The chemical synthesis of human milk oligosaccharides: lacto-N-neotetraose (Galβ1→4GlcNAcβ1→ 3Galβ1→ 4Glc)

Abstract

The discovery of innovative methods that offer new capabilities for obtaining individual oligosaccharides from human milk will help to improve understanding their roles and boost practical applications. The total chemical synthesis of lacto-N-neotetraose (LNnT) has been completed using both linear and convergent strategies. The donor and acceptor protecting and leaving group combinations were found to be of paramount significance to successful couplings.

Graphical Abstract

Carbohydrates are involved in many processes and are referred to as the “essential molecules of life”.¹ Our life begins with fertilization, which takes place via carbohydrate-protein recognition.² Our journey with sugars continues with human milk that becomes the ideal first food.³ Glycans present in human milk can provide prebiotic effects,⁴ function as antimicrobial agents,^5–13 and provide necessary nutrients for the development of the brain and cognition of infants.¹⁴ Thanks to advances in glycosciences, we already know that Human Milk Oligosaccharides (HMO) are a unique and diverse family of glycans.^15–17 Structures of 162 HMO have been elucidated,^18–22 but our understanding of how HMO function is far from complete and is severely hampered by the lack of simple, efficient and cost effective methods for synthesizing glycans. Despite many efforts to prepare HMO enzymatically^23–42 or chemically,^43–54 their availability in pure form remains poor. Adding HMO to infant formulas could be beneficial for infants’ health,^55–58 but HMO are challenging to produce and purify, and exact roles of individual HMO remain unknown.^59–61 Only two simple glycans have been approved for infant formulas in the US and Europe, and three more HMO entered clinical trials.⁶²

One of the two approved HMO structures is lacto-N-neotetraose 1 (LNnT), which is a linear tetrasaccharide comprising a Galβ1→4GlcNAcβ1→3Galβ1→4Glc sequence shown in Scheme 1. More specifically, LNnT contains lactose disaccharide (Galβ1→4Glc) at the reducing end elongated by N-acetyllactosamine (Galβ1→4GlcNAc). During the incipient stage of the synthesis of LNnT 1, we decided to perform a convergent (2+2) synthetic strategy. Thus, per our retrosynthetic analysis, we chose the protected lactosamine donor 2, containing a suitable leaving group (LG), and the regioselectively protected lactose acceptor 3. The latter was designed as a universal precursor for the synthesis of other HMO sequences elongated at C-2 and/or C-6 of the galactose unit. The access to those sequences is enabled by temporary protecting groups, benzoyl and picoloyl (Pico). Another key aspect in our design is the use of benzyl groups as semi-permanent protecting groups, whereas a majority of previous syntheses used acetyl groups that are prone to migration leading to side products. The projected synthesis of lactosamine donor 2 involved coupling of galactosyl trichloroacetimidate donor 4 with glucosamine acceptor 5. To achieve the synthesis of lactose precursor 3, we projected the coupling between the orthogonally protected galactose donor 6 and tetrabenzylated glucose 4-OH acceptor 7. The protecting groups in donor 6 were chosen to provide access to the universal lactose acceptor precursor that would be suitable for glycosylation at C-3 via Fmoc removal (like in case of acceptor 3), C-6 via OPico removal, or provide access to 3,6-branched HMO sequences.

Scheme 1.

Retrosynthesis analysis of LNnT 1.

For the synthesis of lactosamine building block 2 we obtained known trichloroacetimidate donor 4.⁶³ Also known glucosamine thioglycoside acceptor 5 was obtained following a seven-step protocol similar to that previously described method.⁶⁴ Unfortunately, coupling between donor 4 and acceptor 5 in the presence of TMSOTf was not straightforward as anticipated. Instead of the desired product 2, the major product was tetrabenzoylated ethylthio galactoside, presumably generated through an aglycone transfer side reaction. Gildersleeve et al. has done a careful mechanistic study on the aglycone transfer reactions.⁶⁵ According to their mechanistic picture, several factors play a role in determining whether transfer occurs. One is the electronic, armed or disarmed, nature of the donor and the acceptor. Based on that, Gildersleeve et al hypothesized that any thioglycoside could undergo transfer as long as the oxacarbenium ion or the glycosyl intermediate derived from the thioglycoside is more stable than the oxacarbenium ion derived from the glycosyl donor. In further investigation of our coupling reaction, we came to realization that the aglycone transfer practically always takes place in reactions between disarmed galactose donors, regardless of the leaving group, and glucosamine acceptor 5. The same issue was reported by Wang,³¹ and the solution was found in applying galactosyl bromide as the donor that produced the respective lactosamine disaccharide with moderate yield of 70%.

In order to eliminate the aglycone transfer side reaction and to obtain the desired disaccharide 2 in high yield, we first investigated whether galactose trichloroacetimidate donor⁶⁶ equipped with the superarming 2-benzoyl-3,4,6-tri-benzyl pattern^67,68 would be advantageous. This reaction indeed produced disaccharide 2, and no aglycone transfer was detected in this case. However, multiple by-products have been detected that resulted in only a fair yield of disaccharide 2. At this point we decided to screen other leaving groups and determined that the superarmed S-benzoxazolyl (SBox) thioimidate 8^67,68 provides a superior combination of simplicity, efficiency and yields for the synthesis of lactosamine, which is a major constituent in many HMO. Thus, selective activation of the SBox leaving group in glycosyl donor 8 over thioethyl anomeric moiety of glycosyl acceptor 5 was achieved in the presence of silver trifluoromethanesulfonate (AgOTf). This glycosylation afforded the desired β-linked lactosamine disaccharide 2 in excellent yield of 98% (Scheme 2).

Scheme 2.

Convergent synthesis of protected LNnT 12.

