Chemical Synthesis of Human Milk Oligosaccharides: Sialyllacto-N-Tetraose a (LSTa)

Abstract

Being the key factor in safeguarding the growth and development of an infant, human milk oligosaccharides (HMO) have drawn attention from multiple disciplines. The availability of HMO in pure form is needed for facilitating further studies and enhancing our understanding of the exact role of an individual HMO in a pool of more than 200 known compounds. Among those, sialylated HMO are already known to have a pivotal role in the antiadhesive, prebiotic, immunomodulating, and antimicrobial activities along with providing nutritional value for brain development in infants. Reported herein is the first chemical synthesis of a mono sialylated HMO, sialyllacto-N-tetraose a (LSTa).

Graphical Abstract

INTRODUCTION

The studies showcasing the evident difference in health and rich intestinal microbiota present in breastfed infants in comparison to formula-fed infants spiked an interest in human milk research.¹ General constituents of the human milk are fat, protein, and carbohydrates.² Among carbohydrate components, lactose is present in the highest concentration and is often correlated to the requirement of high energy demand of the human brain.² The other significant fraction is human milk oligosaccharides (HMO) that collectively represent the third largest component of human milk.³ Being digestion-resistant, HMO are responsible for the prebiotic effects and the gastrointestinal microbiota.^4,5 HMO structure comprises five basic monosaccharides: d-glucose, d-galactose, N-acetylglucosamine, l-fucose, and N-acetylneuraminic (sialic) acid. Oligosaccharide backbone follows a basic blueprint with a lactose unit at the reducing end extended and/or branched by lacto-N-biose or lactosamine units.⁶ HMO are categorized into type I if connected through Gal(β1–3)GlcNAc linkage (lacto-N-biose) and type II for Gal(β1–4)GlcNAc linkage (lactosamine). The oligosaccharide backbone can be decorated with fucose and/or N-acetylneuraminic acid at various positions.⁷

About 20% of HMO contain sialic acid residue(s) and are hence negatively charged.⁶ It is observed that the binding of sialylated or fucosylated HMO helps inhibiting bacterial adhesion at the epithelial cells. Efforts are being made toward the development of carbohydrate-based drugs with non-immunological functions. These can reduce the interactions of leukocytes with endothelial cells, which helps in the prevention of overreaction during inflammation.^8,9 While the limited availability of HMO continues to be the roadblock to further research and development in the field, it is important to acknowledge the advances in the enzymatic synthesis.¹⁰ These advances enabled industrial synthesis of tri- and tetrasaccharides, boosting preclinical research, and made it possible to include simple HMO, 2′-fucosyllactose (2′-FL) and lacto-N-neotetraose (LNnT), as the main additives to current infant formulas.^4,11–13 The development of robust chemical methods and accessible technologies that will offer new capabilities for synthesizing pure individual HMO will help to improve understanding their roles and boost practical applications.

Our research group has been developing chemical methods for the synthesis of HMO. First synthetic efforts led to the development of convergent and linear synthetic strategies for the synthesis of two common linear core tetrasaccharides i.e., lacto-N-tetraose (LNT)¹⁴ and lacto-N-neotetraose (LNnT).¹⁵ These were then followed by successful syntheses of two branched iso-HMO hexasaccharides i.e., lacto-N-hexaose (LNH)¹⁶ and lacto-N-neohexaose (LNnH).¹⁷ Also synthesized were two linear hexasaccharides, para-lacto-N-hexaose (pLNH) and para-lacto-N-neohexaose (pLNnH) utilizing a convergent 3 + 3 approach.¹⁸ We have also synthesized lacto-N-triose (LNTri II) and 3′-galactosyllactose (β3′-GL) core HMO sequences using an HPLC-based automated platform developed in our laboratory.¹⁹ Recently, we became interested in synthesizing functionalized HMO, and completed the synthesis of two fucosylated HMO, 3-fucosyllactose and lacto-N-fucopentaose V.²⁰ Reported herein is the first chemical synthesis of sialylated HMO, pentasaccharide sialyllacto-N-tetraose a (LSTa).

RESULTS AND DISCUSSION

LSTa (1, Scheme 1) comprises lactose at the reducing end, which is extended by a lacto-N-biose unit at the C-3 position of the galactose through a β-linkage. The lacto-N-biose unit is then extended with N-acetylneuraminic acid at the C-3 position of the galactose through an α-(2–3)-linkage. The chemoenzymatic syntheses of LSTa^21,22 and its fully protected derivative are known,²³ but to our knowledge chemical synthesis of this HMO is not conducted. Per our retrosynthetic analysis of 1 shown in Scheme 1, the fully substituted pentasaccharide 2 can be assembled from two oligosaccharide building blocks 3 and 4 using a convergent 2 + 3 synthetic strategy. To achieve these oligosaccharide components, disaccharide 3 could be assembled using sialyl donor 5 and galactosyl acceptor 6. On the other hand, trisaccharide 4 could be assembled from glucosamine donor 7 and lactose acceptor 8. In turn, lactose 8 could be assembled using known building blocks, galactosyl donor 9 and glucosyl acceptor 10.

Scheme 1.

Retrosynthetic Analysis of Sialyllacto-N-tetraose a (LSTa, 1)

With this plan, we first endeavored to synthesize sialylated disaccharide 3 as outlined in Scheme 2. The synthesis of the desired phosphate donor 5²⁴ began from known per-O-acetylated thioglycoside 11.²⁵ Precursor 11 was de-O-acetylated under acidic conditions in the presence of methanesulfonic acid in refluxing methanol followed by 5-N,4-O-cyclocarbamate introduction yielding intermediate 12 in 67% yield. Subsequent O-acetylation using Ac₂O in the presence of DMAP in pyridine produced per-O-acetylated compound 13²⁶ in 88% yield. N-Acetylation of compound 13 was affected with AcCl in the presence of DIPEA to afford N-acetyl-5-N,4-O-oxazolidinone-protected thiosialoside 14.²⁷ Finally, the phenylthio anomeric group was replaced with a dibutylphosphate moiety in the presence of NIS yielding compound 5 in 88% yield as predominantly β-anomer. Sialyl phosphate donor 5 was chosen for two reasons. First, it has been documented that 5-N,4-O-oxazolidinone and its acetylated counterpart, often provides increased stability toward elimination and high stereoselectivities and yields for a wide range of acceptors.^26–28 Second, per our retrosynthetic analysis, the use of the phosphate group would allow for selective activation of the glycosyl donor 5 over thioglycoside acceptor 6.²⁴ The advantage of such selective activation strategy is that the resulting thioglycoside disaccharide can be used in subsequent steps directly without the necessity of replacing the anomeric leaving group.²⁹

Scheme 2.

Synthesis of Sialyl Galactose Disaccharide Fragment 3

A suitably protected galactosyl acceptor 6 was synthesized from 2,3-diol precursor 15 following the 4-step synthetic sequence as depicted in Scheme 2. Thus, regioselective protection at the C-3 position using TBDMSCl in the presence of imidazole at 0 °C afforded compound 16 in 93% yield. The latter was then subjected to 2-O-benzoylation with benzoyl chloride in the presence of DMAP in pyridine to afford a fully substituted intermediate 17 in 92% yield. The TBDMS group was then selectively removed with HF-Pyridine affording compound 18 in 97% yield. Finally, compound 18 was subjected to regioselective reductive opening of 4,6-O-benzylidene ring using NaCNBH₃/I₂ to afford the desired 3,4-diol acceptor 6. The choice of a 3,4-diol over an acceptor with only one free hydroxyl group is quite common in sialylations: steric hindrance and/or low reactivity of fully protected acceptors often plague sialylation reaction with poor yield and/or stereoselectivities.^30,31 In our experience protecting group pattern employed herein, rather than less bulky groups such as acetates, provides the best results in sialylations.

