Chemoenzymatic Synthesis of a Library of Human Milk Oligosaccharides

Abstract

Human milk oligosaccharides (HMOs) are a family of diverse unconjugated glycans that exist in human milk as one of the major components. Characterization, quantification, and biofunctional studies of HMOs remain a great challenge due to their diversity and complexity. The accessibility of a homogeneous HMO library is essential to solve these issues which have beset academia for several decades. In this study, an efficient chemoenzymatic strategy, namely core synthesis/enzymatic extension (CSEE), for rapid production of diverse HMOs was reported. On the basis of 3 versatile building blocks, 3 core structures were chemically synthesized via consistent use of oligosaccharyl thioether and oligosaccharyl bromide as glycosylation donors in a convergent fragment coupling strategy. Each of these core structures was then extended to up to 11 HMOs by 4 robust glycosyltransferases. A library of 31 HMOs were chemoenzymatically synthesized and characterized by MS and NMR. CSEE indeed provides a practical approach to harvest structurally defined HMOs for various applications.

Graphical abstract

INTRODUCTION

Human milk, as the sole dietary source, contains all the necessary nutrients for an infant to thrive in the first few months. More importantly, human milk can also provide ingredients with health benefits which traditional food cannot do. Human milk oligosaccharides (HMOs) are the third major component in human milk, only after lactose and lipid.¹ Concentrations and components of HMOs vary depending on the stages of lactation.² Particularly, 1 L of mature human milk contains about 12–14 g of HMOs.³ The structures of about 130 discovered HMOs have been elucidated.⁴ The major building blocks of HMOs are 5 monosaccharides, including d-glucose (Glc), d-galactose (Gal), N-acetyl-d-glucosamine (GlcNAc), l-fucose (Fuc), and N-acetylneuraminic acid (Neu5Ac). Even though HMOs were first discovered and confirmed in the 1950s, a comprehensive understanding of their functions is still out of reach, due to their inherit diversity and complexity. Increasing evidence shows that HMOs can provide significant beneficial effects to the health of breast-fed infants through several mechanisms. For instance, HMOs could serve as prebiotics to promote the growth of desired bacteria in an infant’s intestine.^1c,2a,5 In addition, HMOs are antiadhesive antimicrobials by serving as receptors to prevent pathogen attachment to infant mucosal surfaces.⁶ In addition, evidence has demonstrated that HMOs can modulate epithelial and immune cell responses and reduce excessive mucosal leukocyte infiltration and activation, which in turn decreases the risk of necrotizing enterocolitis (NEC), one of the most common fatal disorders in preterm infants.⁷ Furthermore, sialylated HMOs may also provide necessary nutrients for the development of the brain and cognition of infants.⁸

Even though the general functions of HMOs have been explored and discovered, the functional roles of individual HMOs are far less clear because of very limited access to sufficient amounts of structurally defined HMOs. To date, only a handful of short-chain HMOs can be produced on a large scale and the supply of more complicated and branched HMOs is highly demanded.

Until now, only a few approaches have been developed for the synthesis of a small number of well-defined HMOs.^2a,9 For example, Schmidt developed strategies to synthesize some highly branched HMOs by solution-phase and solid-phase synthesis.^9b,d,e Enzymatic methods have also been employed to achieve relatively simple structures.^9a One of the biggest roadblocks in previous syntheses remains the small quantity and limited variety of HMOs needed for biofunctional studies. Recently, we have developed an efficient core synthesis/enzymatic extension (CSEE) strategy for the rapid preparation of N-glycan libraries.¹⁰ In this study, a similar strategy was successfully applied for HMO synthesis. Briefly, 3 core oligosaccharides with 1 or 2 GlcNAc residue(s) at the nonreducing end were first synthesized by the convergent assembly of 3 simple building blocks followed by extension of the cores by 4 robust glycosyltransferases to produce a library of 31 HMOs (Figure 1).

Figure 1.

HMOs synthesized by the core synthesis/enzymatic extension (CSEE) strategy starting with 3 chemically prepared core structures (boxed).

RESULTS AND DISCUSSION

Convergent Core Synthesis

Previous studies highlighted the complexity and challenges associated with synthesizing HMOs via a block coupling strategy. Schmidt developed the sequential synthesis of lactose-containing oligosaccharides, including HMO lacto-N-tetraoside based on the solid-phase synthesis concept.^9d Madsen used one-pot glycosylations to achieve several human milk oligosaccharides.^9c Both methods can synthesize linear and simple oligosaccharides with obvious limitations in achieving more complex HMOs, especially highly branched ones. In this study, we developed an efficient and versatile methodology that utilized oligosaccharyl thioethers and oligosaccharyl bromides as convergent donors for glycosylation, enabling branching assembly in one or two steps of glycosylations with excellent stereoselectivity and yields.

