 0.1 mmol) and triflate acceptor 17 (119 mg, 0.2 mmol) following the general procedure. The resulting crude residue was purified by preparative TLC (Hexanes/EtOAc/CH₂Cl₂: 2/1/1) to furnish 65.2 mg (0.075 mmol, 76%) of compound 21 as a white solid. The J_C1′,H1′ was determined to be 157.9 Hz.${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}=-21.4\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz, CDCl₃) δ 7.46 – 7.22 (m, 23H, H_Ar), 6.92 – 6.86 (m, 2H, H_Ar), 5.04 (d, J = 10.8 Hz, 1H, -OCH₂Ar), 4.91 (d, J = 11.5 Hz, 1H, - OCH₂Ar), 4.88 – 4.80 (m, 2H, -OCH₂Ar), 4.77 (d, J = 12.0 Hz, 1H, -OCH₂Ar), 4.72 (d, J = 11.5 Hz, 1H, -OCH₂Ar), 4.69 (d, J = 12.0 Hz, 1H, -OCH₂Ar), 4.60 (m, 2H, -OCH₂Ar, H-1), 4.57 – 4.49 (m, 2H, -OCH₂Ar), 4.27 (d, J = 1.4 Hz, 1H, H-1′), 4.14 (dd, J = 10.4, 2.1 Hz, 1H, H-6′a), 4.05 (t, J = 9.2 Hz, 1H, H-3), 3.85 (m, 1H, H-5), 3.82 – 3.79 (m, 4H, -OCH₃, H-2′), 3.79 – 3.75 (m, 2H, H-4′, H-6a), 3.73 (dd, J = 10.2, 5.6 Hz, 1H, H-6b), 3.57 – 3.50 (m, 2H, H-2, H-6′b), 3.44 (dd, J = 10.1, 8.8 Hz, 1H, H-4), 3.39 (m, 4H, -OCH₃, H-3′), 3.34 (m, 1H, H-5′), 2.90 (d, J = 2.1 Hz, 1H, C4′-OH). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 159.3, 138.7, 138.4, 138.1, 137.5, 129.8, 129.5, 128.7, 128.5, 128.5, 128.4, 128.2, 128.2, 128.1, 128.0, 128.0, 127.9, 127.7, 127.7, 113.8, 99.7, 97.9, 82.1, 79.9, 79.7, 77.6, 75.8, 74.7, 74.6, 73.5, 72.2, 70.4, 69.6, 68.8, 68.4, 61.4, 55.3, 55.2. HRMS (ESI) m/z: [M + Na]⁺ Calcd for C₄₉H₅₅N₃NaO₁₁ 884.3734; found 884.3704.

Methyl 2-azido-3,4-di-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-β-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranoside (22).

Disaccharide 22 was prepared from lactol donor 14 (50.5 mg, 0.1 mmol) and triflate acceptor 17 (119 mg, 0.2 mmol) following the general procedure. The crude reaction mixture was purified by preparative TLC (Hexanes/EtOAc/CH₂Cl₂ = 3/1/1) to afford compound 22 (79.4 mg, 83%) as colorless solid. The J_C1′,H1′ was determined to be 158.4 Hz.${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}=-3.44\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz, CDCl₃) δ 7.51 (d, J = 2.1 Hz, 1H, H_Ar), 7.42 – 7.16 (m, 28H, H_Ar), 6.83 – 6.76 (m, 1H, H_Ar), 5.00 (d, J = 10.9 Hz, 1H, -OCH₂Ar), 4.90 – 4.80 (m, 4H, -OCH₂Ar), 4.74 – 4.62 (m, 3H, -OCH₂Ar), 4.60 – 4.45 (m, 4H, -OCH₂Ar, H-1), 4.42 (d, J = 11.9 Hz, 1H, -OCH₂Ar), 4.22 (d, J = 1.3 Hz, 1H, H-1′), 4.12 (dd, J = 10.5, 2.1 Hz, 1H, H-6′a), 4.02 (m, 1H, H-3), 3.88 – 3.80 (m, 4H, -OCH₃, H-5), 3.77 (m, 1H, H-2′), 3.71 – 3.62 (m, 3H, H-4′, H-6a/b), 3.58 – 3.46 (m, 3H, H-2, H-3′, H-6′b), 3.41 (t, J = 9.5 Hz, 1H, H-4), 3.36 (s, 3H, -OCH₃), 3.31 (ddd, J = 10.0, 5.4, 2.1 Hz, 1H, H-5′). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 155.4, 138.8, 138.4, 138.2, 138.0, 137.6, 133.1, 131.9, 128.7, 128.6, 128.5, 128.5, 128.3, 128.3, 28.2, 128.2, 128.1, 128.1, 128.1, 128.1, 128.0, 128.0, 127.9, 127.8, 127.8, 113.8, 111.8, 111.5, 99.8, 97.9, 82.2, 80.6, 79.9, 77.6, 75.9, 75.8, 75.7, 75.4, 74.8, 74.4, 73.5, 72.5, 72.2, 70.6, 69.7, 69.7, 69.0, 68.4, 62.0, 56.3, 55.2. HRMS (ESI) m/z: [M + Na]⁺ Calcd for C₅₆H₆₁N₃NaO₁₁ 974.4204; found 974.4174.