With sialyl donor 5 and galactosyl acceptor 6 in hand, we conducted the sialylation reaction. The phosphate leaving group of donor 5 was activated with 1.0 equiv of TMSOTf in CH₂Cl₂ solvent at −78 °C. The low temperature seemed to be critical to ensure a successful regioselectivity at the C-3 position of diol 6. This is because in reactions at higher temperatures (−40 °C) we also obtained the (2→4)-linked regioisomer, which was very difficult to separate from the desired (2→3)-linked product 19. This observation was in accordance with previously reported results.³² Using these precautions, disaccharide 19 was formed in 2 h in 80% yield with complete α-selectivity. The remaining hydroxyl was then protected by acetylation with acetic anhydride in pyridine to avoid possible side reactions at the advanced stages of the pentasaccharide assembly. Disaccharide 3 was obtained in 94%, and in accordance with our strategy, it can directly be used as the disaccharide donor for the 2 + 3 coupling with trisaccharide acceptor 4.

Next, we embarked on the synthesis of glucosamine synthon 7 needed to introduce the middle unit of the target pentasaccharide. As depicted in Scheme 3, this synthesis started from the known 4,6-O-benzylidene-protected precursor 20.³³ The hydroxyl group at C-3 was first silylated with TBDMSCl in the presence of imidazole to afford a fully protected intermediate 21 in 95% yield. The latter was then subjected to the regioselective reductive opening of the 4,6-O-benzylidene ring. In this case, we needed to liberate the hydroxyl at the C-6 position. This transformation was affected in the presence of 1 M BH₃-THF and TMSOTf, and 6-OH derivative 22 was isolated in 80% yield. Subsequently, the hydroxyl group was benzylated with BnBr in the presence of NaH in THF to afford compound 23 in 78% yield. THF was found to be the preferred reaction solvent in this transformation over a more conventional solvent DMF used in benzylations. All attempts to conduct benzylation of 22 in DMF lead to an unidentified side product 27 (refer to the SI for the proton spectrum). This is because of the marginal stability of the phthalimide ring that can be opened or cleaved under basic reaction conditions.

Scheme 3.

Synthesis of the Reducing End Trisaccharide 4

The TBDMS group in building block 23 was then removed with HF-pyridine and the liberated hydroxyl at C-3 was protected with the Fmoc group by reaction with FmocCl in the presence of DMAP in pyridine. As a result, compound 24 was isolated in 71% yield. Finally, the ethylthio anomeric group was replaced with the phosphate leaving group by reaction with dibutyl hydrogen phosphate in the presence of NIS and TfOH to afford glycosyl donor 7 in 98%. Although the S-ethyl leaving group can be activated for glycosylation, we chose to invest into this additional step due to our previous experience with similar building blocks, whereas a highly reactive phosphate leaving group provided superior results.¹⁴

For the preparation of lactosyl acceptor 8, known building blocks, galactosyl donor 9³⁴ and glucosyl acceptor 10¹⁵ were coupled in the presence of NIS and TfOH affording the desired β-linked disaccharide 25 in 98% yield (Scheme 3). The 3-O-TBDMS group in 25 was then removed using HF-pyridine to produce disaccharide acceptor 8 in 92% yield. We then carried out glycosylation reaction between phosphate donor 7 and lactose acceptor 8. The phosphate leaving group was activated with 1.0 equiv of TfOH and trisaccharide 26 was readily obtained in an excellent yield of 90%. The Fmoc group was removed by the treatment with triethylamine over a period of 2 h yielding trisaccharide acceptor 4 in 91% yield.

The synthesis of trisaccharide 26 was optimized following several challenges associated with protecting group strategies and structural modifications. Initially, the lactose acceptor featured a 4,6-O-benzylidene group on the galactose residue. However, glycosylation attempts using a glucosamine donor bearing a benzoyl (OBz) group at the 6-position, instead of a benzyl (OBn) group, consistently failed to yield the desired product. This lack of reactivity was attributed to the possible unfavorable spatial interactions between the phthalimide moiety on the donor and the benzylidene group on the lactose acceptor. Consequently, protecting group adjustments were undertaken, ultimately leading to the removal of the benzylidene ring from the lactose unit. Although the reaction did proceed with the 6-O-benzoyl-protected derivative of glucosamine donor 24, the yields remained poor despite modifications to both the leaving group and glycosylation conditions. To improve the reactivity of the donor, we replaced the electron-withdrawing OBz group with an electron-donating OBn group at the 6-position. This modification significantly enhanced the donor’s reactivity, resulting in a substantial increase in glycosylation yield upon coupling with acceptor 8.

The final assembly of pentasaccharide 2 was achieved as depicted in Scheme 4. Thus, a 2 + 3 glycosylation reaction between donor 3 and acceptor 4 was promoted in the presence of NIS and TfOH at −30 °C, and the protected pentasaccharide 2 was obtained in an excellent yield of 84%. We note that the reaction temperature had a profound impact on the yield. A similar reaction performed at 0 °C have pentasaccharide 2 in a modest yield of 41%. The fully protected pentasaccharide 2 was then subjected to a multistep deprotection sequence. First, the methyl ester was saponified with LiI in pyridine under reflux. The resulting lithium carboxylate was subjected to purification by size-exclusion chromatography. The oxazolidinone ring and some O-acetates were removed by the treatment with NaOMe in MeOH for 1 h. The reaction mixture was reacted with hydrazine hydrate in MeOH at reflux, which removed all remaining ester groups and the phthalimido protecting group. The resulting free amine was N-acetylated with Ac₂O in MeOH in 12 h. The reaction time was critical, as prolonged reaction time could lead to unwanted O-acetylation.

Scheme 4.

Final Assembly and Deprotection of Pentasaccharide 2 to Obtain LSTa 1

In those cases where some O-acetates were introduced/remained, the intermediate was additionally treated with NaOMe in MeOH for 1 h. All benzyl ethers were then removed under hydrogenation conditions over Pd/C yielding the deprotected LSTa as a lithium salt. Lastly, the cation exchange was ensured by the treatment with 1 N aq. NaOH, and the final product was purified by size-exclusion chromatography on Sephadex G-25 to afford LSTa 1 in 30% yield overall (seven steps).

In conclusion, reported herein is the first chemical synthesis of the sialylated HMO LSTa. Using a convergent 2 + 3 strategy, as well as careful selection of leaving groups and strategic placement of protecting groups led to a highly efficient assembly. It should also be noted that practically all reactions required for the synthesis of building blocks, glycan assembly, and deprotection, proceeded with very high to excellent yields. The application of an oxazolidinone-protected sialyl donor was especially impactful: not only did it enable complete regio- and stereoselective sialylation but also allowed the use of DCM as the reaction solvent, facilitating the reaction conducted at low temperature. Further development of new methods and efficient strategies for the synthesis of HMO in solution and using automated approaches are currently underway in our laboratory.

EXPERIMENTAL SECTION

General.

The reactions were performed using commercial reagents and the ACS grade solvents used for reactions were purified and dried in accordance with standard procedures. Column chromatography was performed on silica gel 60 (70–230 mesh), reactions were monitored by TLC on Kieselgel 60 F₂₅₄. The compounds were detected by examination under UV light and by charring with 10% sulfuric acid in methanol. Solvents were removed under reduced pressure at <40 °C. CH₂Cl₂ was distilled from CaH₂ directly prior to application. Molecular sieves (3 or 4 Å) used for reactions, were crushed and activated in vacuo at 390 °C for 8 h in the first instance and then for 2–3 h at 390 °C directly prior to application. Reactions that required heating were carried out using an oil bath as the heat source. Optical rotations were measured at “Jasco P-2000” polarimeter. ¹H NMR spectra were recorded at 400 or 700 MHz, ¹³C NMR spectra were recorded at 101 or 176 MHz. The ¹H NMR chemical shifts are referenced to tetramethyl silane (TMS, δ_H = 0 ppm) or CDCl₃ (CHCl₃ δ_H = 7.26 ppm) for solutions in CDCl₃ or to the signal of the residual HOD (δ_H = 4.80 ppm) for solutions in D₂O. The ¹³C NMR chemical shifts are referenced to the central signal of CDCl₃ (δ_C = 77.00 ppm) for solutions in CDCl_3. Indirect referencing was used for ¹³C NMR for solutions in D₂O. Structural assignments were made with additional information from gCOSY and gHSQC experiments. Mass analysis was performed using Agilent 6230 ESI TOF mass spectrometer.