Having achieved success in the synthesis of lactosamine disaccharide 2, we turned our attention to the synthesis of lactose derivative 3. The synthesis of galactose donor 6 started from known precursor 9⁶⁹ that was converted to the intermediate 10 via the reductive regioselective opening of the benzylidene acetal by reaction with 1 M BH₃ in THF in the presence of catalytic TMSOTf in 91% yield. Subsequently, the 6-hydroxyl derivative 10 was reacted with picolinic acid (PicoOH) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) to afford 6-picoloyl derivative 6 in 94% yield. The synthesis of the 4-OH acceptor 7 followed previously reported procedure.⁷⁰ The coupling of donor 6 and acceptor 7 was carried out by the activation of the thioethyl leaving group with N-iodosuccinimide (NIS) and TfOH in the presence of molecular sieves (3 Å). Subsequent deprotection of the Fmoc group with triethyl amine (Et₃N) in one pot led to the desired disaccharide acceptor 3 in 84% yield.

With the key disaccharide building blocks 2 and 3 in hand, we began to assemble the target tetrasaccharide sequence using the convergent strategy. The coupling of the disaccharide donor 2 bearing the SEt leaving group was very sluggish, and despite all attempts to push the reaction to completion, significant amounts of both the donor and the acceptor remained. To increase the reactivity of the glycosyl donor counterpart, we converted thioglycoside 2 into a phosphate donor 11 in 97% yield via a one-step protocol developed by Seeberger.⁷¹ The coupling of phosphate donor 11 with lactose acceptor 2 was then conducted in the presence of TMSOTf as depicted in Scheme 2. As a result, tetrasaccharide 14 was obtained in a good yield of 70% with complete β-stereoselectivity.

With a very minimal strategic adjustment to our synthetic scheme, we were also able to investigate whether the linear synthetic approach is able to offer any advantage for the synthesis of the target tetrasaccharide sequence. For this purpose, we converted building block 5 into its 4-O-Fmoc derivative 13 that was subsequently converted into phosphate donor 14. The linear synthesis started by glycosylation between donor 14 and lactose acceptor 3 in the presence of TMSOTf to afford the intermediate trisaccharide 15 in 70% yield. The Fmoc protecting group was removed with 30% Et₃N in CH₂Cl₂ and glycosylation of the resulting trisaccharide acceptor 16 with SBox donor 8 in the presence of AgOTf afforded the desired β-linked tetrasaccharide 12 in 87% yield.

With the key tetrasaccharide intermediate 12 which was obtained via both convergent and linear synthesis methods, we then endeavored a series of deprotection steps to obtain the target LNnT tetrasaccharide 1. Deprotection of the phthalimido and the ester groups were achieved by refluxing with NH₂NH₂-H₂O in MeOH, and the treatment with acetic anhydride in MeOH furnished tetrasaccharide 17 in 87% yield. Finally, the remaining benzyl groups were removed by hydrogenation the presence of 10% palladium on charcoal in wet ethanol to obtain the target trisaccharide 1 in 92% yield.

In summary, the total synthesis of lacto-N-neotetraose has been completed using both linear and convergent synthesis approaches. Both approaches employed the universal lactose building block 3 and offered similar efficiency. In accordance with the convergent approach, lactosamine building block 11 needed for glycosylation of acceptor 3 was obtained in three steps from monosaccharide building blocks 5 and 8 followed by the introduction of the phosphate leaving group. As a result, the convergent assembly of tetrasaccharide 12 was achieved in 66% yield over three steps. The linear synthesis of 12 involved glycosylation of acceptor 3 with donor 14, interim Fmoc deprotection, followed by glycosylation with 8. As a result, the linear assembly of tetrasaccharide 12 was also achieved in three synthetic steps in 57% yield overall. Along the way, we have developed new synthetic protocols for different glycosidic linkages. Notably, the donor and acceptor protecting group and the leaving group combinations were found to be of paramount significance to successful coupling. The discovery of innovative methods and accessible technologies that will offer new capabilities for obtaining individual HMO will help to improve understanding their roles and boost practical applications. Further synthetic studies of HMO are underway in our laboratory.

Experimental

General methods.

The reactions were performed using commercial reagents and the ACS grade solvents were purified and dried according to 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 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 (3Å), 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 co-evaporated 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 MHz or 600 MHz, and ¹³C NMR spectra were recorded at 75 MHz 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 CD₃COCD₃ δ_C = 29.84 ppm) for solutions in D₂O. Accurate mass spectrometry determinations were performed using Agilent 6230 ESI TOF LCMS mass spectrometer.

Preparation of monosaccharide building blocks

Ethyl 2-O-benzoyl-4-O-benzyl-3-O-fluorenylmethoxycarbonyl-1-thio-β-D-galactopyranoside (10).

A 1 M solution of BH₃ in THF (48 mL, 48 mmol) was added to a solution of ethyl 2-O-benzoyl-3-O-fluorenylmethoxycarbonyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside⁶⁹ (6.15 g, 9.62 mmol) in CH₂Cl₂ (50 mL). The resulting mixture was cooled to 0°C, TMSOTf (0.87 mL, 4.81 mmol) was added, and the resulting mixture was stirred under argon 2 h. During this time, the reaction was allowed to gradually increase to rt. After that, the reaction was quenched with Et₃N (~2 mL) and MeOH (~5 mL), and the resulting mixture was concentrated in vacuo. The residue was diluted with CH₂Cl₂ (~500 mL) and washed with sat. aq. NaHCO₃ (50 mL) and water (2 × 50 mL). The organic phase was separated, dried over MgSO₄, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate - hexane gradient elution) to afford the title compound as a white foam in 91% yield (5.61 g, 8.75 mmol). Analytical data for 10: R_f = 0.27 (ethyl acetate/hexane, 2/3, v/v); [α]_D²² +31.8 (c 1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ, 1.23 (t, 3H, J = 7.5 Hz, CH₂CH₃), 2.75 (m, 2H, CH₂CH₃), 3.56 (m, 1H, J = 5.4, 8.4, 11.2 Hz, H-6a), 3.67 (m, 1H, J = 6.1 Hz, H-5), 3.85 (m, 1H, H-6b), 4.04 (br d, 1H, H-4), 4.08 (d, 1H, J = 7.4 Hz, OCOCH₂CH), 4.29 (m, 2H, OCOCH₂CH), 4.60 (d, 1H, J_1,2 = 10.0 Hz, H-1), 4.67 (dd, 2H, ²J = 11.6 Hz, CH₂Ph), 5.07 (dd, 1H, J_3,4 = 2.9 Hz, H-3), 5.76 (t, 1H, J_2,3 = 10.0 Hz, H-2), 7.03–8.10 (m, 18H, aromatic) ppm; ¹³C NMR (75 MHz, CDCl₃): δ, 14.9, 24.0, 46.5, 61.7, 68.5, 70.3, 73.5, 74.9, 79.0, 79.2, 84.0, 120.1, 125.0, 125.2, 125.4, 127.2 (×2), 127.9, 128.2, 128.3 (×2), 128.6 (×4), 129.1, 129.6, 130.0, 133.4, 137.6, 141.2, 141.3, 142.9, 143.3, 154.6, 165.4 ppm; ESI TOF LCMS [M+NH₄]⁺ calcd for C₃₇H₄₀NO₈S 658.2475, found 658.2477.

