Chemical Synthesis of Human Milk Oligosaccharides: Lacto‑N‑hexaose Galβ1→3GlcNAcβ1→3 [Galβ1→4GlcNAcβ1→6] Galβ1→4Glc

Abstract

The first synthesis of lacto-N-hexaose (LNH) has been completed using a convergent strategy. The donor–acceptor protecting–leaving group combinations were found to be of paramount significance for achieving successful glycosylations and minimizing side reactions. Lacto-N-tetraose, another common human milk oligosaccharide, was also obtained en route to the target LNH.

Graphical Abstract

INTRODUCTION

Thanks to advances in glycosciences in recent years, the importance of human milk oligosaccharides (HMO) as the essential source of antimicrobials^1,2 and prebiotics³ has come to the fore. Biosynthesis of HMO follows a unique structural blue print. All glycan chains contain lactose (Galβ1→4Glc) at their reducing end, which can be elongated by the addition of β1-3- or β1-6-linked lacto-N-biose (Galβ1→3GlcNAc, type 1 chain) or lactosamine (Galβ1→4GlcNAc, type 2 chain). Elongation of lactose via a β1-6 linkage introduces chain branching, and these structures are designated as iso-HMO, whereas linear structures are commonly designated as para-HMO. Lactose or the elongated oligosaccharide chain can also be fucosylated or sialylated.⁴ Structures of many HMO are known,^5,6 many HMO sequences have already been prepared enzymatically and/or chemically,^7–10 but the exact roles of a majority of individual HMO remain unknown.^11,12

With expectation that the development of reliable synthetic methods for obtaining individual HMO will boost our understanding the roles of these important biomolecules, we previously reported the total syntheses of lacto-N-neo-tetraose¹³ and lacto-N-tetraose (LNT).¹⁴ Our synthetic endeavors made us interested in studying more challenging, branched structures, such as lacto-N-hexaose (LNH),^15,16 whose exact biological roles are yet unknown. LNH 1 represents one of the most common core structures and is structurally derived from LNT 2 (Figure 1).

Figure 1.

Chemical structures of LNH and LNT.

LNH comprises the reducing end lactose disaccharide (AB, Figure 1), common for all HMO, which is branched at the galactose residue (B). More specifically, it is elongated with the lacto-N-biose disaccharide residue (CD) at C-3 of Gal and also with the N-acetyllactosamine residue (EF) at C-6 of Gal. While many chemical^17–19 and enzymatic syntheses²⁰ of LNT are known, to the best of our knowledge, neither chemical nor enzymatic synthesis of LNH have yet been reported. We came across an article by Knerr and Schmidt describing the synthesis of a “branched hexasaccharide related to LNH.”²¹ However, the actual molecule obtained therein is lacto-N-neohexaose, a different HMO core sequence. Human milk isolates of LNH are available on the market, but this common HMO is not yet available in large quantities and at an accessible cost for mainstream research and application. Reported herein is an efficient and versatile method for the scalable synthesis of LNH 1 and, by extension, LNT 2.

RESULTS AND DISCUSSION

Recently, we have disclosed the synthesis of LNT 2 via convergent and linear approaches.¹⁴ The protecting groups in the tetrasaccharide intermediate 3¹⁴ were strategically placed to allow for the synthesis of other derivatives (Scheme 1). Thus, we assumed that selective removal of 6′-O-picoloyl (Pico) substituent in 3 would provide a straightforward access to acceptor 4 suitable for subsequent branching. To pursue this route, the deprotection of 6′-O-Pico in 3 was conducted in the presence of Cu(OAc)₂−H₂O in MeOH/CH₂Cl₂ to afford tetrasaccharide 4 in 96% yield. To our disappointment, all attempts to glycosylate acceptor 4 with the lactosamine thioglycoside donor 5¹³ were largely unsuccessful and practically no desired hexasaccharide was formed. Instead, a large number of unidentified side products derived from the glycosyl donor have been isolated, and the acceptor remained practically intact.

Scheme 1.

Initial Attempt to Assemble LNH

Recalling our previous endeavors with the synthesis of HMO, wherein the nature of the glycosyl donor and the acceptor was found to be of paramount significance, we attempted to employ the corresponding lactosamine phosphate and trichloroacetimidate as glycosyl donors. Despite the extended study, these attempts to access the backbone of LNH were also unsuccessful. In further attempts to enhance the efficiency of this coupling, we applied a less bulky glucosamine thioglycoside donor 6¹³ and its phosphate and trichloroacetimidate analogues. Although the formation of minor amounts of the desired pentasaccharide was observed by mass spectroscopy, these glycosylations were deemed unsuccessful. Having explored all the possibilities available to us, we came to a conclusion that the bulky benzyl group at the 4′-OH position of acceptor 4 is hindering the glycosylation at the 6′-OH position.

In order to decrease the steric hindrance, we changed our strategy and employed building block 7 protected with 4,6-O-benzylidene acetal instead (Scheme 2).²² We envisaged that if the benzylidene acetal is removed at the tetrasaccharide stage, the resulting 4,6-diol will offer a far more accessible glycosyl acceptor site that may be more suitable for glycosylation with bulky disaccharide donor 5. With this strategic adjustment, we coupled donor 7 with 4-OH acceptor 8²³ in the presence of N-iodosuccinimide (NIS) and TfOH. This and all subsequent glycosylations described in this manuscript proceeded with complete 1,2-trans stereoselectivity because of the participating effect of the neighboring substituent. The anomeric configuration and purity of all oligosaccharide products were confirmed by NMR spectroscopy. The resulting disaccharide was subjected to deprotection of the Fmoc group with triethylamine in one pot affording the desired disaccharide acceptor 9 in 87% yield over two steps (Scheme 2). We then attempted to glycosylate acceptor 9 with thioglycoside donor 10. This reaction was sluggish and inefficient, and despite all attempts to push the reaction to completion, significant amounts of acceptor remained. After a quick screening of other leaving groups, we discovered that trichloroacetimidate donor 12 is much more effective in glycosylating acceptor 9. Glycosyl donor 12 was prepared from thioglycoside precursor 10 that was first converted into corresponding hemiacetal 11 via anomeric bromination using Br₂ in CH₂Cl₂ followed by the hydrolysis of the bromide using Ag₂CO₃ in wet acetone in 81% over two steps. Next, the installation of the trichloroacetimidoyl group was performed using trichloroacetonitrile (CCl₃CN) in the presence of 1,8-diazabicylco[5.4.0]undec-7-ene (DBU) to afford the lacto-N-biose trichloroacetimidate 12 in 93% yield. Glycosylation of acceptor 9 with donor 12 was conducted in the presence of catalytic TMSOTf, and the desired tetrasaccharide 13 was obtained in a good yield of 83% with complete β-stereoselectivity. It is noteworthy that the corresponding glycosyl phosphate donor that was found to be advantageous in the synthesis of LNT¹⁴ gave a comparable result in this application.