Synthesis of Monosaccharide Building Blocks 5–7, 9, and 10. Methyl (5-Acetamido-7,8,9-tri-O-acetyl-5-N,4-O-carbonyl-2-(dibutylphohphoryl)-3,5-dideoxy-d-glycero-d-galacto-non-2-ulopyranosyl)onate (5).

Methanesulfonic acid (1.11 mL, 17.1 mmol, 10 equiv) was added to a solution of methyl (phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-d-glycero-β-d-galacto-2-nonulopyranosid)onate (11,²⁵ 1.0 g, 1.71 mmol, 1.0 equiv) in MeOH (40 mL) and the resulting mixture was refluxed for 24 h. After that, the reaction mixture was neutralized with Dowex (⁻OH), the resin was filtered off and rinsed successively with MeOH. The combined filtrate was concentrated under reduced pressure. The residue was dissolved in a mixture of water (10.0 mL) and MeCN (5.0 mL), NaHCO₃ (0.72 g, 8.55 mmol, 5.0 equiv) was added, and the mixture was cooled down to 0 °C. A solution of 4-nitrophenyl chloroformate (NPCC, 1.72 g, 8.55 mmol, 5.0 equiv) in MeCN (5.0 mL) was added dropwise, and the resulting mixture was vigorously stirred for 3 h at 0 °C. The reaction mixture was extracted with EtOAc (3 × 20 mL), and the combined organic extract was washed with brine (20 mL), separated, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (EtOAc-MeOH gradient elution) to afford methyl (phenyl 5-amino-5-N,4-O-carbonyl-3,5-dideoxy-2-thio-d-glycero-β-d-galacto-non-2-ulopyranoside)onate (12, 0.457 g, 1.15 mmol) as a white foam in 67% yield. ¹H NMR (400 MHz, CDCl₃): δ 7.49–7.32 (m, 5H, aromatic), 6.55 (s, 1H, NH), 4.69 (ddd, 1H, J_4,5 = 10.4 Hz, H-4), 4.51 (dd, 1H, J_6,7 = 3.9 Hz, H-6), 3.83–3.65 (m, 7H, H-7, 8, 9a, 9b, COOCH₃), 3.51 (dd, 1H, J_5,6 = 10.5 Hz, H-5), 2.89 (dd, 1H, J_3eq,3ax = 12.9 Hz, J_3eq,4 = 3.8 Hz, H-3eq), 2.35 (dd, J_3ax,4 = 12.8 Hz, H-3ax) ppm. Analytical data for 12 was in agreement with that reported previously.²⁷

Ac₂O (0.44 mL, 4.64 mmol, 4.5 equiv) was added dropwise to a chilled (0 °C) solution of compound 12 (0.41 g, 1.03 mmol, 1.0 equiv) in pyridine (15 mL), and the resulting mixture was stirred under argon for 16 h at rt. The reaction was quenched with methanol (~2 mL), the volatiles were removed under reduced pressure, and the residue was purified by column chromatography on silica gel (acetone-hexane gradient elution) to afford methyl (phenyl 7,8,9-tri-O-acetyl-5-N,4-O-carbonyl-3,5-dideoxy-2-thio-d-glycero-β-d-galactonon-2-ulopyranoside)onate (13, 0.48 g, 0.91 mmol) as a white foam in 88% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.64–8.62 (m, 2H, aromatic), 7.77–7.72 (m, 1H, aromatic), 7.43–7.33 (m, 2H, aromatic), 5.68 (s, 1H, NH), 5.25–5.18 (m, 2H, H-7, 8), 4.71 (ddd, 1H, J_4,5 = 10.4 Hz, H-4), 4.61 (dd, 1H, J_6,7 = 2.5 Hz, H-6), 4.41 (dd, 1H, J_9a,9b = 12.6 Hz, H-9a), 4.22 (dd, 1H, H-9b), 3.60 (s, 3H, COOCH₃), 3.13 (dd, 1H, J_5,6 = 10.5 Hz, H-5), 2.85 (dd, 1H, J_{3eq,3ax =} 13.0 Hz, J_3eq,4 = 3.8 Hz, H-3eq), 2.29 (dd, 1H, J_3ax,4 = 12.8 Hz, H-3ax), 2.15, 2.09, 2.02 (3 s, 9H, 3 × CH₃CO) ppm. HR-ES MS [M + Na]⁺ calcd for C₂₃H₂₇NO₁₁SNa 548.1203, found 548.1209. Analytical data for 13 was in agreement with that reported previously.²⁶

DIPEA (1.57 mL, 9.0 mmol, 10 equiv) and acetyl chloride (0.51 mL, 7.2 mmol, 8.0 equiv) were added sequentially to a chilled (0 °C) solution of compound 13 (0.47 g, 0.90 mmol, 1.0 equiv) in anhydrous CH₂Cl₂ (20 mL), and the resulting mixture was stirred under argon for 3 h at 0 °C. After that, the reaction mixture was diluted with CH₂Cl₂ (~50 mL) and washed with sat. aq. NaHCO₃ (20 mL). and brine (20 mL). The organic phase was separated, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (acetone-hexane gradient elution) to afford methyl (phenyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-carbonyl-3,5-dideoxy-2-thio-d-glycero-β-d-galacto-non-2-ulopyranoside)onate (14, 0.48 g, 0.85 mmol) as a white foam in 94% yield. ¹H NMR (400 MHz, CDCl₃): δ 7.50–7.32 (m, 5H, aromatic), 5.56 (dd, 1H, J_7,8 = 2.4 Hz, H-7), 5.00 (ddd, 1H, J_8,9a = 2.4 Hz, J_8,9b = 8.2 Hz, H-8), 4.88 (dd, 1H, J_6,7 = 2.6 Hz, H-6), 4.77 (ddd, 1H, J_4,5 = 11.3 Hz, H-4), 4.37 (dd, 1H, J_9a,9b = 12 Hz, H-9a), 3.90 (dd, 1H, H-9b), 3.75 (dd, 1H, J_5,6 = 9.1 Hz, H-5), 3.62 (s, 3H, COOCH₃), 2.90 (dd, 1H, J_3eq,3ax = 13 Hz, J_3eq,4 = 3.7 Hz, H-3eq), 2.52 (s, 3H, CH₃CO), 2.33 (dd, 1H, J_3ax,4 = 12.9 Hz, H-3ax), 2.14 (s, 3H, CH₃CO), 2.07 (s, 3H, CH₃CO), 1.96 (s, 3H, CH₃CO) ppm. Analytical data for 14 was in agreement with that reported previously.²⁷

A mixture of compound 14 (0.20 g, 0.35 mmol, 1.0 equiv), dibutyl hydrogen phosphate (0.36 mL, 1.76 mmol, 5.0 equiv), and freshly activated molecular sieves (3 Å, 0.60 g) in DCM (8.0 mL) was stirred under argon for 1 h at rt. Subsequently, NIS (0.236 g, 1.05 mmol, 3.0 equiv) was added, and the resulting mixture was stirred for 16 h at rt. After that, the solids were filtered off and rinsed successively with DCM. The combined filtrate (~100 mL) was washed with sat. aq. NaHCO₃ (2 × 20 mL), and brine (20 mL). The organic phase was separated, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate–hexane gradient elution) to afford the title compound (0.206 g, 0.31 mmol) as amorphous solid in 88% yield as predominantly β-anomer. Analytical data for 5 was in accordance with that previously described.³⁵

Phenyl 2-O-Benzoyl-6-O-benzyl-1-thio-β-d-galactopyranoside (6).