Ethyl 2-O-benzoyl-4-O-benzyl-3-O-fluorenylmethoxycarbonyl-6-O-picoloyl-1-thio-β-D-galactopyranoside (6).

Picolinic acid (1.21 g, 9.83 mmol), EDC (0.90 g, 9.83 mmol), and DMAP (0.18 g, 1.31 mmol) were added to a stirring solution of 10 (4.20 g, 6.55 mmol) in CH₂Cl₂ (60 mL) and the resulting mixture was stirred for 1 h at rt. After that, the reaction mixture was diluted with CH₂Cl₂ (~500 mL) and washed with water (50 mL), 1% aq. HCl (50 mL) and water (2 × 50 mL). The organic phase was separated, dried over MgSO₄, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate - hexane gradient elution) to afford the title compound as a white form in 94% yield (5.30 g, 6.14 mmol). Analytical data for 6: R_f = 0.41 (ethyl acetate/hexane, 3/2, v/v); [α]_D²³ +36.8 (c 1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ, 1.22 (t, 3H, J = 7.5 Hz, CH₂CH₃), 2.73 (m, 2H, CH₂CH₃), 4.06 (m, 2H, H-5, OCOCH₂CH), 4.15 (d,1H, H-4), 4.28 (m, 2H, OCOCH₂CH), 4.47 (dd, 1H, J_5,6a = 11.2 Hz, J_6a,6b = 6.5 Hz, H-6a), 4.61 (dd, 1H, H-6b), 4.68 (d, 1H, J_1,2 = 10.0 Hz, H-1) 4.73 (dd, 2H, ²J = 11.4 Hz, CH₂Ph), 5.13 (dd, 1H, J_3,4 = 2.8 Hz, H-3), 5.79 (dd, 1H, J_2,3 = 10.0 Hz, H-2), 7.00–8.82 (m, 22H, aromatic) ppm; ¹³C NMR (75 MHz, CDCl₃): δ, 15.0, 24.1, 46.5, 63.8, 68.5, 70.3, 73.6, 75.2, 76.0, 79.0, 83.9, 120.1, 125.0, 125.2, 125.4, 127.2 (×3), 127.9 (×2), 128.0, 128.5 (×5), 128.6 (×2), 129.5, 130.0 (×2), 133.4, 137.1, 137.4, 141.2, 141.3, 142.8, 143.3, 147.6, 150.1, 154.6, 164.7, 165.3 ppm; ESI TOF LCMS [M+Na]⁺ calcd for C₄₃H₃₉NNaO₉S 768.2243, found 768.2247.

Ethyl 3,6-di-O-benzyl-2-deoxy-4-O-fluorenylmethoxycarbonyl-2-phthalimido-1-thio-β-D-glucopyranoside (13).

FmocCl (1.06 g, 4.12 mmol) was added to a solution of ethyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside⁶⁴ (5, 1.10 g, 2.06 mmol) in CH₂Cl₂ (20 mL) and pyridine (0.42 mL), and the resulting mixture was stirred under argon for 2 h at rt. After that, the reaction mixture was diluted with CH₂Cl₂ (~250 mL) and washed with 1% aq. HCl (40 mL) and water (2 × 40 mL). The organic phase was separated, dried over MgSO₄, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (ethyl acetate -hexane gradient elution) to afford the title compound as a white form in 87% yield (1.35 g, 1.79 mmol). Analytical data for 13: R_f = 0.53 (ethyl acetate/hexane, 2/3, v/v); [α]_D²³ +75.8 (c 1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ, 1.17 (t, 3H, J = 7.4 Hz, CH₂CH₃), 2.54–2.75 (m, 2H, CH₂CH₃), 3.68 (br d, 2H, J_6a,6b = 4.5 Hz, H-6a, 6b), 3.86 (m, 1H, J_5,6a = J_5,6b = 4.5 Hz, H-5), 4.13 (t, 1H, J = 7.1 Hz, OCOCH₂CH), 4.27–4.44 (m, 2H, OCOCH₂CH), 4.30 (dd, 1H, J_2,3 = 10.5 Hz, H-2), 4.44 (dd, 2H, ²J = 12.2 Hz, CH₂Ph), 4.50 (dd, 1H, J_3,4 = 9.0 Hz, H-3), 4.55 (dd, 2H, ²J = 12.0 Hz, CH₂Ph), 4.99 (dd, 1H, J_4,5 = 10.0 Hz, H-4), 5.27 (d, 1H, J_1,2 = 10.5 Hz, H-1), 6.78–7.83 (m, 22H, aromatic) ppm; ¹³C NMR (75 MHz, CDCl₃): δ, 15.1, 24.2, 46.9, 54.6, 69.9, 70.0, 73.6, 74.4, 77.3 (×2), 77.8, 81.3, 120.2 (×2), 123.4, 123.7, 124.8, 125.1, 125.2, 127.2, 127.3 (×2), 127.5, 127.7 (×3), 127.9 (×2), 128.0, 128.1 (×2), 128.4 (×2), 131.7, 134.1, 137.6, 138.1, 141.4 (×2), 143.2, 143.4, 154.4, 167.3, 168.1 ppm; ESI TOF LCMS [M+Na]⁺ calcd for C₄₅H₄₁NNaO₈S; 778.2451, found 778.2457.