We envisaged that protected lactose 1 (Figure 2) would be a versatile precursor for the synthesis of core structures, including symmetric and asymmetric ones, as C4,C6-hydroxy groups (OH) on Gal are protected by benzylidene and the C3-OH is unprotected for chemical glycosylation. In order to achieve the selective protection of building block 1, the C3-OH group was selectively protected by 4-methoxybenzyl ether (PMB) using standard conditions, followed by C4,C6-OH protection with a benzylidene group. In order to furnish the target cores, two oligosaccharyl thioethers and oligosaccharyl bromide were prepared (Figure 2).

Figure 2.

Three versatile building blocks for the assembly of the three core structures.

Synthesis of Building Blocks

The synthesis of precursor 1 began with lactose peracetate, which was converted to the β-lactoside 4 by reaction with benzyl alcohol in the presence of BF₃· Et₂O, followed by deacetylation with NaOMe/MeOH to furnish compound 5. Then 5 was treated with dibutyltin oxide, followed by a reaction with 4-methoxybenzyl chloride to provide a selectively 3′-O-PMB protected lactoside in fair yield, which has been extensively studied.¹¹ The following benzylidene protection on 4′,6′-OH was conducted with benzaldehyde dimethyl acetal, catalyzed by camphorsulfonic acid (CSA), to give compound 6. The perbenzylation of the remaining hydroxyls of 6 was performed by using sodium hydride and benzyl bromide in anhydrous DMF to give compound 7. After the PMB protecting group of 7 was removed by treatment with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), the synthesis of building block 1 was achieved (Scheme 1).

Scheme 1. Synthesis of Building Block 1^a.

^aReagents and conditions: (a) (i) BF₃·Et₂O, BnOH, DCM, 4 Å MS, (ii) NaOMe, MeOH, 50% over two steps; (b) (i) n-Bu₂SnO, MeOH, reflux, (ii) PMBCl, TBAI, toluene, 4 Å MS reflux, 40%; (c) PhCH(OMe)₂, DMF, CSA, 75%; (d) BnBr, NaH, DMF, 95%; (e) DDQ, DCM/PBS Buffer 9/1, 90%.

The lactosamine building block 3 was initially envisioned to be synthesized using a straightforward fashion by coupling monosaccharides 9 and 10 (Scheme 2A). Unfortunately, the desired product was isolated in minor amounts by silica gel chromatography, and a substantial amount of byproduct 11 was generated through a thioether migration reaction.¹² Therefore, a Koenigs–Knorr reaction coupling approach was carried out by installing the C4 hydroxy group of glucosamine with high yield (Scheme 2B). Building block 2 can be conveniently used in the synthesis of two asymmetric core oligosaccharides.

Scheme 2. Synthesis of Building Block 3: (A) Failed Reaction; (B) Successful Reaction^a.

^aReagents and conditions: (a) Et₃SiH, TfOH, 4 Å MS, −78 °C, 85%; (b) TMSOTf, DCM, 4 Å MS, −20 °C; (c) AgOTf, DCM/toluene, 70%.

Synthesis of Core Oligosaccharides

With all of the building blocks in hand, we began to assemble the three core oligosaccharides using our convergent strategy. The synthesis of HMO1 was depicted in Scheme 3. Disaccharide 13 was obtained in good yield (85%) by selective opening of the benzylidene ring at C6 of building block 1 using Et₃SiH/PhBCl₂. Oligosaccharyl bromide 2 was freshly prepared using HBr/AcOH. Silver triflate (AgOTf) promoted glycosylation of 13 with donor 2 in dichloromethane resulted in the formation of tetrasaccharide 14 in an excellent yield of 85%. Deprotection of 14 with ethylenediamine, followed by treatment with acetic anhydride, furnished the peracetylated tetrasaccharide 15 in 75% yield. O-Deacetylation of compound 15 was performed under Zemplén conditions, followed by the global deprotection of the benzyl group (Bn) by catalytic hydrogenolysis with Pd(OH)₂/H₂ in MeOH/H₂O (10/1). The core structure HMO1 was produced in a total yield of 80% over the two steps.

Scheme 3. Synthesis of HMO1^a.

^aReagents and conditions: (a) Et₃SiH, PhBCl₂, 4 Å MS, DCM, −78 °C, 85%; (b) 2, AgOTf, 2,4,6-collidine, −20 °C, 85%; (c) (i) ethanediamine, n-BuOH, (ii) Ac₂O, pyridine, DMAP; (d) (i) NaOMe/MeOH 75% over two steps, (ii) H₂, Pd(OH)₂/C, 80% over two steps.