Imidazole (3.18 g, 46.7 mmol, 2.4 equiv) was added to a solution of phenyl 4,6-O-benzylidene-1-thio-β-d-galactopyranoside (15,³⁶ 7.0 g, 19.4 mmol, 1.0 equiv) in anhydrous CH₂Cl₂ (100 mL) under argon at rt. The mixture was cooled to 0 °C, a solution of TBDMSCl (4.39 g, 29.2 mmol, 1.5 equiv) in anhydrous CH₂Cl₂ (60 mL) was added dropwise, and the resulting mixture was stirred at for 16 h at rt. The reaction mixture was diluted with CH₂Cl₂ (~100 mL), washed with sat. aq. NaHCO₃ (50 mL), and brine (20 mL). The organic phase was separated, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate- hexane gradient elution) to afford phenyl 4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-1-thio-β-d-galactopyranoside (16, 8.57 g, 18.07 mmol) as a colorless syrup in 93% yield. ¹H NMR (400 MHz, CDCl₃): δ 7.58–7.55 (m, 2H, aromatic), 7.37–7.35 (m, 2H, aromatic), 7.28–7.25 (m, 3H, aromatic), 7.16–7.09 (m, 3H, aromatic), 5.40 (s, 1H, >CHPh), 4.45 (d, 1H, J_1,2 = 9.4 Hz, H-1), 4.29 (dd, 1H, J_6a,6b = 12.4 Hz, H-6a), 3.96 (dd, 1H, J_4,5 = 1.1 Hz, H-4), 3.93 (dd, 1H, H-6b), 3.74 (dd, 1H, J_2,3 = 9.2 Hz, H-2), 3.64 (dd, 1H, J_3,4 = 3.4 Hz, H-3), 3.42 (br d, 1H, H-5), 2.22 (br s, 1H, 2-OH), 0.79 (s, 9H, (CH₃)₃CSi), 0.00, −0.01 (2 s, 6H, 2 × CH₃Si) ppm. Analytical data for 16 was in agreement with that reported previously.³⁶

DMAP (0.31 g, 2.53 mmol, 0.2 equiv) and benzoic anhydride (7.03 g, 31.6 mmol, 2.5 equiv) were added to a chilled (0 °C) solution of compound 16 (6.0 g, 12.7 mmol, 1.0 equiv) in pyridine (150 mL), and the resulting mixture was stirred under argon for 16 h at rt. After that, the reaction mixture was diluted with CH₂Cl₂ (~200 mL), washed with 1 N aq. HCl (3 × 50 mL), sat. aq. NaHCO₃ (2 × 50 mL) and brine (20 mL). The organic phase was separated, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate -hexane gradient elution) to afford phenyl 2-O-benzoyl-4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-1-thio-β-d-galactopyranoside (17, 6.72 g, 11.6 mmol) as a white amorphous solid in 92% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.04–8.02 (m, 2H, aromatic), 7.56–7.37 (m, 10 H, aromatic), 7.24–7.17 (m, 3H, aromatic), 5.52 (s, 1H, >CHPh), 5.49 (dd, 1H, J_2,3 = 9.5 Hz, H-2), 4.82 (d, 1H, J_1,2 = 9.9 Hz, H-1), 4.42 (dd, 1H, J_6a,6b = 12. 3 Hz, H-6a), 4.12−4.01 (m, 3H, H-3, 4, 6b), 3.58 (br d, 1H, H-5), 0.72 (s, 9H, (CH₃)₃CSi), 0.00, −0.16 (2 s, 6H, 2 × CH₃Si) ppm. HR-ES MS [M + Na]⁺ calcd for C₃₂H₃₈O₆SSiNa 601.2056, found 601.2034. Analytical data of 17 was in agreement with that reported previously.³⁶

HF-pyridine (8.6 mL, 8.64 mmol, 2.0 equiv) was added to a chilled (0 °C) solution of compound 17 (2.5 g, 4.32 mmol, 1.0 equiv) in pyridine (15 mL), and the resulting mixture was stirred under argon for 12 h at rt. The reaction mixture was then diluted with EtOAc (~100 mL), washed with 1 N aq. HCl (30 mL), sat. aq. NaHCO₃ (30 mL) and brine (20 mL). The organic phase was separated, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate–hexane gradient elution) to afford phenyl 2-O-benzoyl-4,6-O-benzylidene-1-thio-β-d-galactopyranoside (18, 1.94 g, 4.18 mmol) as a white amorphous solid in 97% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.09–8.06 (m, 2H, aromatic), 7.62–7.24 (m, 13H, aromatic), 5.55 (s, 1H, >CHPh), 5.25 (dd, 1H, J_2,3 = 9.7 Hz, H-2), 4.83 (d, 1H, J_1,2 = 9.8 Hz, H-1), 4.44 (dd, 1H, J_6a,6b = 12.5 Hz, H-6a), 4.29 (dd, 1H, J_4,5 = 1.2 Hz, H-4), 4.08 (dd, 1H, H-6b), 3.90 (ddd, 1H, J_3,4 = 3.7 Hz, J_3,OH = 11.0 Hz, H-3), 3.64 (br d, 1H, H-5), 2.60 (d, 1H, 3-OH) ppm. Analytical data for 18 was in agreement with that reported previously.³⁶

NaCNBH₃ (0.812 g, 13.0 mmol, 5.0 equiv) was added to a mixture of compound 18 (1.2 g, 2.58 mmol, 1.0 equiv) and freshly activated molecular sieves (4 Å, 3.6 g) in CH₃CN (12 mL) under argon at rt. Iodine (1.14 g, 9.03 mmol, 3.5 equiv) was added portionwise over a period of 15 min, and the resulting mixture was stirred for 5 h at rt. The reaction mixture was then diluted with CH₂Cl₂ (~100 mL), washed with 10% aq. Na₂S₂O₃ (15 mL), sat. aq. NaHCO³ (20 mL) and brine (20 mL). The organic phase was separated, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (acetone-toluene gradient elution) to afford the title compound (1.07 g, 2.29 mmol) as a white amorphous solid in 89% yield. Analytical data for 6 was in agreement with that reported previously.³⁵

Di-O-butyl 4,6-Di-O-benzyl-2-deoxy-3-O-flurenylmethoxycarbonyl-2-phthalimido-β-d-glucopyranosyl Phosphate (7).