Di-O-butyl 3,6-di-O-benzyl-2-deoxy-4-O-fluorenylmethoxycarbonyl-2-phthalimido-β-D-glucopyranosyl phosphate (14).

A mixture of thioglycoside 13 (0.30 g, 0.39 mmol), dibutyl hydrogen phosphate (0.23 mL, 1.19 mmol), and freshly activated molecular sieves (3 Å, 0.6 g) in CH₂Cl₂ (7.0 mL) was stirred under argon for 1 h at rt. The reaction mixture was cooled to 0°C, NIS (0.17 g, 0.78 mmol) and TfOH (7.0 μL, 0.08 mmol) were added, and the resulting mixture was stirred for 20 min at 0°C. After that, the solids were filtered off and rinsed successively with CH₂Cl₂. The combined filtrate (~100 mL) was washed with 10% aq. Na₂S₂O₃ (15 mL) and 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 a clear syrup in 90% yield (0.31 g, 0.35 mmol). Analytical data for 14: R_f = 0.45 (ethyl acetate/hexane, 1/4, v/v); [α]_D²³ +55.8 (c 1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ, 0.69, 0.83 (2 t, 6H, 2 × O(CH₂)₃CH₃), 1.03 (m, 2H, J = 7.3, 14.4 Hz, O(CH₂)₂CH₂CH₃), 1.18–1.34 (m, 4H, O(CH₂)₂CH₂CH_3, OCH₂CH₂CH₂CH₃), 1.42–1.56 (m, 2H, OCH₂CH₂CH₂CH₃), 3.61–3.80 (m, 4H, H-6a, 6b, OCH₂CH₂CH₂CH₃), 3.84–4.03 (m, 3H, H-5, OCH₂CH₂CH₂CH₃), 4.13 (t, 1H, J = 7.0 Hz, OCOCH₂CH), 4.29–4.43 (m, 3H, H-2, OCOCH₂CH), 4.44 (dd, 2H, ²J = 12.4 Hz, CH₂Ph), 4.51 (dd, 2H, ²J = 12.0 Hz, CH₂Ph), 4.58 (m, 1H, J_3,4 = 9.4 Hz, H-3), 5.08 (t, 1H, J_4,5 = 9.4 Hz, H-4), 5.84 (t, 1H, J_1,2 = 7.5 Hz, H-1), 6.76–7.82 (m, 22H, aromatic) ppm; ¹³C NMR (75 MHz, CDCl₃): δ, 13.5, 13.6, 18.4, 18.6, 31.9 (d, J = 7.1 Hz), 32.0 (d, J = 7.2 Hz), 46.8, 55.9 (d, J = 8.2 Hz), 67.9 (d, J = 6.2 Hz), 68.1 (d, J = 6.3 Hz), 69.1, 70.1, 73.5, 73.6, 74.4, 76.4, 76.5, 94.1 (d, J = 4.5 Hz), 120.2 (×2), 123.5, 125.0, 125.2, 127.3 (×2), 127.5, 127.7 (×4), 127.8 (×3), 128.0 (×2), 128.1 (×3), 128.4 (×3), 131.6, 134.0 (×2), 137.6, 137.9, 141.4 (×2), 143.1, 143.4, 154.3 ppm; ESI TOF LCMS [M+Na]⁺ calcd for C₅₁H₅₄NNaO₁₂P 926.3281, found 926.3286.

Synthesis of oligosaccharides

Ethyl O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-4,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside (2).

A mixture of benzoxazolyl 2-O-benzoyl-3,4,6-tri-O-benzyl-1-thio-β-D-galactopyranoside^67,68 (8, 0.20 g, 0.29 mmol), acceptor 5 (0.12 g, 0.22 mmol), and freshly activated molecular sieves (3Å, 600 mg) in CH₂Cl₂ (7.0 mL) was stirred under argon for 2 h at rt. The reaction mixture was cooled to −30°C, freshly conditioned AgOTf (0.15 g, 0.58 mmol) was added, and the resulting mixture was stirred for 15 min. The solids were filtered-off 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 98% yield (0.23 g, 0.22 mmol). Analytical data for 2: R_f = 0.54 (acetone/toluene, 1/9, v/v); [α]_D²³ +28.5 (c 1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ, 1.11 (t, 3H, J = 7.4 Hz, CH₂CH₃), 2.45–2.67 (m, 2H, CH₂CH₃), 3.34–3.70 (m, 7H, H-3′, 5, 5′, 6a, 6a′, 6b, 6b′), 4.03 (m, 2H, H-4, 4′), 4.15–4.66 (m, 11H, H-1′, 2, 3, 8 x CHPh), 4.66 (d, 1H, J_1′,2′ = 7.9 Hz, H-1′), 4.91 (d, 1H, ²J = 12.1 Hz, CHPh), 4.97 (d, 1H, ²J = 11.6 Hz, CHPh), 5.13 (d, 1H, J_1,2 = 9.9 Hz, H-1), 5.65 (dd, 1H, J_2′,3′ = 10.1 Hz, H-2′), 6.73–7.98 (m, 34H, aromatic) ppm; ¹³C NMR (75 MHz, CDCl₃): δ, 15.0. 24.0, 54.9, 68.0, 68.2, 71.4, 72.6 (×2), 73.4 (×2), 73.6, 74.6, 74.8, 77.4, 78.1, 79.0, 79.8, 81.1, 100.8, 123.4, 123.5, 126.8, 127.4, 127.6 (×2), 127.7 (×2), 127.8 (×7), 127.9 (×4), 128.0 (×2), 128.2 (×2), 128.4 (×6), 128.5 (×3), 129.9 (×2), 131.7, 133.2, 133.9, 137.8, 138.1, 138.4, 138.8, 138.9, 165.2, 167.7, 168.0 ppm; ESI TOF LCMS [M+NH₄]⁺ calcd for C₆₄H₆₇N₂O₁₂S 1087.4415, found 1087.4426.