Glycosylation of 3′-O-unprotected acceptor 1 with donor 3 proceeded at −20 °C under AgOTf/NIS conditions to furnish the desired tetrasaccharide 16 in 85% yield. Then selective opening of the benzylidene ring at C6 of 16 using Et₃SiH/PhBCl₂ provided 6′-O-unprotected acceptor 17 in 80% yield. The fully protected pentasaccharide was initially attempted to be synthesized by a convergent glycosylation of acceptor 17 and thiol donor 18. Unfortunately, the desired product was not detected by TLC and ESI mass spectrometry analysis (Scheme 4A). Several other donors, including oligosaccharyl trichloroacetimidate and thioether donors, were tried to install the pentasaccharide, but no product was detected. The main challenge should be due to the bulky benzyl group at the 4′-OH position, which has very large steric hindrance and stops the glycosylation on the 6′-OH position, even though the primary alcohol is very nucleophilic. Therefore, 4′,6′-O-unprotected lacto-N-tetraose 19 was proposed as an acceptor for the glycosylation. Removal of the 4′,6′-O-benzylidene of 16 by treatment with ethanethiol in the presence of TsOH afforded acceptor 19. Glycosylation of acceptor 19 with glycosyl bromide 2 to give the protected target pentasaccharide 20 proceeded smoothly and regioselectively by use of AgOTf as a Lewis acid at −20 °C in an excellent yield (85%). The two phthalimides of 20 were then converted into acetamides 21, followed by the global deprotection of Ac and Bn groups. The core oligosaccharide HMO2 was produced in a total yield of 53% over four steps (Scheme 4B).

Scheme 4. Synthesis of HMO2: (A) Failed Reaction; (B) Successful Reaction^a.

^aReagents and conditions: (a) NIS, TfOH, DCM, 4 Å MS, −20 °C, 85%; (b) Et₃SiH, PhBCl₂, DCM, −78 °C, 80%; (c) NIS, AgOTf, DCM, 4 Å MS, −20 °C; (d) TsOH, EtSH, DCM, 90%; (e) 2, AgOTf, 2,4,6-collidine, DCM, 4 Å MS, −20 °C, 85%; (f) (i) ethanediamine, n-buOH, (ii) Ac₂O, pyridine, DMAP 62% over two steps; (g) (i) NaOMe/MeOH, (ii) H₂, Pd(OH)₂/C, 80% over two steps.

AgOTf promoted glycosylation of 3′-O-unprotected acceptor 1 with donor 2 proceeded at −20 °C to furnish the trisaccharide 22 in a good yield of 85%. Then deprotection of the 4′,6′-O-benzylidene of 22 by treatment with EtSH/TsOH provided the dialcohol 23. Glycosylation of acceptor 23 with thiol donor 3 by treatment with AgOTf/NIS at −20 °C gave the protected target pentasaccharide 24 in 70% yield. The two phthalimides of 24 were then converted into acetamides 25. Complete deprotection of 25 was achieved by hydrogenolytic debenzylation (Pd(OH)₂/C, H₂) and complete de-O-acetylation using sodium methoxide in methanol, resulting in core oligosaccharide HMO3 in a total yield of 67% over four steps (Scheme 5).

Scheme 5. Synthesis of HMO3^a.

^aReagents and conditions: (a) AgOTf, 2,4,6-collidine, DCM, 4 Å MS, 85%; (b) TsOH, EtSH, DCM, 80%; (c) 3, NIS, AgOTf, DCM, 4 Å MS, −20 °C, 70%; (d) (i) ethanediamine, n-BuOH, (ii) Ac₂O, pyridine, DMAP, 70% over two steps; (e) (i) NaOMe, MeOH; (ii) Pd(OH)₂, H₂, 95% over two steps.

Enzymatic Extension of Core Structures

A total of 31 HMOs were enzymatically synthesized starting from the 3 core structures (HMO1, HMO2, HMO3) via an enzymatic extension approach using 4 robust GTs: β-1,4-galactosyltransferase from Neisseria meningitidis (LgtB),¹³ α-2,6-sialyltransferase from Photobacterium damselae (Pd2,6ST),¹⁴ C-terminal 66 amino acid truncated α-1,3-fucosyltransferase from Helicobacter pylori (Hpα1,3FT),¹⁵ and α-1,2-fucosyltransferase from Helicobacter mustelae (Hmα1,2FT).¹⁶ All GTs were obtained from bacteria and had high expression levels in Escherichia coli, high activity, and relatively relaxed substrate tolerance. As shown in Figure 3A, glycans HMO11–HMO16 were prepared by starting with the chemically prepared core HMO1. Briefly, in a 2 mL reaction system, 30 mg of HMO1 (20 mM) was incubated with Gal (20 mM), MgCl₂ (20 mM), ATP (20 mM), UTP (20 mM), and varying amounts of BiGalK, BiUSP, and LgtB¹⁷ (Figure 4). After overnight reaction, the reaction was terminated by boiling for 10 min and analyzed by MALDI-MS, which shows a single peak at m/z 1095.748, corresponding to HMO11 [M + Na]⁺. Meanwhile, on the HPLC-ELSD (evaporative light scattering detector) profile, a new peak (TR = 11.946 min) was observed. The reaction mixture was purified by HPLC using a water/acetonitrile gradient elution, giving 40 mg of HMO11 (93% yield). The purified HMO11 (99% pure) was then utilized for the syntheses of HMO12–HMO16 (Figure 3A) catalyzed by Pd2,6ST, and α1,2FT, α1,2FT respectively (see the Supporting Information for details). Interestingly α1,3FT can specifically distinguish the GlcNAc from terminal Galβ1,4GlcNAc and α1,2FT preferentially attaches to terminal Gal. We basically use this feature to biosynthesize Lewis X (Le^x) and Lewis Y (Le^y). In addition, the difucosylated LacNAc motif (Fucα1, 2-Gal-β1, 4-(Fucα1,3-)GlcNAc) was also generated while using Hmα1,2FT. The syntheses of asymmetric biantennary HMO2x and HMO3x (Figure 3B,C) were carried out by enzymatic extension of core HMO2 and HMO3. Asymmetric core HMO2 and HMO3 can more efficiently take advantage of different substrate specificities of GTs over symmetric HMO1 via coupling of Gal to the terminal GlcNAc of one antennary. For example, to obtain HMO311, Gal from the β6GlcNAc branch was sequentially extended by Hmα1,2FT, Hpα1,3FT, and LgtB (Figure 3C). In contrast, HMO310 was sequentially synthesized by Hmα1,2FT, LgtB, and Hpα1,3FT (Figure 3C). Such synthetic routes were designed according to the substrate specificities of GTs to avoid undesirable glycosylation. Each glycosylation step is further described in detail in Figure 4.