Imidazole (2.96 g, 43.5 mmol, 2.4 equiv) was added to a solution of ethyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-1-thio-β-d-glucopyranoside (20,³³ 8.0 g, 18.1 mmol, 1.0 equiv) in anhydrous CH₂Cl₂ (120 mL) under argon at rt. The mixture was cooled to 0 °C, a solution of TBDMSCl (5.47 g, 36.3 mmol, 2.0 equiv) in anhydrous CH₂Cl₂ (50 mL) was added dropwise, and the resulting mixture stirred for 16 h at rt. The reaction mixture then was diluted with CH₂Cl₂ (~50 mL), washed with sat. aq. NaHCO₃ (20 mL) and brine (20 mL). The organic phase was separated, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (acetone - hexane gradient elution) to afford ethyl 4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-2-deoxy-2-phthalimido-1-thio-β-d-glucopyranoside (21, 9.57 g, 17.2 mmol) as a white amorphous solid in 95% yield. ¹H NMR (400 MHz, CDCl₃): δ 7.89–7.73 (m, 4H, aromatic), 7.50–7.34 (m, 5H, aromatic), 5.54 (s, 1H, >CHPh), 5.37 (d, 1H, J_1,2 = 10.6 Hz, H-1), 4.66 (dd, 1H, J_3,4 = 8.7 Hz, H-3), 4.39 (dd, 1H, J_6a,6b = 10.2 Hz, H-6a), 4.32 (dd, 1H, J_2,3 = 9.6 Hz, H-2), 3.80 (dd, 1H, H-6b), 3.71 (ddd, 1H, J_5,6a = 4.7 Hz, J_5,6b = 9.9 Hz, H-5), 3.58 (dd, 1H, J_4,5 = 9.0 Hz, H-4), 2.76–2.59 (m, 2H, SCH₂CH₃), 1.19 (t, 3H, SCH₂CH₃), 0.59 (s, 9H, SiC(CH₃)₃), −0.12, −0.29 (2 s, 6H, 2 × SiCH₃) ppm. Analytical data for 21 was in agreement with that reported previously.¹⁴

A 1 M BH₃-THF (36 mL, 36.0 mmol, 5.0 equiv) and TMSOTf (0.65 mL, 3.61 mmol, 0.5 equiv) were sequentially added to a chilled (0 °C) solution of compound 21 (4.0 g, 7.21 mmol, 1.0 equiv) in CH₂Cl₂ (60 mL), and the resulting solution was stirred under argon for 5 h at rt. The reaction was quenched by the addition of NEt₃ (~2 mL) and MeOH (~5 mL) and the volatiles were removed under reduced pressure. The residue was diluted with CH₂Cl₂ (223C100 mL), washed with sat. aq. NaHCO₃ (20 mL) and brine (20 mL). The organic phase was separated, dried with Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate–hexane gradient elution) to afford ethyl 4-O-benzyl-3-O-tert-butyldimethylsilyl-2-deoxy-2-phthalimido-1-thio-β-d-glucopyranoside (22, 3.21 g, 5.76 mmol) as a white amorphous solid in 80% yield. ¹H NMR (400 MHz, CDCl₃): δ 7.91–7.75 (m, 4H, aromatic), 7.39–7.29 (m, 5H, aromatic), 5.40 (d, 1H, J_1,2 = 10.6 Hz, H-1), 4.88 (d, 1H, ²J = 11.7 Hz, CHPh), 4.71 (d, 1H, ²J = 11.7 Hz, CHPh), 4.56 (dd, 1H, J_3,4 = 7.8 Hz, H-3), 4.27 (dd, 1H, J_2,3 = 10.2 Hz, H-2), 3.94 (ddd, 1H, J_6a,6b = 12.2 Hz, H-6a), 3.74 (ddd, 1H, H-6b), 3.63–3.55 (m, 2H, H-4, 5), 3.75–3.60 (m, 2H, SCH₂CH₃), 1.99 (dd, 1H, 6-OH), 1.21 (t, 3H, SCH₂CH₃), 0.76 (s, 9H, SiC(CH₃)₃), 0.00, −0.38 (2 s, 6H, 2 × SiCH₃) ppm. HR-ES MS [M + Na]⁺ calcd for C₂₉H₃₉NO₆SSiNa 580.2165, found 580.2149. Analytical data for 22 was in agreement with that reported previously.¹⁴

NaH (0.128 g, 5.37 mmol, 3.0 equiv) and BnBr (0.489 mL, 4.12 mmol, 2.3 equiv, dropwise addition) were added to a chilled (0 °C) solution of compound 22 (1.0 g, 1.79 mmol, 1.0 equiv) in THF (15 mL), and the resulting mixture was stirred under argon for 16 h at rt. The reaction was quenched by adding solid NH₄Cl (~20 mg) followed by sat. aq. NH₄Cl (~50 mL), diluted with CH₂Cl₂ (~100 mL), and washed with sat. aq. NaHCO₃ (20 mL) and brine (20 mL). The organic phase was separated, dried with Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford ethyl 4,6-di-O-benzyl-3-O-tert-butyldimethylsilyl-2-deoxy-2-phthalimido-1-thio-β-d-glucopyranoside (23, 0.908 g, 1.40 mmol) as a white amorphous solid in 78% yield. ¹H NMR (400 MHz, CDCl₃): δ 7.93–7.78 (m, 4H, aromatic), 7.42–7.22 (m, 10 H, aromatic), 5.37 (d, 1H, J_1,2 = 10.5 Hz, H-1), 4.86 (d, 1H, ²J = 11.6 Hz, CHPh), 4.73–4.55 (m, 4H, H-3, 3 × CHPh), 4.35 (dd, 1H, J_2,3 = 10.2 Hz, H-2), 3.82–3.78 (m, 2H, H-6a, 6b), 3.74–3.64 (m, 2H, H-4, 5), 2.81–2.66 (m, 2H, SCH₂CH₃), 1.26 (t, 3H, SCH₂CH₃), 0.79 (s, 9H, SiC(CH₃)₃), 0.06, −0.36 (2 s, 6H, 2 × SiCH₃) ppm. Analytical data for 23 was in agreement with that reported previously.¹⁴

One M HF-pyridine (3.0 mL, 3.08 mmol, 2.0 equiv) was added to a chilled (0 °C) solution of compound 23 (1.0 g, 1.54 mmol, 1.0 equiv) in pyridine (10 mL), and the resulting mixture was stirred under argon for 16 h at rt. The reaction mixture was diluted with CH₂Cl₂ (~50 mL), washed sequentially with 1 N aq. HCl (20 mL), sat. aq. NaHCO₃ (20 mL), and brine (~10 mL). The organic phase was separated, dried over Na₂SO₄, and concentrated under reduced pressure and dried in vacuo. The crude residue was dissolved in pyridine (20 mL), FmocCl (0.797 g, 3.08 mmol, 2.0 equiv) and DMAP (0.018 g, 0.154 mmol, 0.1 equiv) were added, and the resulting mixture was stirred under argon for 16 h at rt. The reaction mixture was diluted with CH₂Cl₂ (~100 mL), washed with 1 N aq. HCl (20 mL), sat. aq. NaHCO₃ (20 mL), and brine (20 mL). The organic phase was separated, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate–hexane gradient elution) to afford ethyl 4,6-di-O-benzyl-2-deoxy-3-O-fluorenylmethoxycarbonyl-2-phthalimido-1-thio-β-d-glucopyranoside (24, 0.826 g, 1.09 mmol) as a colorless foam in 71% yield. ¹H NMR (400 MHz, CDCl₃): δ 7.83–7.14 (m, 22H, aromatic), 5.74 (dd, 1H, J_3,4 = 8.9 Hz, H-3), 5.46 (d, 1H, J_1,2 = 10.5 Hz, H-1), 4.70–4.55 (m, 4H, 2 × CH₂Ph), 4.47 (dd, 1H, J_2,3 = 10.4 Hz, H-2), 4.11 (dd, 1H, J_6a,6b = 10.4 Hz, H-6a), 3.99 (dd, 1H, H-6b), 3.93 (dd, 1H, J_4,5 = 9.2 Hz, H-4), 3.86–3.74 (m, 4H, H-5, CH₂ and CH of fmoc), 2.76–2.63 (m, 2H, SCH₂CH₃), 1.22 (t, 3H, SCH₂CH₃) ppm. Analytical data for 24 was in agreement with that reported previously.¹⁴