Benzyl O-(2-O-benzoyl-4-O-benzyl-6-O-picoloyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside (3).

A mixture of donor 6 (0.82 g, 0.96 mmol), benzyl 2,3,6-tri-O-benzyl-β-D-glucopyranoside⁷⁰ (7, 0.40 g, 0.74 mmol), and freshly activated molecular sieves (3Å, 2.0 g) in CH₂Cl₂ (20 mL) was stirred under argon for 2 h at rt. The reaction mixture was cooled to 0°C, NIS (0.43 g, 1.96 mmol) and TfOH (17 μL, 0.19 mmol) were added, and the resulting mixture was stirred for 16 h. Over this time, the reaction temperature was allowed to reach rt. After that, Et₃N (6.0 mL) was added, and the resulting mixture was stirred for 1 h at rt. The solid was filtered-off and rinsed successively with CH₂Cl₂. The combined filtrate (~100 mL) was washed with 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 84% yield (0.61 g, 0.62 mmol). Analytical data for 3: R_f = 0.50 (acetone/toluene, 1/4, v/v); [α]_D²³ −7.8 (c 1.0, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ, 2.39 (d, 1H, J = 9.4 Hz, OH), 3.29 (m, 1H, J_5,6a = 1.4 Hz, J_5,6b = 3.8 Hz, H-5), 3.45 (dd, 1H, J_2,3 = 9.0 Hz, H-2), 3.57 (dd, 1H, J_3,4 = 9.1 Hz, H-3), 3.62 (dd, 1H, J_6a,6b = 10.8 Hz, H-6a), 3.65–3.72 (m, 3H, J_3′,4′ = 3.8 Hz, H-3′, 5′, 6b), 3.93 (br d, 1H, H-4′), 3.96 (dd, 1H, J_4,5 = 9.1 Hz, H-4), 4.33–4.42 (m, 3H, H-6a′, 6b′, CHPh), 4.41 (d, 1H, J_1,2 = 8.0 Hz, H-1), 4.58 (d, 1H, ²J = 12.0 Hz, CHPh), 4.61 (d, 1H, ²J = 12.2 Hz, CHPh), 4.70 (d, 1H, ²J = 11.0 Hz, CHPh), 4.82–4.75 (m, 4H, J_1′,2′ = 8.0 Hz, H-1′, 3 x CHPh), 4.86–4.89 (m, 2H, 2 x CHPh), 5.01 (d, 1H, ²J = 10.9 Hz, CHPh), 5.27 (dd, 1H, J_2′,3′ = 10.0, H-2′), 7.12–8.82 (m, 34H, aromatic) ppm; ¹³C NMR (75 MHz, CDCl₃): δ, 63.1, 68.2, 71.2, 72.1, 73.5, 73.5, 74.6, 74.7, 75.0, 75.4, 75.8, 76.4, 76.8, 81.9, 82.8, 100.2, 102.6, 125.4, 124.4, 127.2, 127.4, 127.7, 127.8 (×3), 127.9 (×2), 128.0 (×2), 128.2 (×6), 128.4 (×2), 128.5 (×2), 128.6 (×4), 128.7 (×2), 129.6, 129.9 (×2), 133.5, 137.1, 137.5, 137.8, 138.3, 138.6, 139.1, 147.6, 150.1, 164.5, 166.7 ppm; ESI TOF LCMS [M+Na]⁺ calcd for C₆₀H₅₉NNaO₁₃ 1024.3884, found 1024.3878.

Di-O-butyl O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-D-galactopyranosyl)- (1→4)-4,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl phosphate (11).

A mixture of compound 2 (0.285 g, 0.282 mmol), dibutyl hydrogen phosphate (0.17 mL, 0.847 mmol), and freshly activated molecular sieves (3 Å, 0.5 g) in CH₂Cl₂ (5.0 mL) was stirred under argon for 1 h at rt. The mixture was cooled to 0°C, NIS (0.126 g, 0.564 mmol) and TfOH (5.0 μL, 0.056 mmol) were added, and the resulting mixture was stirred for 20 min at 0°C. After that, the solids were filtered off and rinsed successively with CH₂Cl_2. The combined filtrate (~100 mL) was washed with 10% aq. Na₂S₂O₃ (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 oily syrup in 97% yield (0.33 g, 0.273 mmol). Analytical data for 11: R_f = 0.35 (acetone/toluene,1/9, v/v); [α]_D²³ +31.7 (c 1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ, 0.71, 0.86 (2 t, 6H, 2 x O(CH₂)₃CH₃), 1.03 (m, 2H, O(CH₂)₂CH₂CH₃), 1.37–1.20 (m, 4H, O(CH₂)₂CH₂CH_3, OCH₂CH₂CH₂CH₃), 1.52 (m, 2H, OCH₂CH₂CH₂CH₃), 3.48–3.59 (m, 6H, H-3′, 5, 6a, 6a′, 6b, 6b′), 3.64–3.80 (m, 3H, H-5′, OCOCH₂CH), 3.84–4.00 (m, 2H, OCOCH₂CH), 4.06 (br d, 1H, J_3′,4′ = 2.8 Hz, H-4′), 4.13 (dd, 1H, J_4,5 = 8.9 Hz, H-4), 4.21–4.63 (m, 8H, H-2, 3, 6 x CHPh), 4.66–4.74 (m, 3H, J_1′,2′ = 7.7 Hz, H-1′, 2 x CHPh), 4.97 (d, 1H, ²J = 12.2 Hz, CHPh), 5.04 (d, 1H, ²J = 11.6 Hz, CHPh), 5.69 (dd, 1H, J_2′,3′ = 9.9 Hz, H-2′), 5.75 (dd, 1H, J = 8.2, 7.1 Hz, H-1), 6.80–8.05 (m, 34H, aromatic); ¹³C NMR (75 MHz, CDCl₃): δ, 13.4, 13.6, 18.3, 18.5, 31.8 (d, J = 7.0 Hz), 32.0 (d, J = 7.2 Hz), 56.0, 56.1, 67.4, 67.7 (d, J = 6.0 Hz), 67.9 (d, J = 6.3 Hz), 68.2, 71.4, 72.4, 72.6, 73.4, 73.5 (x2), 74.5, 74.7, 75.1, 76.4, 76.8, 79.7, 94.1 (d, J = 4.9 Hz), 100.7, 123.3, 125.4, 126.9, 127.4, 127.6 (×2), 127.7, 127.8 (×6), 127.9, 128.0 (×6), 128.2 (×2), 128.3, 128.4 (×2), 128.5 (×6), 129.1, 129.8, 129.9, 131.6, 133.2, 133.9, 137.8, 138.0, 138.1, 138.8 (x2), 165.1, 167.6 (×2) ppm; ESI TOF LCMS [M+Na]⁺ calcd for C₇₀H₇₆NNaO₁₆P 1240.4799, found 1240.4826