Methyl 4-O-acetyl-2-azido-3-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-β-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranoside (23).

Disaccharide 23 was prepared from lactol donor 15 (45.7 mg, 0.1 mmol) and triflate acceptor 17 (119 mg, 0.2 mmol) following the general procedure. The crude reaction mixture was purified by preparative TLC (Hexanes/EtOAc/CH₂Cl₂ = 2/1/1) to afford 64.3 mg (0.071 mmol, 71%) of compound 23 as a white solid. The J_C1′,H1′ was determined to be 159.3 Hz.${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}=-13.9\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz,CDCl₃) δ 7.48 (d, J = 2.1 Hz, 1H, H_Ar), 7.40 – 7.21 (m, 22H, H_Ar), 7.18 (dd, J = 8.4, 2.1 Hz, 1H, H_Ar), 6.80 (d, J = 8.4 Hz, 1H, H_Ar), 5.07 (t, J = 9.5 Hz, 1H, H-4′), 5.00 (d, J = 10.8 Hz, 1H, -OCH₂Ar), 4.87 (d, J = 11.6 Hz, 1H, -OCH₂Ar), 4.85 – 4.79 (m, 2H, -OCH₂Ar), 4.70 – 4.65 (m, 2H, -OCH₂Ar), 4.60 – 4.55 (m, 3H, -OCH₂Ar, H-1), 4.44 – 4.34 (m, 2H, -OCH₂Ar), 4.25 (d, J = 1.3 Hz, 1H, H-1′), 4.11 (dd, J = 10.5, 2.1 Hz, 1H, H-6′a), 4.01 (t, J = 9.2 Hz, 1H, H-3), 3.86 (s, 3H, -OCH₃), 3.82 (m, 1H, H-5), 3.74 (dd, J = 3.7, 1.2 Hz, 1H, H-2′), 3.58 – 3.37 (m, 7H, H-2, H-6a/b, H-3′, H-5′, H-6′b, H-4,), 3.35 (s, 3H, -OCH₃), 1.96 (s, 3H, -OC(O)CH₃). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 169.7, 155.5, 138.8, 138.5, 138.2, 137.4, 133.0, 131.6, 128.7, 128.6, 128.5, 128.5, 128.3, 128.3, 128.2, 128.2, 128.1, 128.1, 128.1, 128.0, 128.0, 127.9, 127.8, 127.8, 111.7, 111.6, 99.6, 97.9, 82.2, 79.9, 77.5, 75.9, 74.7, 74.1, 73.5, 72.6, 72.0, 70.1, 69.6, 68.6, 68.5, 61.6, 56.3, 55.2. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₅₁H₅₈N₃O₁₂ 904.4020; found 904.4047.

Phenyl 2-azido-3,4-di-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-β-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-1-thio-α-D-glucopyranoside (24).