Scheme 2.

Convergent Synthesis of LNH and LNT

Subsequent deprotection of the 4′,6′-O-benzylidene acetal in intermediate 13 was somewhat low yielding in the presence of trifluoroacetic acid in wet DCM. These are traditional conditions for benzylidene cleavage that generally work well, so the outcome of this reaction was somewhat puzzling. Having had no explanation for this unexpected result, we found inspiration in work by Williams and Sit, wherein isopropylidene was removed by ketal exchange with ethanedithiol in the presence of p-toluenesulfonic acid (TsOH).²⁴ When we conducted benzylidene removal reaction with EtSH as an acetal exchange reagent in the presence of TsOH in MeOH/CH₂Cl₂, tetrasaccharide diol 14 was smoothly obtained in an excellent yield of 96% (Scheme 2).

Next, we attempted glycosylation of tetrasaccharide diol 14 with lactosamine thioglycoside donor 5. Even this coupling with a more accessible glycosyl acceptor was not as straightforward as we had initially hoped for. Standard reaction condition employing the NIS/TfOH promoter system produced the desired hexasaccharide 15 in a modest yield of 51%. Although this result was significantly better than our previous attempt to glycosylate acceptor 4, we wanted to make an effort to further improve the outcome of this challenging coupling. With this intention, we identified major side reactions hampering the yield of the product: fairly rapid hydrolysis of lactosamine donor 5 and the formation of the 1-4-linked hexasaccharide 18 alongside the anticipated 1-6-linked counterpart. To suppress these side reactions, we employed milder promoters, dimethyl(thiomethyl)sulfonium triflate^25,26 and NIS/AgOTf.²⁷ The former promoter provided a comparable result that was achieved with NIS/TfOH. With the latter promoter, known for providing a slower release of the iodonium ion, minimal side products resulting from hydrolysis of donor 5 were observed. After careful refinement of the reaction conditions, we achieved the desired hexasaccharide 15 in a commendable yield of 80% (Scheme 2). This result was obtained by conducting the reaction at −40 °C for 30 min and then allowing the reaction temperature gradually increase (to about −18 °C) over the course of the additional 30 min period.

With the protected intermediate 15 in hand, we then endeavored a series of deprotection steps to obtain the target LNH hexasaccharide. Deprotection of the phthalimido and ester groups was achieved by refluxing compound 15 in MeOH in the presence of NH₂NH₂−H₂O to afford intermediate 16 in 74% yield. Column purification was found necessary before the subsequent step: N-acetylation of 16 with acetic anhydride in MeOH. Finally, the benzyl groups were removed by hydrogenation in the presence of 10% palladium on charcoal in wet ethanol to obtain target LNH 1 in 80% over two steps (Scheme 2).

We also performed deprotection of the key tetrasaccharide intermediate 14 to obtain LNT 2. This was achieved via deprotection of the phthalimido and ester groups in the presence of NH₂NH₂−H₂O in refluxing MeOH followed by N-acetylation with acetic anhydride in MeOH to furnish the partially protected tetrasaccharide intermediate 17 in 84% yield. Subsequently, benzyl ethers were removed by hydrogenation in the presence of 10% Pd/C in wet ethanol to afford target LNT 2 in 87% yield (Scheme 2).

In summary, the first total synthesis of LNH has been completed using a convergent 2 + 2 + 2 strategy. This approach employed preassembled lactose, lactosamine, and lacto-N-biose building blocks. Along the way, we have also obtained the LNT core sequence. It has been acknowledged that including HMO to infant formulas could be beneficial for infants’ health,^28–31 but HMO are challenging to produce and purify, and exact roles of individual HMO remain unknown.^11,12,32 Only two simple HMO have been approved for infant formulas, and a few additional HMO are currently in clinical trials.³³ With expectation that new methods for reliable synthesis of individual HMO will boost practical applications of these important biomolecules, further synthetic studies of HMO are underway in our laboratory.

EXPERIMENTAL SECTION

General.

Reactions were performed using commercial reagents, and the ACS grade solvents were purified and dried according to the standard procedures. Column chromatography was performed on silica gel 60 (70–230 mesh) and Sephadex G-25 size exclusion resin; reactions were monitored by TLC on Kieselgel 60 F₂₅₄. The compounds were detected by examination under UV light and by charring them 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 Å), used for reactions, were crushed and activated in vacuo at 390 °C during 8 h in the first instance and then for 2–3 h at 390 °C directly prior to application. AgOTf was coevaporated with toluene (3 × 10 mL) and dried in vacuo for 2–3 h directly prior to application. Optical rotations were measured using a Jasco polarimeter. ¹H NMR spectra were recorded at 300 or 600 MHz, and ¹³C{1H} NMR spectra were recorded at 75 or 151 MHz. The ¹H chemical shifts are referenced to the signal of the residual TMS (δ_H = 0.00 ppm) for solutions in CDCl₃ or the signal of the residual D₂O (δ_H = 4.79 ppm) for solutions in D₂O. The ¹³C chemical shifts are referenced to the central signal of CDCl₃ (δ_C = 77.16 ppm) for solutions in CDCl₃ or the central signal of added CD₃COCD₃ (δ_C = 29.84 ppm) for solutions in D₂O. Accurate mass spectrometry determinations were performed using an Agilent 6230 ESI TOF LCMS mass spectrometer.