A mixture containing compound 24 (0.970 g, 1.28 mmol, 1.0 equiv), Bu₂PO₄H (0.79 mL, 3.85 mmol, 3.0 equiv), and freshly activated molecular sieves (4 Å, 2.0 g) in CH₂Cl₂ (~100 mL) was stirred under argon for 2 h at rt. The mixture was cooled to 0 °C, NIS (0.577 g, 2.56 mmol, 2.0 equiv) and TfOH (22 μL, 0.26 mmol, 0.2 equiv) were added, and the resulting mixture was stirred for 30 min at 0 °C. The reaction was then quenched with triethylamine (0.5 mL), the solids were filtered off through a pad of Celite and rinsed successively with CH₂Cl₂. The combined filtrate (~100 mL) was washed with 10% aq. Na₂S₂O₃ (10 mL) and water (2 × 10 mL). The organic phase was separated, dried with Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (acetone – hexane gradient elution) to afford the title compound as a colorless foam in 98% yield (1.13 g, 1.25 mmol). HR-ES MS [M + Na]⁺ calcd for C₅₁H₅₄NO₁₂PNa 926.3281, found 926.3235. Analytical data for 7 was in agreement with that reported previously.¹⁴

Ethyl 2,6-di-O-benzoyl-4-O-benzyl-3-O-tert-butyldimethylsilyl-1-thio-β-d-galactopyranoside (9) was synthesized as reported previously, and its analytical data was in accordance with that previously described.³⁴

Benzyl 2,3,6–tri–O–benzyl–β-d-glucopyranoside (10) was synthesized as reported previously, and its analytical data was in accordance with that previously described.¹⁵

Synthesis of Oligosaccharides 1–4 and 8. Phenyl O-(Methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-carbonyl-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonate)-(2→3)-4-O-acetyl-2-Obenzoyl-6-O-benzyl-1-thio-β-d-galactopyranoside (3).

A mixture of glycosyl donor 5 (0.41 g, 0.62 mmol, 1.2 equiv), glycosyl acceptor 6 (0.25 g, 0.54 mmol, 1.0 equiv), and freshly activated molecular sieves (4 Å, 1.2 g) in CH₂Cl₂ (5.0 mL) were stirred under argon for 2 h at rt. The mixture was cooled to −78 °C, TMSOTf (0.29 mL, 1.62 mmol, 3.5 equiv) was added, and the resulting mixture was stirred for 2 h at −78 °C. The reaction was then quenched with triethylamine (~1 mL), the solids were filtered off through a pad of Celite and rinsed successively with CH₂Cl₂. The combined filtrate (~100 mL) was washed with 10% aq. Na₂S₂O₃ (20 mL), sat. aq. NaHCO₃ (20 mL), and brine (10 mL). The organic phase was separated, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (acetone-hexane gradient elution) to afford phenyl O-(methyl 5-acetamido-7,8,9-tri-Oacetyl-5-N,4-O-carbonyl-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonate)-(2→3)-2-O-benzoyl-6-O-benzyl-1-thio-β-d-galactopyranoside (19, 0.40 g, 0.43 mmol) as a white amorphous solid in 80% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.16–8.14 (m, 2H, aromatic), 7.60–7.56 (m, 1H, aromatic), 7.49–7.45 (m, 4H, aromatic), 7.36–7.21 (m, 8H, aromatic), 5.54–5.46 (m, 2H, H-7′, 8′), 5.41 (dd, 1H, J_2,3 = 9.7 Hz, H-2), 4.94 (d, 1H, J_1,2 = 9.9 Hz, H-1), 4.58 (m, 2H, CH₂Ph), 4.52–4.46 (m, 2H, H-3, 6′), 4.41 (dd, 1H, J_9a’,9b’ = 12.3 Hz, H-9a’), 3.93 (dd, 1H, H-9b’), 3.87–3.76 (m, 5H, H-4, 4′, 5, 6a, 6b), 3.71 (s, 3H, COOCH₃), 3.52 (dd, 1H, J_5′6′ = 11.2 Hz, H-5′), 2.83 (dd, 1H, J_{3eq’,3ax’} = 12.0 Hz, J_3eq’,4′ = 3.4 Hz, H-3′eq), 2.68 (br d, 1H, 4-OH), 2.42, 2.11 (2 s, 6H, 2 × CH₃CO), 2.07 (dd, 1H, J_3ax’,4′ = 11.4 Hz, H-3′ax) 1.99, 1.47 (2 s, 6H, 2 × CH₃CO) ppm. HR-ES MS [M + Na]⁺ calcd for C₄₅H₄₉NO₁₈SNa 946.2468, found 946.2472. Analytical data for 19 was in agreement with that reported previously.³⁵

DMAP (0.026 g, 0.022 mmol, 0.2 equiv) and Ac₂O (20.4 μL, 2.0 equiv) were added to a solution of compound 19 (0.100 g, 0.108 mmol, 1.0 equiv) in pyridine (5.0 mL), and the resulting mixture was stirred under argon for 16 h at rt. The reaction was quenched by the addition of MeOH (~1 mL), the volatiles were removed under reduced pressure, and the residue was coevaporated with toluene (3 × 5 mL). The residue was purified by column chromatography on silica gel (acetone-hexane gradient elution) to afford the title compound as an amorphous solid in 94% yield (0.098 g, 0.101 mmol). Analytical data for 3: R_f 0.35 (acetone/hexane, 1:2, v/v); [α]_D²³ + 4.2 (c 1.0, CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ 8.22–8.19 (m, 2H, aromatic), 7.50–7.22 (m, 13H, aromatic), 5.66–5.62 (m, 1H, H-8′), 5.45 (dd, 1H, J_7′,8′ = 9.2 Hz, H-7′), 5.34 (dd, 1H, J_2,3 = 9.8 Hz, H-2), 5.08–5.02 (m, 2H, H-1, 4), 4.76 (dd, 1H, J_3,4 = 3.0 Hz, H-3), 4.55–4.45 (m, 2H, CH₂Ph), 4.42 (dd, 1H, H-9b’), 4.21 (dd, 1H, J_6′,7′ = 9.4 Hz, H-6′), 3.99 (dd, 1H, J_5,6a = J_5,6b = 6.2 Hz, H-5), 3.94 (dd, 1H, J_9a′,9b′ = 12.3 Hz, H-9a’), 3.82–3.78 (m, 4H, H-4′, COOCH₃), 3.60–3.56 (m, 1H, H-6b), 3.51–3.42 (m, 2H, H-5′, 6a), 2.90 (dd, 1H, J_3′eq,4′ = 3.4 Hz, H-3_′eq), 2.39, 2.14, 2.08, 1.96 (4 s, 12H, 4 × CH₃CO), 1.87 (dd, 1H, J_3′ax,4′ = 13.0 Hz, J_{3′eq,3′ax} = 11.8 Hz, H-3_′ax), 1.23 (s, 3H, COCH₃) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 171.5, 170.7 (× 2), 170.2, 170.0, 167.6, 165.6, 153.5, 149.6, 138.0, 133.4, 132.9, 132.6 (× 2), 130.5, 130.2, 128.7 (× 2), 128.5 (× 2), 128.3 (× 2), 127.8, 127.7 (× 3), 97.4, 86.8, 77.2, 75.8, 75.7, 74.3, 73.4, 72.8, 71.4, 69.5, 68.5, 68.1, 63.5, 58.8, 53.4, 34.9, 24.5, 21.5, 20.8, 20.7, 19.9 ppm; HR-ES MS [M + Na]⁺ calcd for C₄₇H₅₁NO₁₉SNa 988.2674, found 988.2673.

Benzyl O-(2,6-Di-O-benzoyl-4-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (8).