Disaccharide 24 was prepared from lactol donor 14 (50.5 mg, 0.1 mmol) and triflate acceptor 18 (135 mg, 0.2 mmol) following the general procedure. The resulting crude residue was purified by flash column chromatography (Hexanes/EtOAc: 10/1) to furnish 93.1 mg (0.090 mmol, 90%) of compound 24 as a white solid. The J_C1′,H1′ was determined to be 158.2 Hz.${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}=+33.6\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz,CDCl₃) δ 7.57 – 7.50 (m, 2H, H_Ar), 7.47 – 7.23 (m, 30H, H_Ar), 7.24 – 7.17 (m, 2H, H_Ar), 6.90 – 6.84 (m, 2H, H_Ar), 5.66 (d, J = 5.3 Hz, 1H, H-1), 5.09 (d, J = 10.8 Hz, 1H, -OCH₂Ar), 4.97 (d, J = 11.4 Hz, 1H, -OCH₂Ar), 4.90 – 4.86 (m, 2H, -OCH₂Ar), 4.83 (d, J = 11.6 Hz, 1H, - OCH₂Ar), 4.79 – 4.74 (m, 2H, -OCH₂Ar), 4.71 (d, J = 11.8 Hz, 1H, -OCH₂Ar), 4.65 (d, J = 11.5 Hz, 1H, -OCH₂Ar), 4.59 (d, J = 11.7 Hz, 1H, -OCH₂Ar), 4.56–4.50 (m, 2H, -OCH₂Ar), 4.48 (m, 1H, H-5), 4.20 (d, J = 1.2 Hz, 1H, H-1′), 4.17 (dd, J = 11.1, 1.9 Hz, 1H, H-6′a), 4.01 (t, J = 9.2 Hz, 1H, H-3), 3.93 (dd, J = 9.7, 5.3 Hz, 1H, H-2), 3.84 (s, 3H, -OCH₃) 3.76 – 3.64 (m, 4H, H-4′, H-6a/b, H-6′b), 3.58 – 3.51 (m, 2H, H-4, H-3), 3.34 (ddd, J = 9.7, 5.4, 2.1 Hz, 1H, H-5′). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 159.2, 138.6, 138.3, 138.0, 137.9, 137.6, 137.6, 137.5, 134.2, 133.1, 132.1, 132.0, 131.9, 130.3, 129.6, 128.9, 128.7, 128.7, 128.6, 128.5, 128.5, 128.4, 128.4, 128.4, 128.4, 128.3, 128.3, 128.2, 128.2, 128.1, 128.1, 128.0, 128.0, 128.0, 127.9, 127.9, 127.8, 127.7, 127.2, 127.2, 113.7, 99.5, 86.8, 86.8, 82.4, 82.4, 80.9, 79.8, 75.8, 75.74, 75.3, 74.8, 74.5, 73.2, 72.6, 72.1, 71.1, 68.7, 68.3, 61.8, 55.3. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₆₁H₆₅N₃O₁₀S 1030.4312, found 1030.4348.

Synthesis of tetrasaccharide core of M. leutens

2-Azido-3,4-di-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-β-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-D-glucopyranose (27).

To a solution of Phenyl 2-azido-3,4-di-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-β-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-1-thio-α-D-glucopyranoside 24 (240 mg, 0.23 mmol) in 4.3 mL acetone and 287 μL water cooled at 0 °C was added 1,3-dibromo-5,5-dimethylhydantoin (DBDMH) (72 mg, 0.25 mmol). The resulting mixture was stirred at 0 °C for 10 minutes before being quenched with saturated NaHCO₃ and saturated Na₂S₂O₃ solution (50 μL each). Acetone was removed under reduced pressure, and the remaining aqueous mixture was extracted with EtOAc (3×10 mL). Combined extracts were sequentially washed with water (3×20 mL) and brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude residue was purified by flash column chromatography (Hexanes/EtOAc =5/1) to furnish 200 mg (0.21 mmol, 91%) of compound (27) as a mixture of anomers (α/β =1.3/1).${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}=+3.25\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz, CDCl₃) δ 7.45 – 7.25 (m, 37H), 7.21 – 7.17 (m, 4H), 6.90 – 6.84 (m, 4H), 5.24 (s, 1H), 5.07 – 4.47 (m, 24H), 4.41 (d, J = 1.3 Hz, 1H), 4.30 (d, J = 1.2 Hz, 1H), 4.23 – 4.11 (m, 4H), 4.07 (t, J = 9.2 Hz, 1H), 4.02 – 3.95 (m, 1H), 3.85 (dd, J = 3.6, 1.2 Hz, 1H), 3.80 (s, 3H), 3.79 (s, 3H), 3.76 – 3.55 (m, 12H), 3.54 – 3.32 (m, 5H). ¹³C{¹H} NMR (150 MHz, CDCl₃) 159.2, 159.2, 138.7, 138.5, 138.4, 138.4, 138.1, 138.0, 138.0, 137.9, 137.6, 137.6, 130.1, 130.0, 129.7, 129.7, 128.6, 128.6, 128.6, 128.5, 128.5, 128.4, 128.4, 128.2, 128.1, 128.1, 128.1, 128.1, 128.0, 128.0, 128.0, 127.9, 127.9, 127.9, 127.9, 127.8, 127.8, 127.8, 127.7, 127.7, 113.8, 113.8, 100.0, 99.5, 97.3, 91.0, 84.6, 83.5, 81.8, 81.1, 80.9, 80.2, 77.8, 77.8, 75.7, 75.7, 75.74, 75.61, 75.3, 75.3, 74.9, 74.8, 74.8, 74.4, 74.4, 74.4, 73.3, 73.1, 72.1, 72.0, 69.5, 69.3, 68.8, 68.7, 68.7, 61.8, 61.6, 55.3, 55.3. HRMS (ESI) m/z: [M + Na]⁺ Calcd for C₅₅H₈₉N₃NaO₁₁ 960.4047; found 960.4044.