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

Cu(OAc)₂−H₂O (15.3 mg, 0.084 mmol) was added to a solution of benzyl O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-(1→3)-O-(4,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)-(1→3)-O-(2-O-benzoyl-4-O-benzyl-6-O-picoloyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside¹⁴ (3, 113.0 mg, 0.056 mmol) in MeOH/CH₂Cl₂ (8.0 mL, 1/3, v/v), and the resulting mixture was stirred for 20 min at rt. Next, the reaction mixture was diluted with CH₂Cl₂ (~100 mL), and washed with H₂O (15 mL), sat. aq. NaHCO₃ (15 mL), and water (2 × 15 mL). The organic phase was separated, dried over MgSO₄, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (acetone–toluene gradient elution) to afford the title compound as an off-white amorphous solid in 96% yield (102.3 mg, 0.053 mmol). Analytical data for 4: R_f = 0.60 (ethyl acetate/hexane, 1/1, v/v); ${\left[\alpha \right]}_{\text{D}}^{21}+5.8$ (c 1.0, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 2.84 (ddd, 1H, J = 8.9 Hz, H-5), 3.10–3,45 (m, 11H, H-2, 3, 3‴, 5″, 5‴, 6a, 6a″, 6a‴, 6b, 6b″, 6b‴), 3.54–3.60 (m, 2H, H-3′, 4″), 3.69–3.77 (m, 3H, 4, 5″, 6a″), 3.80 (br d, 1H, J_3′,4′ = 2.2 Hz, H-4′), 3.87–3.89 (m, 2H, H-4‴, 6b′), 4.13–4.18 (m, 2H, H-1′, 2″), 4.20–4.30 (m, 6H, H-1, 1‴, 2 × CH₂Ph), 4.42–4.59 (m, 10H, 5 × CH₂Ph), 4.67 (d, 1H, ²J = 11.1 Hz, CHPh), 4.78–4.91 (m, 6H, H-3″, 5 × CHPh), 4.98 (d, 1H, J_1″,2″ = 8.4 Hz, H-1″), 5.08 (d, 1H, ²J = 10.5 Hz, CHPh), 5.12 (dd, 1H, J_1′,2′ = 8.1, J_2′,3′ = 10.0 Hz, H-2′), 5.46 (dd, 1H, J_1‴,2‴ = 8.7, J_2‴,3‴ = 9.4 Hz, H-2‴), 6.73–7.66 (m, 64H, aromatic) ppm; ¹³C{1H} NMR (151 MHz, CDCl₃): δ 56.0, 62.0, 67.3, 67.8, 69.7, 71.0, 71.7, 72.0, 72.5, 72.7, 73.2, 73.5 (×2), 73.6 (×2), 74.3, 74.6, 74.8, 74.9, 75.0, 75.2, 75.4, 75.8, 75.9, 76.0, 77.6, 79.8, 80.2, 81.2, 82.7, 99.3, 100.5 (×2), 102.6, 114.0, 122.7 (×2), 127.5 (×3), 127.6 (×4), 127.7 (×2), 127.8 (×5), 127.9 (×4), 128.0 (×2), 128.1 (×6), 128.2 (×9), 128.3 (×9), 128.4 (×2), 128.5 (×4), 128.6 (×5), 129.0 (×2), 129.6, 129.8, 130.0, 130.1, 130.8, 131.3, 132.7, 133.0, 133.4, 133.6, 137.5, 137.6, 138.0, 138.2, 138.5, 138.6, 138.7, 138.8, 164.3, 165.5, 166.3, 168.4 ppm; ESI TOF LCMS: [M + Na]⁺ calcd for C₁₁₆H₁₁₃NNaO₂₄, 1927.7584; found, 1927.7613.

Benzyl O-(2-O-Benzoyl-4,6-O-benzylidene-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (9).

A mixture of ethyl 2-O-benzoyl-4,6-O-benzylidene-3-O-fluorenylmethyloxycarbonyl-1-thio-β-d-galactopyranoside²² (7, 0.30 g, 0.45 mmol), benzyl 2,3,6-tri-O-benzyl-β-d-glucopyranoside³⁴ (8, 0.19 g, 0.36 mmol), and freshly activated molecular sieves (3 Å, 1.0 g) in CH₂Cl₂ (10 mL) was stirred under argon for 2 h at rt. The reaction mixture was cooled to 0 °C, NIS (0.21 g, 0.94 mmol) and TfOH (18 μL, 0.09 mmol) were added, and the resulting mixture was stirred for 30 min. After that, the reaction mixture was warmed to rt, Et₃N (~4.5 mL) was added, and the resulting mixture was stirred for 1 h at rt to achieve complete deprotection of the Fmoc group. Next, the solids were filtered off and rinsed successively with CH₂Cl₂. The combined filtrate (~100 mL) was washed with sat. aq. NaHCO₃ (15 mL), 10% aq. Na₂S₂O₃ (15 mL), and water (2 × 15 mL). The organic phase was separated, dried over MgSO₄, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (acetone–toluene gradient elution) to afford the title compound as a white foam in 87% yield (0.28 g, 0.31 mmol). Analytical data for 9: R_f = 0.35 (acetone/toluene, 1/4, v/v); ${\left[\alpha \right]}_{\text{D}}^{23}-15.3$ (c 1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 2.51 (d, 1H, J = 11.4 Hz, OH), 3.11 (br s, 1H, H-5′), 3.28 (ddd, 1H, H-5), 3.49 (dd, 1H, J_2,3 = 9.1 Hz, H-2), 3.59–3.67 (m, 3H, H-3, 3′, 6a), 3.71 (dd, 1H, J_5,6b = 4.2, J_6a,6b = 11.0, Hz, H-6b), 3.87 (dd, 1H, J_5′,6a′ = 1.5, J_6a′,6b′ = 12.5 Hz, H-6a′), 3.96 (dd, 1H, J_3,4 = J_4,5 = 9.0 Hz, H-4), 4.11 (br d, 1H, J_3′,4′ = 3.6 Hz, H-4′), 4.21 (d, 1H, J_6a′,6b′ = 12.5 Hz, H-6b′), 4.45 (dd, 2H, ²J = 12.2 Hz, CH₂Ph), 4.43 (d, 1H, J_1,2 = 7.8 Hz, H-1), 4.45 (dd, 2H, ²J = 12.2 Hz, CH₂Ph), 4.74 (dd, 2H, ²J = 12.0 Hz, CH₂Ph), 4.76 (d, J_1′,2′ = 8.1 Hz, H-1′), 4.81 (dd, 2H, ²J = 10.9 Hz, CH₂Ph), 4.97 (dd, 2H, ²J = 10.8 Hz, CH₂Ph), 5.32 (dd, 1H, J_2′,3′ = 9.9 Hz, H-2′), 5.51 (s, 1H, CHPh), 7.16–8.05 (m, 30H, aromatic) ppm; ¹³C{1H} NMR (75 MHz, CDCl₃): δ 66.7, 68.4, 68.8, 71.2, 72.0, 72.2, 72.4, 73.4, 73.6, 74.6, 75.0, 75.6, 75.8, 77.8, 100.8, 101.6, 102.5, 126.6 (×2), 127.4, 127.6, 127.8 (×2), 127.9 (×2), 128.0 (×2), 128.1 (×2), 128.2 (×2), 128.3 (×4), 128.4 (×2), 128.5 (×4), 128.6 (×2), 129.3, 129.8, 129.9 (×2), 133.4, 137.5, 137.6, 138.4, 138.6, 139.1, 166.0 ppm; ESI TOF LCMS: [M + Na]⁺ calcd for C₅₄H₅₄NaO₁₂, 917.3513; found, 917.3521.