Figure 3.

Enzymatic extension of human milk oligosaccharides.

Figure 4.

(a) LgtB, BiGalK, AtUSP, Gal, ATP, UTP, Mg²⁺. (b) Pd2,6ST, NmCSS, Neu5Ac, CTP, Mg²⁺. (c) Pd2,6ST, NmCSS, Neu5Gc, CTP, Mg²⁺. (d) Hpα1,3FT, GDP-Fuc, Mn²⁺. (e) Hmα1,2FT, GDP-Fuc, Mn²⁺. Abbreviations: LgtB, Neisseria meningitidis β-1,4-galactosyltransferase; BiGalk, Bifidobacterium infantis galactokinase; AtUSP, Arabidopsis thaliana pyrophosphorylase; Pd2,6ST, Photobacterium damselae α-2,6-sialyltransferase; NmCSS, Neisseria meningitidis CMP-sialic acid synthetase; Hpα1,3FT, C-terminal 66 amino acids truncated Helicobacter pylori α-1,3-fucosyltransferase; Hmα1,2FT, Helicobacter mustelae α-1,2-fucosyltransferase.

To avoid tedious purification steps of producing much more complex HMO structures, one-pot multienzyme systems (OPME) were used for LgtB and Pd2,6ST catalyzed glycosylation steps (Figure 4a–c).¹⁸ LgtB, which can specifically recognize terminal GlcNAc, was combined with enzymes (BiGalK and AtUSP) involved in the biosynthesis of their corresponding sugar nucleotides (UDP-Gal) to produce the target glycan with the desired β1,4 linkage (Figure 4a). Similarly, Pd2,6ST was combined with NmCSS, a CMP-sialic acid synthetase, to form the α-2,6 configuration (Figure 4b,c). Both Hmα1,2FT and Hpα1,3FT simply used pure GDP-Fuc to form α-1,2 and α-1,3 linkages, respectively (Figure 4d,e).

CONCLUSION

In summary, we have utilized our well-developed CSEE strategy for the efficient synthesis of a library of structure-defined HMOs, which was assisted by rapid HPLC purification. The combination of CSEE and HPLC purification allowed us to deliver 31 diverse, high-purity homogeneous HMOs. These HMOs are valuable materials for bioactivity evaluations as well as glycan analyses. In this work, oligosaccharyl thioethers and oligosaccharyl bromide were consistently utilized as chemical glycosylation donors for the convergent installation of branched lactose-terminated antennae. This general and efficient method furnished 3 core oligosaccharides with high stereoselectivity and excellent yields. This work further confirmed that any GlcNAc-terminated glycan could be extended to 5 or more glycans, including Le^X and SLe^X, which are very important epitopes in glycobiology. The CSEE strategy demonstrated a practical way to harvest diverse and complex HMOs with defined structures for various applications. The “mass” production of more homogeneous HMOs and bioactivity evaluations are underway.

EXPERIMENTAL SECTION

General Methods

All chemicals were purchased as reagent grade and used without further purification. Anhydrous dichloromethane (CH₂Cl₂), acetonitrile (CH₃CN), tetrahydrofuran (THF), N,N-dimethylformamide (DMF), toluene, and methanol (MeOH) were purchased from a commercial source without further distillation. Pulverized molecular sieves MS-4 Å (Aldrich) for glycosylation was activated by heating at 350 °C for 3 h. All reactions were performed with dry solvents and under nitrogen unless otherwise stated. Thin-layer chromatography (TLC) with 60 F₂₅₄ silica gel plastic plates was visualized under UV (254 nm) and/or by staining with a solution of 10 mL of anisaldehyde and 10 mL of 95% H₂SO₄ in 400 mL of ethanol, followed by heating on a hot plate. Column chromatography was carried out on silica gel (EMD 230–400 mesh ASTM) and P2 gel. Optical rotation values were measured using a polarimeter at ambient temperature in the specified solvents. ¹H NMR spectra were recorded on a 400 or 500 MHz NMR spectrometer at 25 °C. All ¹H chemical shifts (in ppm) were assigned according to CDCl₃ (δ 7.24 ppm) or D₂O (δ 4.79 ppm). ¹³C NMR spectra were obtained with a 400 MHz NMR spectrometer and calibrated with CDCl₃ (δ 77.00 ppm). Coupling constants (J) are reported in hertz (Hz). Splitting patterns are described using the following abbreviations: s, singlet; br s, broad singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; m, multiplet. ¹H NMR spectra are reported in the following order: chemical shift, multiplicity, coupling constant(s), and number(s) of protons. All NMR signals were assigned on the basis of ¹H NMR and ¹³C NMR experiments. High-resolution MALDI mass spectra were recorded on an ESI-Orbitrap or MALDI-TOF spectrometer.