Methyl 2-azido-3,4-di-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-β-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-2-azido-3-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-β-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranoside (29).

To a solution of 2-azido-3,4-di-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-β-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-D-glucopyranose 27 (200 mg, 0.21 mmol) in dichloromethane (1 mL) cooled at 0 °C was added 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) (3.2 μL, 0.021 mmol) and trichloroacetonitrile (84 μL, 0.84 mmol). The resulting mixture was stirred at 0 °C for 2 hours before being quenched with H₂O. The resulting solution was diluted with 5 mL of dichloromethane and were sequentially washed with water (3×20 mL) and brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude residue was purified by flash column chromatography (Hexanes/EtOAc =7/1) to furnish 196 mg (0.18 mmol, 86%) of 2-azido-3,4-di-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-β-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranosyl trichloroacetimidate 28 (α only) which was found to be unstable for characterization and thus used immediately for the next reaction. A mixture of 2-azido-3,4-di-O-benzyl-2-deoxy-6-O-(para-methoxybenzyl)-β-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranosyl trichloroacetimidate 28 (52 mg, 0.048 mmol) and disaccharide acceptor 21 (35 mg, 0.04 mmol) was dissolved in mixed solvent containing Et₂O (0.48 mL) and CH₂Cl₂ (0.24 mL) (2/1, v/v) and freshly activated 4 Å molecular sieves (50 mg) was added. The resulting mixture was stirred at room temperature for 10 minutes under argon atmosphere before being cooled down to −60 °C and stirred for another 30 minutes. Trimethylsilyl trifluoromethanesulfonate (1.0 µL) was added, and the resulting mixture was stirred for 3 hours. The reaction mixture was quenched with triethylamine and filtered through celite. The filtrate was concentrated and the crude residue was purified by preparative thin layer chromatography (Hexanes/EtOAc/ CH₂Cl₂ = 3/1/1) to furnish desired tetrasaccharide 29 (58.4 mg, 82 %) and its β-anomer (6.2 mg, 8.7%) (α/β = 9.4/1).${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}=+8.6\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz, CDCl₃) δ 7.51 – 7.12 (m, 49 H), 6.88 – 6.82 (m, 2H), 6.81–6.72 (m, 2H), 5.54 (s, 1H, H-1_Glc), 5.01 (d, J = 10.9, 1H, -OCH₂Ph), 4.94 (d, J = 10.9, -OCH₂Ph), 4.91 – 4.75 (m, 7H, -OCH₂Ph), 4.72 (dd, J = 11.7, 2.2 Hz, 1H), 4.71 – 4.40 (m, 16H, -OCH₂Ph, H-1_Glc), 4.31 (s, 1H, H-1_Man), 4.21 (s, 1H, H-1_Man), 4.13 (d, J = 10.6 Hz, 1H, H-6_Glc), 4.08 – 3.97 (m, 3H), 3.93 (m, 1H), 3.86 – 3.3 (m, 9H), 3.72–3.64 (m, 7H), 3.61 – 3.37 (m, 8H), 3.36 – 3.30 (m, 4H). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 159.3, 159.1, 138.8, 138.8, 138.6, 138.4, 138.2, 138.1, 138.1, 137.7, 137.6, 130.5, 130.4, 129.6, 129.5, 128.7, 128.6, 128.6, 128.5, 128.5, 128.5, 128.5, 128.4, 128.3, 128.2, 128.2, 128.1, 128.0, 128.0, 127.9, 127.9, 127.9, 127.8, 127.7, 127.7, 127.4, 113.8, 113.7, 99.7, 99.6, 99.6, 97.9, 96.9, 82.2, 81.8, 81.2, 81.0, 80.0, 79.6, 77.7, 77.6, 75.9, 75.8, 75.6, 75.5, 75.3, 74.9, 74.8, 74.6, 73.5, 73.3, 73.2, 73.1, 72.1, 71.6, 71.2, 71.0, 69.7, 69.6, 68.8, 68.4, 68.2, 61.9, 61.2, 56.3, 55.3, 55.3, 55.2. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₁₀₄H₁₁₃N₆O₂₁ [M+H]⁺ 1781.7959; found 1781.7935.