O-(2-O-Benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-(1→3)-4,6-di-O-benzyl-2-deoxy-2-phthalimido-α/β-d-glucopyranose (11).

A freshly prepared solution of Br₂ in DCM (6.5 mL, 1/165, v/v) was added to a prechilled solution of ethyl O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-(1→3)-4,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-d-glucopyranoside¹⁴ (10, 0.73 g, 0.68 mmol) in CH₂Cl₂ (9.0 mL), and the resulting mixture was stirred for 15 min at 0 °C. After that, the volatiles were evaporated under reduced pressure. The residue was coevaporated with CH₂Cl₂ (3 × 10 mL) and dried in vacuo for 2 h. The crude residue was dissolved in acetone (20 mL), water (1.0 mL) and Ag₂CO₃ (0.09 g, 0.34 mmol) were added, and the resulting mixture was stirred in the absence of light for 16 h at rt. After that, the solids were filtered off and rinsed successively with CH₂Cl₂. The combined filtrate was concentrated in vacuo. The residue was purified by column chromatography on silica gel (acetone–toluene gradient elution) to afford the title compound as a white foam in 81% yield (0.56 g, 0.55 mmol). Analytical data for β-11: R_f = 0.35 (acetone/toluene, 1/4 v/v); ¹H NMR (600 MHz, CDCl₃): δ 2.86 (d, 1H, J = 8.8 Hz, OH), 3.30 (m, 1H, H-5′), 3.38–3.45 (m, 3H, H-3′, 6a′, 6b′), 3.60–3.65 (m, 2H, H-4, 5), 3.72–3.74 (m, 2H, H-6a, 6b), 3.94 (br d, 1H, H-4′), 4.06 (dd, 1H, J = 8.7, 10.7 Hz, H-2), 4.19 (d, 1H, ²J = 11.6 Hz, CHPh), 4.26–4.33 (m, 2H, CH₂Ph), 4.36–4.61 (m, 7H, H-1′, 3 × CH₂Ph), 4.85 (dd, 1H, H-3), 4.90 (d, 1H, ²J = 11.4 Hz, CHPh), 5.07 (m, 1H, H-1), 5.47–5.52 (dd, 1H, H-2′), 6.94–7.70 (m, 34H, aromatic) ppm; ¹³C{1H} NMR (151 MHz, CDCl₃): δ 57.8, 67.8, 68.9, 71.7, 72.6, 73.3, 73.6, 73.7, 74.8 (×2), 75.0, 75.2, 76.4, 77.2, 80.2, 93.0, 100.7, 127.3, 127.5 (×2), 127.6 (×2), 127.8, 127.9, 128.0 (×3), 128.1 (×11), 128.3 (×5), 128.5 (×3), 128.6 (×3), 130.0, 130.2, 131.5, 132.8, 134.1, 137.6, 138.0, 138.1, 138.6, 138.8, 165.4 ppm; ESI TOF LCMS: [M + Na]⁺ calcd for C₆₂H₅₉NNaO₁₃, 1048.3884; found, 1048.3893.

O-(2-O-Benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-(1→3)-4,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl Trichloroacetimidate (12).

CCl₃CN (1.01 mL, 10.14 mmol) and DBU (7.2 μL, 0.05 mmol) were added to a solution of compound 11 (0.52 g, 0.51 mmol) in CH₂Cl₂ (15 mL), and the resulting mixture was stirred under argon for 1 h at rt. After that, the reaction mixture was concentrated in vacuo. The residue was purified by column chromatography on silica gel (acetone–toluene gradient elution) to afford the title compound as a white foam in 93% yield (0.47 g, 0.40 mmol). Analytical data for 12: R_f = 0.60 (acetone/toluene, 1/4 v/v); ${\left[\alpha \right]}_{\text{D}}^{24}+52.1$ (c 1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 3.27–3.50 (m, 4H, H-3′, 5′, 6a′, 6b′), 3.79 (m, 4H, H-4, 5, 6a, 6b), 3.94 (dd, 1H, J = 2.6 Hz, H-4′), 4.25 (dd, 2H, ²J = 11.5 Hz, CH₂Ph), 4.30 (d, 1H, ²J = 12.0 Hz, CHPh), 4.43–4.53 (m, 5H, H-1′, 2, 3 × CHPh), 4.58 (dd, 2H, ²J = 12.2 Hz, CH₂Ph), 4.88–4.97 (m, 2H, H-3, CHPh), 5.10 (d, 1H, ²J = 10.7 Hz, CHPh), 5.53 (dd, 1H, J_1′,2′ = 7.9, J_2′,3′ = 10.0 Hz, H-2′), 6.22 (d, 1H, J_1,2 = 8.9 Hz, H-1), 6.83–7.87 (m, 34H, aromatic), 8.48 (s, 1H, NH) ppm; ¹³C{1H} NMR (75 MHz, CDCl₃): δ 54.7, 67.8, 68.4, 71.7, 72.5, 72.8, 73.3, 73.4, 73.5, 74.8, 74.9, 76.1, 76.6, 76.8, 80.1, 90.4, 94.0, 100.8, 123.5, 127.3, 127.5 (×3), 127.6 (×2), 127.7, 127.9, 128.0 (×4), 128.1 (×6), 128.2 (×3), 128.3 (×3), 128.4 (×3), 128.5 (×3), 129.1, 130.0, 130.2, 131.3, 132.8, 134.1, 137.5, 138.0, 138.1, 138.6 (×2), 160.8, 163.4, 165.3, 167.6 ppm; ESI TOF LCMS: [M + Na]⁺ calcd for C₆₄H₅₈Cl₃N₂NaO₁₃, 1191.2980/1193.2951; found, 1193.2968.

Benzyl O-(2-O-Benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-(1→3)-O-(4,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)-(1→3)-O-(2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (13).