Neu5Ac and Neu5Gc were purchased from Carbosynth Limited. ATP and CTP were purchased from Sigma. Thermosensitive alkaline phosphatase from shrimp (FastAP) was purchased from Thermo Scientific. Other enzymes, including Neisseria meniningitidis β1,4-galactosyltransferase (NmLgtB), α-2,6-sialyltransferase from Photobacterium damselae (Pd2,6ST), C-terminal 66 amino acid truncated Helicobacter pylori α-1,3-fucosyltransferase (Hpα1,3FT), Helicobacter mustelae α-1,2-fucosyltransferase (Hmα1,2FT), and CMP-sialic acid synthetase from N. meningitides (NmCSS), were expressed and purified as previously described. Enzymes were then desalted against 50 mM Tris-HCl, 100 mM NaCl, and 50% glycerol and stored at −20 °C for long-term use. Sugar nucleotide guanoside 5′-diphospho-l-fucose (GDP-Fuc) was prepared as described in our previous paper.¹⁹

General Procedures

A. Transformation of N-Phth to NHAc

A mixture of N-Phth protected oligosaccharide was dissolved in n-BuOH at room temperature, followed by addition of ethylenediamine (n-BuOH/ethylenediamine 2/1). After it was stirred at 90 °C for 12 h, the mixture was evaporated in vacuo to give a residue for the next step without further purification. To a solution of the residue in pyridine was added Ac₂O. After it was stirred at room temperature for 12 h, the solution was diluted with EtOAc and washed with aqueous 1 M HCl, saturated aqueous NaHCO₃, and brine solution. The organic layer was dried over Na₂SO₄, filtered, and evaporated in vacuo to give a residue, which was purified by silica gel column chromatography to give the NHAc compound.

B. Deacetylation

An Ac-protected oligosaccharide was dissolved in MeOH, and NaOMe in MeOH was added until the pH was about 10. After it was stirred at room temperature for 12 h, the solution was neutralized with ion exchange resin (H⁺) and then filtered. The residue was concentrated in vacuo to afford the desired deacetylated product.

C. Deprotection of Benzyl Group

Pd(OH)₂ on carbon was added to a solution of protected oligosaccharide in MeOH/H₂O (10/1). The mixture was stirred under 1 atm of hydrogen. After it was stirred for 24 h, the mixture was filtered through a PTFE syringe filter and concentrated in vacuo. The residue was purified by Bio-Gel P-2 (BIO-RAD) column chromatography using water as eluent. The product was then lyophilized to give the target compound as a white powder.

D. Production of Oligosaccharyl Bromide

Peracetylated oligosaccharide was added portionwise to a stirred solution of HBr (33%) in glacial acetic acid (20.0 mL) at 0 °C. After all the sugar had been added, the reaction mixture was stirred at room temperature for 45 min. TLC analysis (hexanes/ethyl acetate 1/1) indicated formation of the product and consumption of starting material. Then the reaction was quenched with ice water (200 mL) and the product extracted with DCM (2 × 200 mL). The combined organic extracts were washed with a solution of NaHCO₃ (aqueous saturated, 2 × 200 mL), dried with Na₂SO₄, filtered, and then concentrated in vacuo. The crude product was used without further purification.

E. Enzyme Treatment and HPLC Purification

In general, 31 HMOs were enzymatically synthesized by 4 glycosyltransferases (NmLgtB, Pd2,6ST, Hpα 1,3FT, Hmα 1,2FT) under the nearly same reaction conditions. Reaction mixtures contain 50 mM Tris-HCl (pH 8.0), 10 mM acceptor HMOs, 12 mM sugar nucleotide (or its corresponding synthetase), 10 mM MnCl₂, and varying amounts of glycotransferases. FastAP (1 U/200 µL) was also added to digest the reaction byproduct UDP to drive the reaction forward. Reaction mixtures were incubated at 37 °C overnight and monitored by HILIC-ELSD (Waters XBridge BEH amide column, 130 Å, 4.6 mm × 250 mm under a gradient running condition (solvent A, 100 mM ammonium formate, pH 3.4; solvent B, acetonitrile; flow rate 1 mL/min; B% 65–50% within 25 min)). The desired products were detected by a highly efficient ELSD (evaporative light scattering detector), which increases the sample concentration to minimize the noise and deliver higher sensitivity. After over 90% of the acceptor was converted, the reaction was quenched by boiling for 10 min, followed by concentration with a rotary evaporator. HPLC-A210 nm was then used to purify target HMOs using a semipreparative column (Waters XBridge BEH amide column, 130 Å, 5 µm, 10 mm × 250 mm) under the following gradient running conditions: solvent A, 100 mM ammonium formate, pH 3.4; solvent B, acetonitrile; flow rate 4 mL/min; B%: 65–50% within 25 min.¹⁰ MS data for purified HMOs were obtained by ESI-MS and MALDI-MS.