Methyl 2-azido-3,4-di-O-benzyl-2-deoxy-β-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-2-azido-3-O-benzyl-2-deoxy-β-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranoside (30).

To tetrasaccharide 29 (179 mg, 0.1 mmol) was added 1.8 mL of CH₂Cl₂ and 200 µL of trifluoroacetic acid and the resulting mixture was stirred at room temperature for 20 minutes. The reaction mixture was quenched with saturated NaHCO₃ solution and organic layer was separated. The aqueous solution was extracted with CH₂Cl₂ (3 × 5 mL), and combined organic extracts were sequentially washed with water and brine. The resulting organic solution was dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude residue was purified by flash column chromatography (Hexanes/EtOAc = 2/1) to furnish 120 mg (0.077 mmol, 78%) of desired compound 30.${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}=+7.4\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz, CDCl₃) δ 7.41 – 7.18 (m, 45 H), 5.44 (d, J = 3.7 Hz, 1H, H-1_Glc), 5.02 – 4.50 (m, 19H), 4.32 (s, 1H, H-1_Man), 4.30 (s, 1H, H-1_Man), 4.08 – 3.56 (m, 18H), 3.54 – 3.45 (m, 2H), 3.42 (t, J = 9.4 Hz, 1H, H-4_Man), 3.38 – 3.32 (m, 5H), 3.20 (ddd, J = 9.7, 4.9, 2.6 Hz, 1H, H-5_Man). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 138.8, 138.6, 138.5, 138.4, 138.3, 138.3, 138.2, 138.2, 138.1, 138.1, 137.7, 137.6, 128.7, 128.6, 128.6, 128.6, 128.6, 128.6, 128.5, 128.5, 128.4, 128.4, 128.3, 128.2, 128.2, 128.1, 128.1, 128.1, 128.0, 128.0, 128.0, 128.0, 128.0, 127.9, 127.9, 127.9, 127.9, 127.8, 127.8, 127.4, 99.9, 99.6, 99.6, 98.0, 97.7, 82.2, 81.7, 80.9, 80.9, 80.0, 80.0, 79.4, 77.8, 77.5, 75.9, 75.9, 75.9, 75.7, 75.5, 75.5, 75.5, 75.0, 74.9, 74.8, 74.3, 73.6, 73.6, 73.1, 72.2, 71.9, 71.9, 71.7, 71.3, 69.8, 69.0, 68.6, 62.2, 62.1, 61.7, 61.2, 55.3. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₈₈H₉₇N₆O₁₉ 1541.6808; found 1541.6881.

Methyl (methyl 2-azido-3,4-di-O-benzyl-2-deoxy-β-D-mannopyranosyl uronate)-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-(methyl 2-azido-3-O-benzyl-2-deoxy-β-D-mannopyranosyl uronate)-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranoside (32).