A mixture of donor 12 (0.23 g, 0.19 mmol), acceptor 9 (0.13 g, 0.15 mmol), and freshly activated molecular sieves (3 Å, 600 mg) in CH₂Cl₂ (7.0 mL) was stirred under argon for 2 h at rt. The reaction mixture was cooled to −30 °C, TMSOTf (8.20 μL, 0.04 mmol) was added, and the resulting mixture was stirred for 10 min, while the reaction temperature was allowed to increase gradually. After that, the solids were filtered off and rinsed successively with CH₂Cl₂. The combined filtrate (~50 mL) was washed with sat. aq. NaHCO₃ (10 mL) and water (2 × 10 mL). The organic phase was separated, dried over MgSO₄, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (acetone–toluene gradient elution) to afford the title compound as a white foam in 83% yield (0.27 g, 0.12 mmol). Analytical data for 13: R_f = 0.55 (acetone/toluene, 1/4 v/v); ${\left[\alpha \right]}_{\text{D}}^{22}-4.0$ (c 1.0, CHCl₃): ¹H NMR (600 MHz, CDCl₃): δ 2.89 (m, 1H, J = 8.8 Hz, H-5), 3.04 (br s, 1H, H-5′), 3.21–3.58 (m, 11H, H-2, 3, 3′, 3‴, 5‴, 6a, 6a″, 6a‴, 6b, 6b″, 6b‴), 3.70–3.84 (m, 3H, H-4, 4″, 5″), 3.89–3.90 (m, 2H, H-4′, 4‴), 4.11–4.32 (m, 8H, H-1‴, 2″, 3″, 6a′, 6b′, 3 × CHPh), 4.34 (d, 1H, ²J = 11.6 Hz, CHPh), 4.40 (d, 1H, ²J = 8.1 Hz, CHPh), 4.43–4.49 (m, 2H, H-1′, CHPh), 4.51 (d, 1H, ²J = 12.0 Hz, CHPh), 4.55–4.70 (m, 5H, 5 × CHPh), 4.77–4.88 (m, 4H, H-1, 3 × CHPh), 4.91 (d, 1H, ²J = 11.4 Hz, CHPh), 5.02 (d, 1H, ²J = 10.7 Hz, CHPh), 5.06–5.09 (m, 2H, H-1″, CHPh), 5.17 (dd, 1H, J_1′,2′ = 8.2, J_2′,3′ = 10.0 Hz, H-2′), 5.44–5.51 (m, 2H, H-2‴, CHPh), 6.93–7.72 (64H, m, aromatic) ppm; ¹³C{1H} NMR (151 MHz, CDCl₃): δ 55.5, 66.7, 67.7 (×2), 68.5, 70.0, 71.0, 71.1, 71.7, 72.5, 73.2, 73.4, 73.5 (×2), 73.6, 74.3, 74.8 (×2), 74.9, 75.2, 75.7, 75.8, 75.9, 76.7, 77.6, 77.8, 80.0, 81.7, 83.0, 99.2, 100.3, 100.6, 100.7, 102.4, 125.4, 126.5 (×2), 127.2, 127.4 (×2), 127.5 (×3), 127.7, 127.8 (×6), 127.9 (×2), 128.0 (×8), 128.1 (×4), 128.2 (×10), 128.3 (×3), 128.4 (×4), 128.5 (×8), 128.6 (×2), 129.1 (×2), 129.5, 129.6 (×2), 130.0 (×2), 130.1, 132.7, 132.8, 137.5 (×2), 137.9, 138.0 (×2), 138.2, 138.3, 138.4, 138.6 (×2), 139.0, 163.3, 164.1, 165.4 ppm; ESI TOF LCMS: [M + 2Na]²⁺ calcd for C₁₁₆H₁₁₁NNa₂O₂₄, 974.3663; found, 974.3645.

Benzyl O-(2-O-Benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-(1→3)-O-(4,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)-(1→3)-O-(2-O-benzoyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (14).

TsOH (15.49 mg, 0.09 mmol) and EtSH (57 μL, 0.81 mmol) were added to a solution of compound 13 (0.25 g, 0.13 mmol) in MeOH/CH₂Cl₂ (10 mL, 1/1, v/v), and the reaction mixture was stirred for 3 h at rt. The reaction was then quenched with triethylamine (~0.5 mL), and the volatiles were removed under reduced pressure. The residue was purified by column chromatography on silica gel (acetone - toluene gradient elution) to afford the title compound as a white amorphous solid in 96% yield (0.23 g, 0.12 mmol). Analytical data for 14: R_f = 0.55 (acetone/toluene, 1/4, v/v); ${\left[\alpha \right]}_{\text{D}}^{22}+23.4$ (c 1.0, CHCl₃); ¹H NMR (600 MHz, CDCll₃): δ 2.66 (br s,1H, H-5′), 2.87 (m, 1H, H-5), 3.19–3.38 (m, 9H, H-2, 3, 3‴, 5‴, 6a, 6a′, 6a″, 6b, 6b′), 3.42–3.46 (m, 3H, H-3′, 6a‴, 6b‴), 3.50 (dd, 1H, J_2″,3″ = 9.1 Hz, H-2″), 3.61–3.71 (m, 3H, H-3″, 5″, 6b″), 3.76–3.78 (m, 2H, H-4, 4″), 3.87 (br d, 1H, J = 1.5 Hz, H-4‴), 3.96 (br s, 1H, H-4′), 4.12–4.31 (m, 9H, H-1, 1′, 1‴, 3 × CH₂Ph), 4.42–4.68 (m, 6H, 3 × CH₂Ph), 4.77–4.89 (m, 5H, H-1″, 2 × CH₂Ph), 5.00–5.05 (m, 2H, CH₂Ph), 5.08 (dd, 1H, J_2′,3′ = 8.9 Hz, H-2′), 5.45 (1H, dd, J_2‴,3‴ = 8.9 Hz, H-2‴), 6.89–7.74 (m, 59 H, aromatic) ppm; ¹³C{1H} NMR (151 MHz, CDCl₃): δ 55.5, 62.2, 67.4, 67.8, 69.0, 69.4, 71.0, 71.3, 71.8, 72.6, 73.3, 73.5, 73.6 (×2), 73.7 (×2), 74.3, 74.8, 74.9, 75.0, 75.2, 75.6, 76.0, 76.3, 77.3, 80.1, 80.2, 81.4, 82.6, 98.8, 100.3 (×2), 102.6, 127.5 (×3), 127.6 (×4), 127.7, 127.8 (×3), 127.9 (×4), 128.0, 128.1 (×7), 128.2 (×10), 128.3 (×8), 128.4 (×8), 128.5 (×3), 128.6 (×5), 129.4, 129.6 (×2), 130.0 (×2), 130.2, 132.8, 137.5, 137.6, 138.0 (×2), 138.2, 138.4, 138.6, 138.7, 164.2, 165.4 ppm; ESI TOF LCMS: [M + 2Na]²⁺ calcd for C₁₀₉H₁₀₇NNa₂O₂₄, 930.3506; found, 930.3488.