A mixture of glycosyl donor 9 (0.313 g, 0.49 mmol, 1.33 equiv), glycosyl acceptor 10 (0.20 g, 0.37 mmol, 1 equiv), and freshly activated molecular sieves (3 Å, 1.0 g) in CH₂Cl₂ (3.0 mL) was stirred under argon for 2 h at rt. The mixture was cooled to 0 °C, NIS (0.221 g, 0.98 mmol, 2.6 equiv) and TfOH (8.7 μL, 0.98 mmol, 0.25 equiv) were added, and the resulting mixture was stirred for 30 min at 0 °C. The reaction was then quenched with triethylamine (~0.5 mL), the solids were filtered off through a pad of Celite and rinsed successively with CH₂Cl₂ (~50 mL). The combined filtrate was washed with 10% aq. Na₂S₂O₃ (10 mL), sat. aq. NaHCO₃ (2 × 10 mL), and brine (10 mL). The organic phase was separated, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate - hexane gradient elution) to afford benzyl O-(2,6-di-O-benzoyl-4-O-benzyl-3-O-tert-butyldimethylsilyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (25, 0.405 g, 0.363 mmol) as a colorless syrup in 98% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.01–7.95 (m, 4H, aromatic), 7.59–7.09 (m, 31H, aromatic), 5.59 (dd, 1H, J_2′3′ = 9.8 Hz, H-2′), 5.13 (d, 1H, ²J = 11.2 Hz, CHPh), 5.03 (d, 1H, ²J = 10.9 Hz, CHPh), 4.88–4.84 (m, 2H, CH₂Ph), 4.75–4.68 (m, 3H, H-1′, CH₂Ph), 4.61 (d, 1H, ²J = 12.3 Hz, CHPh), 4.57–4.54 (m, 2H, CH₂Ph), 4.39–4.32 (m, 3H, H-1, H-6a’, CHPh), 4.22 (dd, 1H, H-6b’), 3.96 (dd, 1H, J_4,5 = 9.3 Hz, H-4), 3.79 (dd, 1H, J_3′4′ = 9.8 Hz, H-3′), 3.73 (br d, 1H, H-4′), 3.64–3.55 (m, 4H, H-3, 5′, 6a, 6b), 3.43 (dd, 1H, J_2,3 = 7.8 Hz, H-2), 3.23–3.19 (m, 1H, H-5), 0.77 (s, 9H, SiC(CH₃)₃), 0.10, –0.09 (2 s, 6H, 2 × SiCH₃) ppm. HR-ES MS [M + Na]⁺ calcd for C₆₇H₇₄O₁₃SiNa 1137.4796, found 1137.4767. Analytical data of 25 was in agreement with that reported previously.¹⁹

HF-pyridine (0.81 mL, 0.81 mmol, 5.0 equiv) was added to a chilled (0 °C) solution of compound 25 (0.180 g, 0.162 mmol, 1.0 equiv) in pyridine (2.0 mL), and the resulting mixture was stirred for 12 h at rt. The reaction mixture was then diluted with EtOAc (60 mL), washed with 1N aq. HCl (30 mL), sat. aq. NaHCO₃ (2 × 20 mL), and brine (20 mL). The organic phase was separated, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate-toluene gradient elution) to afford the title compound as an amorphous solid in 92% yield (0.148 g, 0.148 mmol). Analytical data for 8 was in agreement with that reported previously.¹⁹

Benzyl O-(4,6-Di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)-(1→3)-O-(2,6-di-O-benzoyl-4-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (4).

A mixture of glycosyl donor 7 (0.132 g, 0.18 mmol, 2.0 equiv), glycosyl acceptor 8 (0.088 g, 0.09 mmol, 1.0 equiv), and freshly activated molecular sieves (4 Å, 0.500 g) in CH₂Cl₂ (3.0 mL) was stirred under argon for 2 h at rt. The mixture was cooled to −30 °C, TfOH (15.6 μL, 0.18 mmol, 2.0 equiv) was added, and the resulting mixture was stirred for 2 h at −30 °C. The reaction was then quenched with triethylamine (~0.5 mL), the solids were filtered off through a pad of Celite and rinsed successively with CH₂Cl₂. The combined filtrate (~50 mL) was washed with sat. aq. NaHCO₃ (2 × 10 mL) and brine (10 mL). The organic phase was separated, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate - hexane gradient elution) to afford benzyl O-(4,6-di-O-benzyl-2-deoxy-3-O-fluorenylmethoxycarbonyl-2-phthalimido-β-d-glucopyranosyl)-(1→3)-O-(2,6-di-O-benzoyl-4-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (26, 0.135 g, 0.079 mmol) as a colorless foam in 90% yield. ¹H NMR (400 MHz, CDCl₃): δ 7.97–7.94 (m, 2H, aromatic), 7.66–7.08 (m, 55H, aromatic), 5.66 (dd, 1H, J_3″4″ = 8.6 Hz, H-3″), 5.44 (d, 1H, J_1″2″ = 8.2 Hz, H-1″), 5.35 (dd, 1H, J_2′3′ = 7.9 Hz, H-2′), 5.12 (d, 1H, ²J = 11.5 Hz, CHPh), 4.92 (d, 1H, ²J = 10.6 Hz, CHPh), 4.82–4.79 (m, 2H, CH₂Ph), 4.68–4.35 (m, 11H, H-1′, 2″, 9 × CHPh), 4.29–4.15 (m, 4H, H-1, 6a′, 6b′, CHPh), 4.08 (br d, 1H, H-4′), 4.03 (dd, 1H, J_6a″,6b″ = 10.5 Hz, H-6a″), 3.92–3.70 (m, 8H, H-3′, 4, 4″, 5″, 6b″, OCOCH₂CH, OCOCH₂), 3.60–3.59 (m, 1H, H-5′), 3.43–3.28 (m, 4H, H-2, 3, 6a, 6b), 2.93 (m, 1H, H-5) ppm. Analytical data for 26 was in agreement with that reported previously.¹⁹

NEt₃ (0.18 mL, 1.28 mmol, 20 equiv) was added to a solution of compound 26 (0.109 g, 0.064 mmol, 1.0 equiv) in CH₂Cl₂ (5.0 mL), and the resulting mixture was stirred under argon for 2 h at rt. The reaction mixture was the concentrated under reduced pressure and the residue was purified by column chromatography on silica gel (acetone-hexane gradient elution) to afford the title compound as a white amorphous solid in 91% yield (0.868 g, 0.059 mmol). Analytical data for 4: R_f 0.35 (acetone/hexane, 1:2, v/v); [α]_D²³ −26.4 (c 1.0, CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ 7.96–7.94 (m, 2H, aromatic), 7.53–7.07 (m, 47H, aromatic), 5.34 (dd, 1H, J_2′,3′ = 10 Hz, H-2′), 5.20 (d, 1H, J_1″,2″ = 8.3 Hz, H-1″), 5.12 (d, 1H, ²J = 11 Hz, CHPh), 4.92 (d, 1H, ²J = 11 Hz, CHPh), 4.83–4.79 (m, 2H, CH₂Ph), 4.73–4.38 (m, 11H, H-1′, 3″, 9 × CHPh), 4.28–4.12 (m, 5H, H-1, 2″, 6a′, 6b′, CHPh), 4.06 (br. d, 1H, H-4′), 3.85 (dd, 1H, J_4,5 = 9.2 Hz, H-4), 3.76–3.66 (m, 4H, H-3′, 5″, 6a″, 6b″), 3.62–3.54 (m, 2H, H-4″, 5′), 3.43–3.29 (m, 4H, H-2, 3, 6a, 6b), 2.95 (m, 1H, H-5), 2.25 (br. s, 1H, OH) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 166.0, 164.3, 139.0, 138.8, 138.7, 138.3, 138.1, 137.9, 137.6, 134.1, 133.6, 133.0, 132.8, 131.7, 131.2, 130.0, 129.7, 129.6, 129.4, 128.9, 128.6 (× 4), 128.5 (× 7), 128.4 (× 2), 128.3 (× 4), 128.2, 128.1 (× 3), 128.0 (× 8), 127.9 (× 5), 127.8 (× 4), 127.7, 127.6 (× 2), 127.5 (× 2), 127.1, 102.5, 100.3, 99.7, 82.7, 81.7, 80.5, 79.4, 76.2, 76.1, 75.4, 75.1, 74.9, 74.9, 74.8, 74.4, 73.6, 73.4, 72.1, 72.0, 71.1, 71.0, 69.3, 67.7, 63.0, 56.8 ppm; HR-ES MS [M + Na]⁺ calcd for C₈₉H₈₅NO₁₉Na 1494.5613, found 1494.5612.