To a mixture of methyl 2-azido-3,4-di-O-benzyl-2-deoxy-β-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-2-azido-3-O-benzyl-2-deoxy-β-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranoside (30) (100 mg, 0.064 mmol) in 1.0 mL of dichloromethane and 0.1 mL water were added 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO) (2 mg, 0.013 mmol), and phenyliodine(III) diacetate (PIDA) (82 mg, 0.256 mmol). The resulting mixture was stirred at room temperature for 2 hours before being quenched with saturated NaHCO₃ solution. The crude mixture was extracted with CH₂Cl₂ (3 × 5 mL), and combined extracts were sequentially washed with water and brine, dried over anhydrous Na₂SO₄, filtered and concentrated. The crude residue was purified by flash column chromatography (3% MeOH in CH₂Cl₂) to furnish 78 mg (0.049 mmol, 76%) of methyl (2-azido-3,4-di-O-benzyl-2-deoxy-β-D-mannopyranosyl uronic acid)-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-(2-azido-3-O-benzyl-2-deoxy-β-D-mannopyranosyl uronic acid)-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranoside (31). This dicarboxylic acid 31 (38.7 mg, 0.024 mmol) was subsequently dissolved in 480 µL of DMF followed by addition of K₂CO₃ (10 mg, 0.072 mmol) and CH₃I (3 µL, 0.05 mmol). The mixture was stirred at room temperature for 4 hours before being quenched with water. The reaction mixture was extracted with EtOAc (3 × 5 mL), and combined organic extracts were sequentially washed with water and brine. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude residue was purified by flash column chromatography (3% MeOH in CH₂Cl₂) to furnish 36 mg (0.022 mmol, 94%) of desired product 32.${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}=+54.0\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz, CDCl₃) δ 7.57 – 6.98 (m, 45H), 5.23 (s, 1H), 5.02 – 4.50 (m, 19H), 4.39 – 4.18 (m, 3H), 4.14 – 4.04 (m, 1H), 4.02 – 3.83 (m, 6H), 3.80 – 3.55 (m, 12H), 3.52 – 3.29 (m, 5H), 3.28 (s, 3H, -OMe). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 168.4, 168.3, 138.8, 138.7, 138.6, 138.3, 138.2, 138.1, 138.0, 137.5, 128.7, 128.6, 128.6, 128.5, 128.5, 128.5, 128.3, 128.2, 128.2, 128.2, 128.1, 128.1, 128.0, 128.0, 128.0, 128.0, 127.9, 127.9, 127.8, 127.8, 127.6, 100.0, 99.9, 97.8, 82.2, 81.5, 80.0, 79.7, 78.8, 77.9, 75.9, 75.7, 75.5, 75.4, 75.1, 75.0, 74.9, 74.8, 73.7, 73.5, 73.2, 72.3, 72.2, 71.0, 69.8, 68.9, 67.7, 61.6, 55.2, 52.8, 52.6. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₉₀H₉₆N₆O₂₁ 1596.6629, found 1597.6725.

Methyl (methyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-β-D-mannopyranosyl uronate)-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-(methyl 2-acetamido-3-O-benzyl-2-deoxy-β-D-mannopyranosyl uronate)-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranoside (33).

To tetrasaccharide 32 (20 mg, 0.0125 mmol) was added 125 µL of pyridine and 125 µL of thioacetic acid. The resulting mixture was warmed to 40 °C and stirred at this temperature for 12 hours. Solvents were removed under reduced pressure and the crude residue was purified by flash column chromatography (3% MeOH in CH₂Cl₂) to furnish 18 mg (0.011 mmol, 88%) of desired compound 33.${\left[\mathit{\alpha }\right]}_{\mathbf{D}}^{\mathbf{25}}=-8.6\left(c\hspace{0.17em}1.0,{\text{CHCl}}_{\text{3}}\right)$; ¹H NMR (600 MHz, CDCl₃) δ 7.63 (d, J = 9.8 Hz, 1H, -NHAc), 7.40 – 7.17 (m, 45H), 5.95 (d, J = 9.9 Hz, 1H, -NHAc), 5.57 (d, J = 3.7 Hz, 1H, H-1_Glc), 5.12 – 4.70 (m, 12H), 4.66 (d, J = 11.9 Hz, 2H), 4.62 – 4.41 (m, 8H), 4.38 – 4.30 (m, 2H), 4.20 (t, J = 8.2 Hz, 1H), 4.08 (dd, J = 10.7, 1.8 Hz, 1H, H-6_Glc), 4.08 – 4.00 (m, 2H), 3.98 (t, J = 9.2 Hz, 1H, H-3_Glc), 3.91 – 3.82 (m, 1H), 3.83 – 3.75 (m, 4H), 3.73 (s, 3H, -COOCH₃), 3.68 (s, 3H, -COOCH₃), 3.65 (dd, J = 8.9, 4.4 Hz, 1H, H-1_Man), 3.52 – 3.44 (m, 3H), 3.40 (dd, J = 9.8, 3.6 Hz, 1H, H-1_Glc), 4.38 – 4.30 (m, 4H), 3.16 (dd, J = 10.2, 8.8 Hz, 1H, H-4_Glc), 2.10 (s, 3H, -NHC(O)CH₃), 2.01 (s, 3H, -NHC(O)CH₃). ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 172.1, 171.1, 169.3, 169.0, 138.8, 138.7, 138.4, 138.3, 138.2, 138.0, 137.9, 137.6, 137.6, 128.6, 128.5, 128.5, 128.5, 128.4, 128.4, 128.4, 128.2, 128.2, 128.2, 128.1, 128.0, 128.0, 128.0, 128.0, 127.9, 127.9, 127.8, 127.8, 127.7, 127.7, 127.7, 127.6, 127.6, 101.4, 100.1, 97.8, 95.4, 82.2, 81.2, 80.0, 79.2, 79.2, 78.4, 78.2, 75.9, 75.7, 75.7, 75.3, 75.1, 74.8, 74.4, 73.5, 72.3, 71.0, 70.9, 70.5, 70.1, 70.0, 69.2, 69.1, 55.0, 52.7, 52.6, 48.9, 47.9, 29.8, 23.6, 23.0. HRMS (ESI) m/z: [M + H]⁺ Calcd for C₉₄H₁₀₅N₂O₂₃ 1629.7108, found 1629.7175.