Benzyl O-(2-O-Benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-(1→3)-O-(4,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)-(1→3)-[O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-(1→4)-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)-(1→6)]-O-(2-O-benzoyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (15).

A mixture of donor 5¹³ (15.3 mg, 0.0143 mmol), acceptor 14 (20.0 mg, 0.011 mmol), and freshly activated molecular sieves (3 Å, 100 mg) in CH₂Cl₂ (2.0 mL) was stirred under argon for 2 h at rt. The reaction mixture was cooled to −40 °C, NIS (4.95 mg, 0.022 mmol) and freshly conditioned AgOTf (1.4 mg, 0.005 mmol) were added, and the resulting mixture was stirred for 30 min at −40 °C. The external cooling was removed, and the reaction mixture was stirred for additional 30 min during which the reaction temperature was allowed to increase gradually (to −18 °C). After that, the solids were filtered off and rinsed successively with CH₂Cl₂. The combined filtrate (~30 mL) was washed with sat. aq. NaHCO₃ (7 mL), Na₂S₂O₃ (7 mL), and water (2 × 7 mL). The organic phase was separated, dried over MgSO₄, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (acetone–toluene gradient elution) to afford the title compound as a white foam in 80% yield (24.8 g, 0.0088 mmol). Analytical data for 15: R_f = 0.65 (acetone/toluene, 1/4 v/v); ${\left[\alpha \right]}_{\text{D}}^{22}+22.9$ (c 1.0, CHCl₃); ¹H NMR (600 MHz, CDCl₃, monosaccharide residues A–F are designated in the order shown in Figure 1; the assignment of signals for terminal galactose residues D and F can be interchanged): δ 2.67 (br d, 1H, J = 1.5 Hz, H−5^B), 2.70–2.76 (ddd, 1H, 5^C), 2.85 (ddd, 1H, J = 9.8, J = 2.8 Hz, H-5^A), 3.02 (ddd, 1H, J = 9.6 Hz, H-5^E), 3.14–3.18 (m, 2H, H-6a^A, 6b^A), 3.23–3.77 (m, 20H, H-2^A, 3^A, 3^B, 3^D, 3^F, 4^A, 4^B, 4^C, 5^D, 5^F, 6a^B, 6a^C, 6a^D, 6a^E, 6a^F, 6b^B, 6b^C, 6b^D, 6b^E, 6b^F), 3.86 (br d, 1H, J = 2.4 Hz, H-4^D), 3.97–4.08 (m, 5H, H-2^C, 2^E, 3^E, 4^E, 4^F), 4.20–4.36 (m, 11H, H-1^A, 1^B, 1^D, 4 × CH₂Ph), 4.39–4.68 (m, 16H, H−1^C, 1^F, 3^C, 13 × CHPh), 4.74 (d, 1H, ²J = 10.4 Hz, CHPh), 4.80 (m, 2H, CH₂Ph), 4.88 (m, 3H, H-1^E, CH₂Ph), 4.96 (m, 2H, CH₂Ph), 5.05 (dd, 1H, J = 8.3, 9.5 Hz, H-2^B), 5.42 (dd, 1H, J = 8.4, 9.3 Hz, H-2^D), 5.63 (dd, 1H, J = 8.0, 10.0 Hz, H-2^F), 6.75–7.98 (m, 93H, aromatic) ppm; ¹³C{1H} NMR (151 MHz, CDCl₃): δ 55.3, 55.8, 65.9, 67.1, 67.6, 67.8, 67.9, 68.2, 68.8, 71.0, 71.5, 71.6, 71.8 (×2), 72.6, 72.7 (×2), 73.3 (×3), 73.6 (×6), 74.3, 74.6 (×4), 74.7, 74.8, 74.2 (×2), 75.4, 75.7, 75.9, 77.6, 79.9, 80.2 (×2), 81.7, 82.8, 97.8, 98.2, 100.0, 100.2, 100.8, 102.6, 123.2, 123.8, 126.8, 127.2, 127.3, 127.4, 127.5 (×2), 127.6, 127.7 (×4), 127.8(×10), 127.9 (×4), 128.0 (×6), 128.1 (×16), 128.2 (×13), 128.3 (×6), 128.4 (×6), 128.5 (×6), 128.6 (×9), 129.5, 130.0 (×2), 130.2, 131.4, 132.1, 132.6, 132.7, 133.3, 134.0, 134.5, 137.5, 137.8, 137.9 (×2), 138.1, 138.2 (×2), 138.4, 138.7 (×2), 138.9, 139.0 (×2), 139.1, 164.0, 165.2, 165.4, 167.6, 168.0 ppm; ESI TOF LCMS: [M + Na]⁺ calcd for C₁₇₁H₁₆₄N₂NaO₃₆, 2845.0995; found, 2845.0975. Also isolated were minor quantities of benzyl O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-(1→3)-O-(4,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)-(1→3)-[O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-(1→4)-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)-(1→4)]-O-(2-O-benzoyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (18). Analytical data for 18: R_f = 0.55 (acetone/toluene, 1/4, v/v); ${\left[\alpha \right]}_{\text{D}}^{22}+22.9$ (c 1.0, CHCl₃); ¹H NMR (600 MHz, CDCl₃, monosaccharide residues A–F are designated as in 15; the assignment of signals for terminal galactose residues D and F can be interchanged): δ 2.78 (ddd, 1H, J = 1.4, 8.1 Hz, H-5^A), 3.04–3.68 (m, 24H, H-2^A, 2^C, 3^A, 3^B, 3^D, 3^F, 4^C, 5^B, 5^C, 5^D, 5^E, 5^F, 6a^A, 6b^A, 6a^B, 6b^B, 6a^C, 6b^C, 6a^D, 6b^D, 6a^E, 6b^E, 6a^F, 6b^F), 3.73 (dd, 1H, J_3A,4A = 9.2 Hz, H-4A), 3.77–3.81 (m, 2H, H-4^D, 4^E), 3.98 (d, 1H, J_1D,2D = 7.8 Hz, H-1^D), 4.05 (br d, 1H, J_3F,4F = 1.1 Hz, H−4^F), 4.04 (d, 1H, ²J = 11.8 Hz, CHPh), 4.10–4.14 (m, 2H, CH₂Ph), 4.16–4.24 (m, 7H, H-1^A, 1^B, 2^E, 2 × CH₂Ph), 4.25–4.34 (m, 4H, H-4^B, 3 × CHPh), 4.36–4.47 (m, 6H, 3 × CH₂Ph), 4.50–4.57 (m, 3H, H-3^C, CH₂Ph), 4.60–4.71 (m, 5H, H-1^F, 3^E, 3 × CHPh), 4.75–4.84 (m, 3H, H-2^B, CH₂Ph), 4.86–4.95 (m, 3H, H-1^C, CH₂Ph), 4.96–5.02 (m, 3H, 3 × CHPh), 5.44 (dd, 1H, J_2D,3D = 10.1 Hz, H-2^D), 5.46 (d, 1H, J_1E,2E = 8.5 Hz, H-1^E), 5.64 (dd, 1H, J_1F,2F = 8.4, J_2F,3F = 9.5 Hz, H-2^F), 6.66–7.91 (m, 93H, aromatic); ¹³C NMR{1H} (151 MHz, CDCl₃): δ 55.3, 55.9, 67.7, 67.8, 68.2, 68.7, 69.1, 71.0, 71.1, 71.4, 71.6, 72.4, 72.5 (×3), 72.8, 73.9, 73.4 (×2), 73.6 (×4), 73.8, 74.2, 74.3, 74.4, 74.7, 74.8, 75.0 (×2), 75.6, 75.7, 75.9, 77.2, 77.3, 77.6, 78.6, 80.0, 80.1, 81.6, 82.1, 82.6, 98.8, 99.9, 100.0 (×2), 101.2, 102.6, 122.3 (×2), 122.8 (×2), 126.6 (×2), 127.0, 127.3 (×4), 127.5 (×2), 127.6 (×3), 127.7 (×2), 127.8 (×4), 127.9 (×4), 128.0 (×11), 128.1 (×9), 128.2 (×7), 128.3 (×5), 128.4 (×12), 128.5 (×5), 128.6 (×6), 129.2, 129.5, 129.9, 130.0, 130.3, 130.6, 130.7, 132.2, 132.7, 133.1, 133.2, 133.4, 133.5, 137.5, 137.7, 137.8, 138.0 (×2), 138.3, 138.5, 138.7, 138.9 (×2), 139.4 (×2), 163.1, 164.6, 165.0, 165.3, 168.4 ppm; ESI TOF LCMS: [M + Na]⁺ calcd for C₁₇₁H₁₆₄N₂NaO₃₆, 2845.0995; found, 2845.1057.