Benzyl O-(3,6-di-O-benzyl-2-deoxy-4-O-fluorenylmethoxycarbonyl-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-glucopyranoside (15).

A mixture of donor 14 (0.20 g, 0.22 mmol), acceptor 2 (0.17 g, 0.17 mmol), and freshly activated molecular sieves (3Å, 600 mg) in CH₂Cl₂ (10 mL) was stirred under argon for 2 h at rt. The mixture was cooled to −30°C, TMSOTf (80 μL, 0.44 mmol) was added, and the resulting mixture was stirred for 15 min. The reaction mixture was then diluted with CH₂Cl₂, the solid was 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 an off-white amorphous solid in 70% yield (0.20 g, 0.12 mmol). Analytical data for 15: R_f = 0.52 (acetone/toluene, 1/4 v/v); [α]_D²³ +10.0 (c 1.0, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ, 2.90 (m, 1H, J = 9.2 Hz, H-5), 3.25 (dd, 1H, J = 10.0 Hz, H-6a), 3.32 (dd, 1H, J_2,3 = 8.9 Hz, H-2), 3.36–3.39 (m, 2H, H-3, 6b), 3.63–3.72 (m, 4H, H-3′, 5′, 6a′′, 6b′′), 3.81(dd, 1H, H-4), 3.88 (m, 1H, H-5′′), 4.03 (br d, 1H, J_3′,4′ = 1.9 Hz, H-4′), 4.11 (t, 1H, J = 6.9 Hz, OCOCH₂CH), 4.15–4.27 (m, 5H, H-1, 2′′, 6a′, 2 x CHPh), 4.31–4.37 (m, 3H, H-6b, OCOCH₂CH), 4.42 (d, 1H, J_1′,2′ = 8.0 Hz, H-1′), 4.44–4.49 (m, 4H, H-3′′, 3 x CHPh), 4.56–4.68 (m, 5H, 5 x CHPh), 4.78–4.82 (m, 2H, 2 x CHPh), 4.89–4.94 (m, 2H, H-4′′, CHPh), 5.05 (d, 1H, ²J = 11.5 Hz, CHPh), 5.19 (d, 1H, J_{1′′,2′′} = 8.4 Hz, H-1′′), 5.30 (dd, 1H, J_2′,3′ = 9.8 Hz, H-2′), 6.62–8.86 (m, 56H, aromatic) ppm; ¹³C NMR (151 MHz, CDCl₃): δ, 46.0, 55.6, 63.7, 67.4, 69.7, 69.9, 70.9, 71.9 (x2), 72.9, 73.4, 73.6 (×2), 74.1, 74.2, 74.8, 74.9, 75.4, 75.7, 76.0, 76.3, 80.0, 81.5, 82.5, 99.4, 100.1, 102.4, 120.0 (×2), 124.9, 125.0, 125.3, 126.7, 127.1 (×2), 127.2, 127.3 (×2), 127.4, 127.5, 127.6 (×4), 127.7 (×3), 127.8, 127.9 (×11), 128.0 (×3), 128.1, 128.2 (×11), 128.4 (×3), 128.5 (×2), 128.6 (×3), 129.3, 129.6, 132.8, 136.8, 137.4 (×2), 137.6, 138.2, 138.4, 138.5, 138.9, 141.2, 141.3, 143.0, 143.2, 147.7, 149.8, 154.2, 164.2, 164.3 ppm; ESI TOF LCMS [M+Na]⁺ calcd for C₁₀₃H₉₄N₂NaO₂₁ 1718.6280, found 1718.6223.

Benzyl O-(3,6-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-glucopyranoside (16).