Methyl (2-acetamido-2-deoxy-β-D-mannopyranosyl uronic acid)-(1→6)-α-D-glucopyranosyl-(1→4)-(2-acetamido-2-deoxy-β-D-mannopyranosyl uronic acid)-(1→6)-α-D-glucopyranoside (6).

A 100 mL three-necked round bottom flask containing a glass stir bar was equipped with a “U” shape condenser (for cooling ammonia gas) and cooled at −78 °C under argon. After acetone and dry ice were added into the “U” shape condenser, liquid ammonia (ca. 5 mL) was collected in the three-necked flask. A piece of sodium metal (125 mg, 5.43 mmol) was added to the flask and stirred for 15 minutes. Extra sodium was added until the solution remained dark blue in color. A solution of methyl (2-azido-3,4-di-O-benzyl-2-deoxy-β-D-mannopyranosyl uronic acid)-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-(2-azido-3-O-benzyl-2-deoxy-β-D-mannopyranosyl uronic acid)-(1→6)-2,3,4-tri-O-benzyl-α-D-glucopyranoside 31 (55 mg, 0.035 mmol) in 2.5 mL of THF was added and the resulting mixture was stirred at −78 °C for 1 hour before being quenched with solid NH₄Cl. The mixture was slowly warmed to room temperature, and liquid ammonia and THF were removed by nitrogen flow. The residue was desalted using size-exclusion chromatography (Bio-Gel^® P-2 Media, eluted with water). The obtained crude zwitterionic oligosaccharide was dissolved in a mixture of H₂O (2 mL) and THF (0.2 mL), and solid NaHCO₃ was added to adjust the pH to 9. The reaction mixture was cooled at 0 °C before Ac₂O (32 µL) was added. The reaction was stirred for another 1 hour before being neutralized with 1 N HCl and concentrated. The residue was purified through size-exclusion chromatography (Bio-Gel^® P-2 Media, eluted with water) to furnish 11 mg (0.013 mmol, 40%) of desired compound 6. ¹H NMR (600 MHz, D₂O) δ 5.28 (d, J = 3.9 Hz, 1H), 4.65 (d, J = 3.9 Hz, 1H), 4.43 – 4.36 (m, 1H), 4.35– 4.32 (m, 1H), 4.07 – 3.04 (m, 23H), 1.95 (s, 6H). ¹³C{¹H} NMR (150 MHz, D₂O) δ 175.3, 175.3, 99.5, 99.5, 99.0, 98.0, 77.3, 77.0, 73.4, 72.9, 72.5, 72.4, 71.6, 71.4, 71.1, 70.5, 70.2, 69.4, 69.2, 68.7, 68.5, 67.6, 54.8, 53.5, 52.9, 21.9, 21.9.. HRMS (ESI) m/z: [M – H]⁻Calcd for C₉₄H₁₀₅N₂O₂₃ 789.2413, found 789.2400.