Benzyl O-(3,4,6-Tri-O-benzyl-β-d-galactopyranosyl)-(1→3)-O-(2-amino-4,6-di-O-benzyl-2-deoxy-β-d-glucopyranosyl)-(1→3)-[O-(3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-(1→4)-O-(2-amino-3,6-di-O-benzyl-2-deoxy-β-d-glucopyranosyl)-(1→6)]-O-(β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (16).

Compound 15 (65.0 mg, 0.023 mmol) was dissolved in NH₂NH₂−H₂O/MeOH (3.0 mL, 1/2, v/v), and the resulting mixture was kept for 36 h at reflux. After that, the volatiles were removed under reduced pressure. The residue was purified by column chromatography on silica gel (methanol–dichloromethane gradient elution) to afford the title compound as an off-white amorphous solid in 74% yield (37.2 mg, 0.016 mmol). Selected analytical data for 16: R_f = 0.50 (MeOH/CH₂Cl₂, 1/9, v/v); ${\left[\alpha \right]}_{\text{D}}^{22}+9.3$ (c 1.0, CHCl₃); ¹H NMR (600 MHz, CDCl₃, monosaccharide residues A–F are designated in the order shown in Figure 1; the assignment of signals for terminal galactose residues D and F can be interchanged): δ 2.65–4.11 (m, 37H, H-1^A, 2^A, 2^B, 2^C, 2^D, 2^E, 2^F, 3^A, 3^B, 3^C, 3^D, 3^E, 3^F, 4^A, 4^B, 4^C, 4^D, 4^E, 4^F, 5^A, 5^B, 5^C, 5^D, 5^E, 5^F, 6a^A, 6a^B, 6a^C, 6a^D, 6a^E, 6a^F, 6b^A, 6b^B, 6b^C, 6b^D, 6b^E, 6b^F), 4.17–4.23 (m, 3H, 3 × CHPh), 4.28 (d, 1H, CHPh), 4.43–4.51 (m, 9H, H-1^B, 1^C, 1^D, 1^E, 1^F, 2 × CH₂Ph), 4.55–4.74 (m, 12H, 6 × CH₂Ph), 4.79–4.93 (m, 6H, 3 × CH₂Ph), 5.02–5.08 (m, 4H, 2 × CH₂Ph), 7.11–7.38 (m, 70H, aromatic) ppm; ¹³C{1H} NMR (151 MHz, CDCl₃): δ 56.8, 57.3, 67.4, 68.1 (×2), 68.4, 68.6, 68.8, 71.3, 72.2, 72.3 (×3), 72.6, 73.0, 73.6 (×2), 73.4 (×2), 73.5 (×3), 73.7, 73.8, 74.6 (×3), 74.7 (×3), 74.7 (×2), 75.0, 75.1, 75.2, 75.7, 76.5, 76.8, 82.1 (×3), 82.4, 83.6, 83.9, 102.8, 103.0, 103.2 (×2), 103.8, 107.2, 127.3 (×3), 127.4 (×3), 127.5 (×3), 127.6 (×3), 127.8 (×17), 127.9 (×3), 128.0 (×8), 128.1 (×3), 128.2 (×8), 128.4 (×9), 128.5 (×8), 128.6 (×4), 137.7, 137.9, 138.0, 138.1 (×2), 138.4, 138.7, 138.8, 138.9, 139.0, 139.3 (×2) ppm; ESI TOF LCMS: [M + Na]⁺ calcd for C₁₃₄H₁₄₈N₂NaO₂₉, 2273.0099; found, 2273.0114.

O-(β-d-Galactopyranosyl)-(1→3)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1→3)-[O-(β-d-galactopyranosyl)-(1→4)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1→6)]-O-(β-d-galactopyranosyl)-(1→4)-β-d-glucopyranose (1).