Benzyl O-(4,6-O-Benzylidene-3-O-(4-methoxybenzyl)-β-d-galactopyranosyl)-(1→4)-β-d-glucopyranoside (6)

A suspension of benzyllactose 5 (12.0 g, 27.78 mmol) and Bu₂SnO (7.6 g, 30.54 mmol) in anhydrous MeOH (100 mL) was heated to reflux and stirred for 8 h. The solvent was removed in vacuo. Then the residue was dissolved in dry toluene (100 mL). p-Methoxybenzyl chloride (3.76 mL, 20.37 mmol), tetrabutylammonium iodide (2.05 g, 11.10 mmol), and 4 Å molecular sieves (5 g) were added. The resulting mixture was heated to reflux for another 8 h and then cooled to room temperature. The suspension was filtered through a Celite pad, and the filtrate was concentrated and chromatographied (dichloromethane/methanol 6/1) to afford 9.2 g of crude product (60% yield).

Benzaldehyde dimethyl acetal (2.75 mL, 18.33 mmol) was added to a solution of the above crude product (7.8 g, 14.10 mmol) in anhydrous dimethylformamide (100 mL), and then camphorsulfonic acid was added to adjust the pH to about 2.0–3.0. The reaction mixture was stirred overnight and then quenched with triethylamine. The mixture was concentrated under vacuum. The residue was purified by flash column chromatography (dichloromethane/methanol 10/1) to give 6 as a white solid (8.47 g, 87.0%). [α]_D²⁰ = +6.7 (c 1.0, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 7.48–7.51 (dd, 2H), 7.28–7.38 (m, 10 H), 6.85–6.87 (m, 2 H), 5.34 (s, 1 H), 4.87 (d, J = 11.95 Hz, 1 H), 4.59–4.63 (m, 4 H), 4.45 (d, J = 7.8 Hz, 1 H), 4.36 (d, J = 8.1 Hz, 1 H), 4.21 (d, J = 14.0 Hz, 1 H), 4.10 (s, 1 H), 3.92–4.00 (m, 3 H), 3.79–3.89 (m, 2 H), 3.78 (s, 3 H), 3.60–3.69 (m, 3 H), 3.40–3.50 (m, 2 H), 3.27–3.40 (m, 4 H). ¹³C NMR (CDCl₃, 100 MHz): δ 137.6,137.2, 130.0,129.5, 128.5, 128.3, 128.2, 127.9, 126.3, 113.9, 103.6, 101.8, 101.1, 78.8, 74.9,74.7, 73.5, 72.7, 71.3, 71.2, 69.1, 69.0, 66.9, 61.9, 55.3. HRMS (ESI-Orbitrap): [M + Na]⁺ calcd for C₃₄H₄₀NaO₁₂ 663.2417, found 663.2420.

Benzyl O-(2-O-Benzyl-4,6-O-benzylidene-3-O-(4-methoxybenzyl)-β-d-galactopyranos-yl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (7)

NaH (60%; 2.25 g, 56.25 mmol) and BnBr (6.66 mL, 56.25 mmol) were added to a stirred solution of 6 (6.0 g, 9.38 mmol) in DMF (60 mL) cooled to 0 °C. The solution turned light yellow. The reaction mixture was maintained at room temperature for 4 h. Then the solution was quenched with MeOH. The mixture was diluted with EtOAc and washed with water. The organic layer was dried with Na₂SO₄ and concentrated. The residue was purified on a silica gel column (hexanes/EtOAc 6/1) to afford the product 7 (1.85 g, 92.5%) as a white powder. [α]_D²⁰ = +7.4 (c 1.0, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 7.57–7.59 (m, 2 H), 7.51–7.54 (m, 2 H), 7.42–7.45 (m, 2 H), 7.31–7.38 (m,23 H), 7.23–7.24 (m, 2 H), 6.90–6.92 (m, 2 H), 5.51 (s, 1H), 5.25 (d, J = 11.0 Hz, 1 H), 4.98–5.03 (m, 2 H), 4.90 (d, J = 11.1 Hz, 1 H), 4.79–4.85 (m, 3 H),4.71–4.74 (m, 3 H), 4.63 (d, J = 12.0 Hz, 1 H), 4.52–4.55 (m, 2 H), 4.43 (d, J = 12.0 Hz, 1 H), 4.26 (dd, J = 1.4 Hz, 12.4 Hz, 1 H), 4.04–4.08 (m, 2 H), 3.96 (dd, J = 4.2, 11.3 Hz, 1 H), 3.90 (dd, J = 1.8, 12.5 Hz, 1 H), 3.85 (s, 3 H), 3.79–3.83 (m, 2 H), 3.69 (t, J = 8.8 Hz, 1 H), 3.56–3.60 (m, 1 H), 3.40–3.46 (m, 2 H). ¹³C NMR (CDCl₃, 100 MHz): δ 159.3, 139.0, 139.0, 138.7, 138.6, 138.2, 137.6, 130.5, 129.4, 128.9, 128.7, 128.4, 128.3, 128.1, 128.0, 127.8, 127.8, 127.6, 127.6, 127.5, 127.4, 127.3, 126.6, 113.8, 103.0, 102.6, 101.4, 83.1, 81.9, 79.4, 78.9, 77.7, 75.9, 75.3, 75.2, 75.1, 73.8, 73.0, 71.4, 71.1, 69.0, 68.3,66.4, 55.3. HRMS (ESI-Orbitrap): [M + Na]⁺ calcd for C₆₂H₆₄NaO₁₂ 1023.4295, found 1023.4285.