Compound 15 (200 mg, 0.118 mmol) was dissolved in a solution of Et₃N in CH₂Cl₂ (7.0 mL, 1/165, v/v), and the resulting solution was stirred for 2 h at rt. The resulting mixture was concentrated in vacuo, and 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 98% yield (170.3 mg, 0.116 mmol). Analytical data for 16: R_f = 0.55 (acetone/toluene, 1/4, v/v); [α]_D²² −5.7 (c 1.0, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ, 2.92 (m, 1H, J = 1.8, 3.4, 10.0 Hz, H-5), 3.28 (dd, 1H, H-6a), 3.34 (dd, 1H, J_1,2 = 7.8 Hz, J_2,3 = 9.2 Hz, H-2), 3.37–3.42 (m, 2H, H-3, 6b), 3.67–3.84 (m, 7H, H-3′, 4, 4′′, 5′, 5′′, 6a′′, 6b′′), 4.02 (br d, 1H, J_3′,4′ = 2.2 Hz, H-4′), 4.16–4.29 (m, 5H, H-1, 2′′, 6a′, 6b′, CHPh), 4.37 (dd, 1H, J = 6.2, 11.2 Hz, H-3′′), 4.41 (d, 1H, ²J = 12.1 Hz, CHPh), 4.45–4.70 (m, 9H, J_1′,2′ = 8.0 Hz, H-1′, 8 x CHPh), 4.81 (d, 1H, ²J = 12.0 Hz, CHPh), 4.82 (d, 1H, ²J = 11.4 Hz, CHPh), 4.93 (d, 1H, ²J = 10.4 Hz, CHPh), 5.08 (d, 1H, ²J = 11.5 Hz, CHPh), 5.22 (d, 1H, J_{1′′,2′′} = 8.2 Hz, H-1′′), 5.33 (dd, 1H, J_2′,3′ = 10.1 Hz, H-2′), 6.63–8.81 (m, 48H, aromatic) ppm; ¹³C NMR (151 MHz, CDCl₃): δ, 55.7, 63.8, 67.6, 70.7, 71.0, 72.0, 73.4, 73.9 (×4), 74.3, 74.5, 74.9, 75.0, 75.5, 75.9, 76.1, 78.6, 80.1, 81.6, 82.6, 99.7, 100.3, 102.5, 122.8, 123.4, 125.4, 126.9, 127.2, 127.5, 127.5, 127.6 (×3), 127.7 (×3), 127.8 (×2), 128.0 (×5), 128.1 (×4), 128.2 (×5), 128.3 (×6), 128.4 (×2), 128.6 (×2), 128.7 (×5), 129.4, 129.7, 130.9, 131.4, 132.9, 133.4, 133.5, 137.0, 137.5, 138.1, 138.3, 138.6, 139.0, 147.8, 149.9, 164.4, 164.4, 167.2, 168.1 ppm; ESI TOF LCMS [M+Na]⁺ calcd for C₈₈H₈₄N₂NaO₁₉ 1495.5566, found 1495.5579.

Benzyl 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→3)-O-(2-O-benzoyl-4-O-benzyl-6-O-picoloyl-β-D-galactopyranosyl)- (1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside (12).

A convergent (2+2) approach. A mixture of donor 11 (128.3 mg, 0.105 mmol), acceptor 3 (80.0 mg, 0.081 mmol), and freshly activated molecular sieves (3Å, 400 mg) in CH₂Cl₂ (7.0 mL) was stirred under argon for 2 h at rt. The mixture was cooled to −60°C, TMSOTf (29 μL, 0.162 mmol) was added, and the resulting mixture was stirred for 30 min. During this time the reaction temperature was allowed to gradually increase to −30°C. The reaction mixture was then diluted with CH₂Cl₂, the solid was 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 an off-white amorphous solid in 70% yield (146.6 mg, 0.073 mmol). A linear approach. A mixture of donor 8^67,68 (42.5 mg, 0.062 mmol), acceptor 16 (70.0 mg, 0.0475 mmol), and freshly activated molecular sieves (3Å, 250 mg) in CH₂Cl₂ (4.0 mL) was stirred under argon for 2 h at rt. The mixture was cooled to −30°C, freshly conditioned AgOTf (31.8 mg, 0.124 mmol) was added, the external cooling was removed, and the resulting mixture was stirred for 15 min. During this time the reaction temperature was allowed gradually increase to −23°C. The reaction mixture was then diluted with CH₂Cl₂, the solid was 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 an off-white amorphous solid in 84% yield (80.0 mg, 0.0398 mmol). Analytical data for 12: R_f = 0.50 (acetone/toluene, 1/4, v/v); [α]_D²² +11.5 (c 1.0, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ, 2.89 (m, 1H, J = 1.9, 3.4, 9.9 Hz, H-5), 3.24 (dd, 1H, H-6a), 3.28–3.46 (m, 7H, H-2, 3, 5′′, 5′′′, 6a′′′, 6b, 6b′′′), 3.49 (dd, 1H, J = 2.8, 10.1 Hz, H-3′′′), 3.53 (dd, 1H, J_{5′′,6a′′} = 0.8 Hz, H-6a′′), 3.56–3.61 (m, 2H, H-3′, 5′), 3.66 (dd, 1H, J_{5′′,6b′′} = 3.8 Hz, H-6b′′, 3.78 (dd, 1H, J_4,5 = 9.2 Hz, H-4), 3.95–3.99 (m, 3H, H-4′, 4′′, 4′′′), 4.12–4.32 (m, 10H, H-1, 2′′, 3′′, 6a′, 6b′, 5 x CHPh), 4.40–4.49 (m, 4H, H-1′, 3 x CHPh), 4.50–4.58 (m, 4H, 4 x CHPh), 4.60–4.67 (m, 3H, J_{1′′′,2′′′} = 7.9 Hz, H-1′′′, 2 x CHPh), 4.79 (m, 2H, 2 x CHPh), 4.85 (d, 1H, ²J = 12.0 Hz, CHPh), 4.90 (d, 1H, ²J = 10.4 Hz, CHPh), 4.95 (d, 1H, CHPh), 5.08 (m, 2H, J_{1′′,2′′} = 8.1 Hz, H-1′′, CHPh), 5.28 (dd, 1H, J_2′,3′ = 10.0, H-2′), 5.61 (dd, 1H, J_{2′′′,3′′′} = 10.0 Hz, H-2′′′), 6.59–8.70 (m, 68H, aromatic) ppm; ¹³C NMR (151 MHz, CDCl₃): δ, 29.8, 56.0, 63.9, 67.6, 68.0, 68.3, 71.0, 71.4, 71.9, 72.0, 72.5, 72.6, 73.4, 73.5, 73.6, 74.3, 74.6 (x2), 74.0, 75.0, 75.1, 75.6, 76.1, 76.2, 76.8, 77.8, 79.8, 80.1, 80.3, 81.7, 82.6, 99.7, 100.4, 101.0, 102.5, 122.8, 123.3, 125.5, 126.7, 126.8, 127.2, 127.4 (×2), 127.5 (×2), 127.6, 127.7 (×7), 127.8 (×8), 127.9 (×4), 128.0 (×4), 128.1 (×6), 128.3 (×6), 128.4 (×5), 128.5 (×5), 128.6 (×3), 129.4, 129.7, 130.0 (×2), 131.0, 131.5, 132.9, 133.3, 137.0, 137.6, 137.8, 138.0 (×2), 138.3, 138.7, 138.8 (×2), 138.9, 139.0, 147.8, 149.9, 164.3, 164.4, 165.2, 167.2, 167.8 ppm; ESI TOF LCMS [M+H]⁺ calcd for C₁₂₂H₁₁₇N₂O₂₅ 2010.7979, found 2010.7988.