Compound 16 (23.0 mg, 0.009 mmol) was dissolved in a mixture of Ac₂O/MeOH (2.0 mL, 1/1, v/v), and the resulting mixture was stirred for 16 h at rt. Then, the volatiles were removed under the reduced pressure. The residue was diluted with CH₂Cl₂ (~50 mL) and washed with sat. aq. NaHCO₃ (10 mL) and water (2 × 10 mL). The organic phase was separated, dried over MgSO₄, concentrated under reduced pressure, and dried in vacuo for 3 h. The crude residue was dissolved in EtOH/H₂O (3.0 mL, 4/1, v/v), 10% Pd on charcoal (75 mg) was added, and the resulting mixture was stirred under hydrogen for 24 h at rt. After that, the solids were filtered off and rinsed successively with methanol and water. The combined filtrate (~40 mL) was concentrated in vacuo. The residue was purified by column chromatography on Sephadex G-25 (water elution) to afford the title compound as a white amorphous solid in 80% yield (7.6 mg, 0.007 mmol). The spectral data for LNH 1 was in agreement with that reported previously.¹⁶ Selected analytical data for 1: R_f = 0.50 (chloroform/methanol/water, 2/1/0.4, v/v/v); ¹H NMR (600 MHz, D₂O): δ 2.00, 2.03 (2 s, 6H, 2 × CH₃CO), 3.27 (dd, 1H, J = 8.1, 9.0 Hz), 3.43–3.99 (m, 46H), 4.12 (d, 1H, J = 3.3 Hz), 4.41 (dd, 2H, J = 5.8, 7.8 Hz), 4.44 (d, 1H, J = 7.8 Hz), 4.62 (dd, 1H, J = 4.2, 7.9 Hz), 4.64 (d, 1H, J = 8.0 Hz), 4.70 (dd, 1H, J = 2.7, 8.4 Hz), 5.19 (d, 1H, J = 3.7 Hz) ppm; ¹³C{1H} NMR (151 MHz, D₂O): δ 22.6, 22.8, 55.1, 55.4, 60.4, 60.9, 61.4, 68.8 (×2), 69.0 (×2), 70.2 (×2), 70.4, 71.0, 71.3, 71.6, 71.8, 72.8 (×2), 73.8, 74.2, 74.7, 75.1 (×2), 75.6 (×2), 75.7, 78.7, 79.2, 79.4, 82.0, 82.4, 92.2, 96.1, 101.3, 102.9, 103.2, 103.3, 103.8, 174.8, 175.3 ppm; ESI TOF LCMS: [M + Na]⁺ calcd for C₄₀H₆₈N₂NaO₃₁, 1095.3704; found, 1095.3705.

Benzyl O-(3,4,6-Tri-O-benzyl-β-d-galactopyranosyl)-(1→3)-O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-β-d-glucopyranosyl)-(1→3)-O-(β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (17).

Compound 14 (30.0 mg, 0.016 mmol) was dissolved in NH₂NH₂−H₂O/MeOH (2.5 mL, 1/4, v/v), and the resulting mixture was kept for 24 h at reflux. After that, the volatiles were removed under the reduced pressure. The residue was dissolved in MeOH/CH₂Cl₂ (~5 mL, 1/9, v/v) and filtered through a pad of silica gel eluting with MeOH/CH₂Cl₂ (1/9, v/v). The combined filtrate (~50 mL) was concentrated under the reduced pressure and dried in vacuo for 3 h. The crude residue was dissolved in Ac₂O/MeOH (2.0 mL, 1/1, v/v), and the resulting mixture was stirred for 12 h at rt. Then, the volatiles were removed under reduced pressure. The residue was diluted with CH₂Cl₂ (50 mL) and washed with sat. aq. NaHCO₃ (10 mL) and brine (2 × 10 mL). The organic phase was separated, dried over MgSO₄, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (acetone–toluene gradient elution) to afford the title compound as an off-white amorphous solid in 84% yield (20.3 mg, 0.013 mmol). Analytical data for 17: R_f = 0.65 (acetone/toluene, 2/3, v/v); ${\left[\alpha \right]}_{\text{D}}^{22}+28.2$ (c 1.0, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 1.91 (3H, s, CH₃CO), 3.15–4.13 (m, 24H, H-2, 2′, 2″, 2‴, 3, 3′, 3″, 3‴, 4, 4′, 4″, 4‴, 5, 5′, 5″, 5‴, 6a, 6a′, 6a″, 6a‴, 6b, 6b′, 6b″, 6b‴), 4.20 (dd, 2H, ²J = 11.7 Hz, CH₂Ph), 4.35–4.49 (m, 7H, H-1, 1′, 1‴, 2 × CH₂Ph), 4.52–4.74 (m, 7H, 7 × CHPh), 4.82 (d, 1H, ²J = 12.1 Hz, CHPh), 4.85–4.94 (m, 4H, H-1″, 3 × CHPh), 4.98 (d, 1H, ²J = 10.6 Hz, CHPh), 6.44 (d, 1H, J = 7.0 Hz, NH), 7.04–7.37 (m, 45H, aromatic) ppm; ¹³C{1H} NMR (151 MHz, CDCl₃): δ 23.7, 56.6, 62.4, 68.3, 68.4, 68.7, 69.5, 70.5, 71.1, 71.5, 72.5, 73.0, 73.4, 73.5, 73.6, 73.8, 74.2 (×2), 74.7, 74.8, 74.9, 75.1, 75.5, 77.9, 80.4, 82.3 (×2), 83.1, 83.8, 100.7, 102.8, 103.0, 104.3, 127.3, 127.5 (×2), 127.7 (×2), 127.8 (×8), 127.9 (×4), 128.0 (×3), 128.1 (×2), 128.2 (×6), 128.3 (×2), 128.4 (×4), 128.5 (×7), 128.6 (×4), 137.8, 138.0, 138.1, 138.3 (×2), 138.4, 138.6, 138.7, 139.2, 172.2 ppm; ESI TOF LCMS: [M + Na]⁺ calcd for C₈₉H₉₉NNaO₂₁, 1540.6607; found, 1540.6627.

O-(β-d-Galactopyranosyl)-(1→3)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1→3)-O-(β-d-galactopyranosyl)-(1→4)-d-glucopyranose (2).

10% Pd on charcoal (75 mg) was added to a solution of 17 (20.0 mg, 0.013 mmol) in EtOH/H₂O (3.0 mL, 4/1, v/v), and the resulting mixture was stirred under hydrogen for 24 h at rt. After that, the solids were filtered off and rinsed successively with methanol and water. The combined filtrate (~40 mL) was concentrated in vacuo, and the residue was purified by column chromatography on Sephadex G-25 (water elution) to afford the title compound as a white amorphous solid in 87% yield (7.9 mg, 0.011 mmol). Analytical data for 2 was in agreement with that reported previously:¹⁴ R_f = 0.30 (chloroform/methanol/water, 2/1/0.4, v/v/v); ESI TOF LCMS: [M + Na]⁺ calcd for C₂₆H₄₅NNaO₂₁, 730.2382; found, 730.2392.