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

2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (2.75 g, 12.10 mmol) was added to a solution of 7 (6.0 g, 6.05 mmol) in 9/1 CH₂Cl₂/phosphate-buffered saline (200 mL). The solution was stirred for 1.5 h at room temperature and diluted with CH₂Cl₂. The solution was washed with aqueous NaHCO₃ and brine, dried over Na₂SO₄, and concentrated. The residue was purified on a silica gel column (hexanes/EtOAc 5/1) to afford the product 1 (5.02 g, 95%) as a white powder. [α]_D²⁰ = +15.4 (c 1.0, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 7.52–7.55 (m, 4 H), 7.20–7.45 (m, 26 H), 5.56 (s, 1 H), 5.21 (d, J = 8.0 Hz 1 H), 4.97–5.02 (m, 2 H), 4.78–4.83 (m, 4 H), 4.65–4.73 (m, 2 H), 4.51–4.54 (m, 2 H), 4.46 (d, J = 12.3 Hz, 1 H), 4.30 (d, J = 12.3 Hz, 1 H), 4.06–4.13 (m, 2 H), 3.93–3.97 (m, 2 H), 3.79 (dd, J = 1.3, 11.0 Hz, 1 H), 3.68 (m, 1 H), 3.55–3.60 (m, 3 H), 3.38–3.41 (m, 1 H), 3.14 (s, 1 H). ¹³C NMR (CDCl₃, 100 MHz): δ 138.9, 138.7, 138.6, 138.5, 137.9, 137.5, 129.2, 128.8, 128.4, 128.2, 128.0, 127.8, 127.6, 127.4, 126.5, 102.8, 102.6, 101.5, 83.1, 81.9, 80.2, 77.6, 75.9, 75.7, 75.2, 75.1, 73.1, 72.9, 71.0, 68.9, 68.2, 66.5. HRMS (ESI-Orbitrap): [M + Na]⁺ calcd for C₅₄H₅₆NaO₁₁ 903.3720, found 903.3725.

Ethyl 3,6-Di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-d-glucopyranoside (9)

A mixture of compound 8 (2 g, 4.09 mmol) and 4 Å molecular sieves (2 g) in dry CH₂Cl₂ was stirred at room temperature under nitrogen for 2 h. Triethylsilane (2.1 mL, 13.1 mmol) and TfOH (1.05 mL, 11.9 mmol) were sequentially added at −78 °C. The reaction mixture was stirred at −78 °C for 2 h and then quenched with MeOH (2 mL) and Et₃N (2 mL). The resulting mixture was filtered. The filtrate was diluted with CH₂Cl₂, washed with aqueous NaHCO₃ and brine, dried over Na₂SO₄, and concentrated. The residue was purified on a silica gel column (hexanes/EtOAc 5/1) to afford the product 9 (1.85 g, 92.5%) as a white powder. [α]_D²⁰ = +91.0 (c 1.0, CHCl₃). ¹H NMR (CDCl₃, 400 MHz): δ 7.82 (d, J = 5.9 Hz, 1 H), 7.66–7.72 (m, 3 H), 7.28–7.42 (m, 5 H), 7.03–7.10 (m, 2 H), 6.92–7.00 (m, 3 H), 5.32 (d, J = 9.9 Hz, 1 H), 4.80 (d, J = 12.0 Hz, 1 H), 4.54–4.70 (m, 3 H), 4.25–4.34 (m, 2 H), 3.80–3.89 (m, 3 H), 3.70–3.73 (m, 1 H), 2.59–2.72 (m, 2 H), 1.19 (t, J = 7.3 Hz, 3 H). ¹³C NMR (CDCl₃, 100 MHz): δ 168.1, 167.6, 138.2, 137.7, 134.0, 133.9, 131.6, 128.5, 127.9, 127.9, 127.8, 127.5, 123.5, 123.3, 81.2, 79.7, 78.0, 74.5, 74.2, 73.8, 70.7, 54.5, 24.0, 15.0. HRMS (ESI-Orbitrap): [M + Na]⁺ calcd for C₃₀H₃₁NNaO₆S 556.1770, found 556.1760.