Deprotection of tetrasaccharide 12 Benzyl O-(3,4,6-tri-O-benzyl-β-D-galactopyranosyl)- (1→4)-O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl)- (1→3)-O-(4-O-benzyl-β-D-galactopyranosyl)- (1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside (17).

Compound 12 (78.0 mg, 0.038 mmol) was dissolved in MeOH (5.0 mL), NH₂NH₂-H₂O (167 μL, 3.42 mmol) was added, and the resulting mixture was kept for 24 h at reflux. After that, the volatiles were removed under reduced pressure, and the residue was dried in vacuo for 3 h. The crude residue was dissolved in a mixture of Ac₂O/MeOH (2.0 mL, 1/1, v/v) and the resulting mixture was stirred for 12 h at rt. After that, the volatiles were removed under reduced pressure. The residue was diluted with CH₂Cl₂ (50 mL), and washed with water (10 mL), 1 M HCl (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 an off-white amorphous solid in 87% yield (53.1 mg, 0.033 mmol). Analytical data for 17: R_f = 0.67 (acetone/toluene, 2/3, v/v); [α]_D²² +12.0 (c 1.0, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ, 1.75 (s, 3H, CH₃CO). 2.94–4.03 (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′′′), 4.30 (dd, 2H, ²J = 11.7 Hz, CH₂Ph), 4.43–4.50 (m, 3H, H-1, 1′, 1′′′), 4.52–4.73 (m, 11H, 11 x CHPh), 4.80–4.96 (m, 7H, 7 x CHPh), 4.99 (d, 1H, J_{1′′,2′′} = 7.8 Hz, H-1′′), 5.61 (d, 1H, J = 7.4 Hz, NHCOCH₃), 7.16–7.40 (m, 50H, aromatic) ppm; ¹³C NMR (151 MHz, CDCl₃): δ, 23.6, 57.2, 61.8, 68.3, 68.8, 69.0, 71.3, 71.9, 72.1, 72.3, 72.8, 73.5, 73.6 (×3), 73.7, 74.3 (×2), 74.7, 74.8, 74.9, 75.0, 75.1, 75.3, 77.1, 77.2, 80.2, 82.0, 82.2, 83.2 (×2), 102.2, 102.9, 103.2, 103.4, 127.4, 127.5 (×3), 127.6 (×2), 127.7 (×3), 127.8 (×3), 127.9 (×5), 128.0 (×7), 128.1 (×2), 128.3 (×6), 128.4 (×5), 128.5 (×4), 128.6 (×7), 137.6 (×2), 137.9 (×2), 138.1, 138.2, 138.4, 138.6, 138.8 (x2), 138.9, 139.1, 170.9 ppm; ESI TOF LCMS [M+Na]⁺calcd for C₉₆H₁₀₅NNaO₂₁ 1631.7110, found 1631.7130.

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

A 10% Pd on charcoal (150 mg) was added to a solution of 17 (53.0 mg, 0.033 mmol) in EtOH/H₂O (5.0 mL, 4/1, v/v), and the resulting mixture was stirred under hydrogen atmosphere 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 92% yield (21.4 mg, 0.030 mmol). Selected analytical data for 1:⁷² R_f = 0.32 (chloroform/methanol/water, 2/1/0.4, v/v/v); ¹H NMR (600 MHz, D₂O): δ, 2.01 (s, 3H), 3.24–3.28 (m, 1H), 3.88–3.49 (m, 29H), 3.89–3.96 (m, 4H), 4.14 (d, 1H, J = 3.3 Hz), 4.42 (d, 1H, J = 7.9 Hz), 4.46 (d, J = 7.8 Hz, 1H), 4.64 (d, 1H, J = 8.0 Hz), 4.68 (dd, 1H, J = 2.1, 8.4 Hz,), 5.20 (d, 1H, J = 3.8 Hz) ppm; ¹³C NMR (151 MHz, D₂O): δ, 22.5, 55.5, 60.2, 60.3, 60.4, 61.3, 61.4, 68.7 (x2), 68.9, 70.3 (x2), 70.4, 70.5, 71.3, 71.5 (×2), 71.7, 72.5, 72.8, 74.1, 74.7, 74.9, 75.1, 75.2, 75.7, 78.5, 78.6, 78.7, 82.4, 92.2, 96.1, 103.1, 103.2 (x2), 103.3, 103.4, 175.2 ppm; ESI TOF LCMS [M+Na]⁺ calcd for C₂₆H₄₅NNaO₂₁ 730.2382, found 730.2392.

Scheme 3.

The linear synthesis of tetrasaccharide 12 and its deprotection to obtain LNnT 1.

• The total synthesis of lacto-N-neotetraose has been completed using both linear and convergent synthesis approaches;

• New synthetic protocols for different glycosidic linkages have been developed and refined;

• Aglycone transfer was encountered, and has been successfully solved by tuning the donor-acceptor protecting-leaving group combinations;

• New methods for obtaining individual HMO help to improve understanding their roles and boost practical applications