Benzyl O-(Methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-carbonyl-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonate)-(2→3)-O-(4-O-acetyl-2-O-benzoyl-6-O-benzyl-1-thio-β-d-galactopyranosyl)-(1→3)-O-(4,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)-(1→3)-O-(2,6-di-O-benzoyl-4-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (2).

A mixture of glycosyl donor 3 (0.098 g, 0.101 mmol, 1.5 equiv), glycosyl acceptor 4 (0.100 g, 0.067 mmol, 1.0 equiv), and freshly activated molecular sieves (4 Å, 0.300 g) in CH₂Cl₂ (3.0 mL) was stirred under argon for 2 h at rt. The mixture was cooled to −30 °C, NIS (0.045 g, 0.202 mmol, 3.0 equiv) and TfOH (5.5 μL, 0.062 mmol, 0.9 equiv) were added, and the resulting mixture was stirred for 2 h at −30 °C. The reaction was then quenched with triethylamine (~0.5 mL), the solids were filtered off through a pad of Celite and rinsed successively with CH₂Cl₂. The combined filtrate (~100 mL) was washed with 10% aq. Na₂S₂O₃ (10 mL), sat. aq. NaHCO₃ (2 × 10 mL), and brine (10 mL). The organic phase was separated, dried with Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (acetone – hexane gradient elution) to afford the title compound as a colorless foam in 84% yield (0.132 g, 0.057 mmol). Analytical data for 2: R_f 0.40 (acetone/hexane, 1:2, v/v); [α]_D²³ −14.7 (c 1.0, CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ 7.93–6.07 (m, 59H, aromatic), 5.52 (dd, 1H, J_7E,8E = 9.4 Hz, H-7E), 5.22–5.14 (m, 4H, H-2B, 2D, 3C, 8E), 5.03–4.88 (m, 5H, H-1C, 4D, 3 × CHPh), 4.81–4.78 (m, 2H, CH₂Ph), 4.73 (d, 1H, J_1D,2D = 7.7 Hz, H-1D), 4.66 (d, 1H, ²J = 11 Hz, CHPh), 4.58–4.37 (m, 8H, 4 × CH₂Ph), 4.32–4.25 (m, 4H, H-1A, 1B, 3D, CHPh), 4.22–4.12 (m, 4H, H-2C, 6Ba, 6Bb, CHPh), 4.07–4.03 (m, 2H, H-6E, 9Eb), 3.94 (br. d, 1H, H-4B), 3.82–3.58 (m, 10H, H-3B, 4A, 4E, 5C, 6Ca, 6Cb, 9Ea, COOCH₃), 3.53–3.42 (m, 4H, H-4C, 5B, 5D, 6 Da), 3.39–3.19 (m, 6H, H-2A, 3A, 5E, 6Aa, 6Ab, 6Db), 2.87–2.84 (m, 1H, H-5A), 2.70 (dd, 1H, J_3Eeq,4E = 3.5 Hz, H-3E_eq), 2.36, 2.06, 1.92, 1.78 (4 s, 12H, 4 × CH₃CO), 1.73 (dd, 1H, J_3Eax,3Eeq = 12.0 Hz, J_3Eax,4E = 13.0 Hz, H-3_axE), 1.25 (s, 3H, CH₃CO) ppm; ¹³C NMR (101 MHz, CDCl₃): δ 170.8 (× 2), 169.9, 169.5 (× 2), 167.2, 165.9, 165.8, 164.1, 153.4, 139.0, 138.7, 138.6, 138.4, 138.2, 138.0, 137.8, 137.5, 133.3, 132.9, 132.8, 130.2, 130.0, 129.9, 129.7 (× 8), 129.6 (× 2), 129.4, 128.6 (× 3), 128.5 (× 3), 128.4 (× 3), 128.3 (× 8), 128.2 (× 2), 128.1 (× 8), 127.9 (× 9), 127.7 (× 8), 127.6 (× 2), 127.4 (× 2), 127.0, 102.5, 100.3, 99.3, 98.6, 97.5, 82.6, 81.5, 79.3, 77.2, 76.0, 75.9 (× 2), 75.4, 74.9, 74.8 (× 2), 74.7, 74.3, 73.7, 73.4, 73.3 (× 2), 72.0 (× 2), 71.9, 71.8, 71.6, 70.9, 70.3, 69.4, 68.2, 67.7, 67.4, 67.3, 62.9, 61.4, 58.9, 56.4, 53.2, 35.4, 29.7, 24.4, 21.1, 20.9, 20.6, 20.0 ppm; HR-ES MS [M + Na]⁺ calcd for C₁₃₀H₁₃₀N₂O₃₈Na 2349.8199, found 2349.8196.

O-(5-N-Acetamido-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosyl)-(2→3)-O-(β-d-galactopyranosyl)-(1→3)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1→3)-O-(β-d-galactopyranosyl)-(1→4)-d-glucopyranose (1).

LiI (0.021 g, 0.161 mmol, 15 equiv, dried at 90 °C for 18 h in vacuo) was added to a solution of compound 2 (0.025 g, 0.0107 mmol, 1.0 equiv) in pyridine (5.0 mL), and the resulting mixture kept under argon for 4 h at reflux. The reaction mixture was allowed to cool to rt and passing through a filter column (Sephadex G-25, water elution) and the combined eluate was concentrated under reduced pressure. The residue was dissolved in MeOH (5.0 mL), 1 M soln. of NaOMe in MeOH (0.5 mL) was added, and the resulting mixture was stirred for 1 h at rt. The reaction was neutralized with Amberlite (H⁺), the resin was filtered off and rinsed successively with MeOH. The combined filtrate was concentrated under reduced pressure. The crude residue was dissolved in MeOH (5.0 mL), NH₂NH₂–H₂O (2.0 mL) was added, and the resulting mixture was kept for 24 h at reflux. The reaction mixture was allowed to cool to rt, and the volatiles were removed under reduced pressure. The crude residue was redissolved in a solution of Ac₂O/MeOH (6.0 mL, 1:2, v/v) and the resulting mixture was stirred at for 12 h at rt. After that, the volatiles were removed under reduced pressure. The residue was dissolved in MeOH (5.0 mL), a 1 M soln. of NaOMe in MeOH (2.0 mL) was added, and the resulting mixture was stirred for 6 h at rt. The reaction was neutralized with Amberlite (H⁺), the resin was filtered off and rinsed successively with MeOH. The combined filtrate was concentrated under reduced pressure. The residue was dissolved in 20% aq. EtOH (5.0 mL), Pd/C (15 mg) was added, and the resulting mixture was stirred under H₂ atmosphere for 24 h at rt. The solids were filtered off through a pad of Celite and rinsed successively with water. The combined filtrate was concentrated under reduced pressure. The residue was dissolved in water (2.0 mL), 1 M aq. NaOH (50 μL) was added, and the resulting mixture was stirred for 15 min at rt. The reaction mixture was then concentrated under reduced pressure, and the residue was purified by size exclusion chromatography of Sephadex G-25 to afford 1 as a white amorphous solid in 30% yield (3.2 mg, 0.0032 mmol). Analytical data for 1 was in agreement with that reported previously.²¹

Supplementary Material

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.joc.5c02428.

NMR spectra for all compounds (PDF)

Data Availability Statement

The data underlying this study are available in the published article and its Supporting Information.