Ethyl 2,3,4,6-Tetra-O-acetyl-β-d-glucopyranosyl-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-d-glucopyranoside (3)

2,3,4,6-Tetra-O-acetyl-β-d-galactosyl bromide (3.35 g, 8.17 mmol) was prepared by following general procedure D. Then the bromide donor (3.35 g, 8.17 mmol) and 3,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-d-glucopyranoside 9 (2.64 g. 5.43 mmol) were dissolved in a mixture of dry toluene and CH₂Cl₂ (1/1, 30 mL). Powdered molecular sieves (4 Å) were added, and the mixture was stirred under nitrogen for 1 h. The flask was wrapped in aluminum foil and cooled to −45 °C. AgOTf (2.79 g, 10.86 mmol) dissolved in dry toluene (20 mL) was added during 1 h with the exclusion of light. After additional stirring for 30 min at −45 °C, the reaction mixture was quenched by aqueous Na₂S₂O₃. The mixture was transferred to a separatory funnel via a Celite-packed glass filter funnel. The organic phase was separated, dried with Na₂SO₄, filtered, and concentrated. Purification of the residue by silica gel column chromatography (hexanes/EtOAc 4/1) gave compound 3 (5.81 g, 80%). [α]_D²⁰ = +31.0 (c 1.0, CHCl₃). ¹H NMR (CDCl₃, 400 MHz): δ 7.77 (d, J = 6.3 Hz, 1 H), 7.62–7.66 (m, 3 H), 7.29–743(m, 5 H), 7.01(d, J = 7.0 H), 6.82–6.92 (m, 3 H), 5.13–5.28 (m, 3 H), 4.78–4.87 (m, 3 H), 4.62 (d, J = 7.9 Hz, 1 H), 4.43–4.51 (m, 2 H), 4.20–4.28 (m, 2 H), 4.09 (t, J = 9.5 Hz, 1 H), 3.89–4.02 (m, 2 H), 3.79 (s, 2 H), 3.64 (t, J = 6.8 Hz, 1 H), 3.56 (d, J = 10.0 Hz, 1 H), 2.55–2.75 (m, 2 H), 2.06 (s, 3 H), 2.02 (s, 6 H), 1.97 (s, 3 H). ¹³C NMR (CDCl₃, 100 MHz): δ 170.3, 170.2, 170.0, 169.2, 167.9, 167.4, 138.5, 137.9, 133.9, 133.7, 131.6, 128.6, 128.0, 127.9, 127.9, 127.1, 123.4, 123.3, 100.3, 81.1, 79.1, 77.8, 77.6, 74.5, 73.6, 70.4,69.5, 67.7, 66.9, 60.7, 54.7, 23.9, 20.8, 20.7, 20.6, 20.6, 14.9. HRMS (ESI-Orbitrap): [M + Na]⁺ calcd for C₄₄H₄₉NNaO₁₅S 886.2721, found 886.2729.

Benzyl O-(2,4-Di-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (13)

A mixture of compound 1 (2 g, 2.27 mmol) and 4 Å molecular sieves (2 g) in dry CH₂Cl₂ was stirred at room temperature under nitrogen for 2 h. Triethylsilane (0.69 mL, 4.34 mmol) and PhBCl₂ (0.56 mL, 4.34 mmol) were sequentially added at −78 °C. The reaction mixture was stirred at −78 °C for 2 h and then quenched by the addition of MeOH (2 mL) and Et₃N (2 mL). The resulting mixture was filtered. The filtrate was diluted with CH₂Cl₂, washed with aqueous NaHCO₃ and brine, dried over Na₂SO₄, and concentrated. The residue was purified on a silica gel column (hexanes/EtOAc 5/1) to afford the product 13 (1.70 g, 85%) as a white powder. [α]_D²⁰ = +8.9 (c 1.0, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 7.35–7.52 (m, 27 H), 7.26–7.34 (m, 3 H), 5.03–5.16 (m, 3 H), 4.78–4.97 (m, 6 H), 4.70–4.75 (m, 2 H), 4.59–4.65 (m, 2 H), 4.52 (d, J = 7.0 Hz, 1 H), 4.08 (t, J = 8.7 Hz, 1 H), 3.92 (d, J = 2.6 Hz, 2 H). ¹³C NMR (CDCl₃, 100 MHz): δ 138.9, 138.7 (2 C), 138.5, 137.6, 128.6, 128.6, 128.5, 128.5, 128.4, 128.1, 127.9, 127.8, 127.7, 127.6, 102.8, 102.6, 82.8, 81.9, 80.5, 77.0, 75.8, 75.5, 75.3, 75.2 (2 C), 75.1, 74.4, 73.4, 71.1, 68.4,61.7. HRMS (ESI-Orbitrap): [M + Na]⁺ calcd for C₅₄H₅₈NaO₁₁ 905.3877, found 905.3867.

Benzyl O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)-(1→3)-[3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl]-(1→6)-(2,4-di-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (14)

3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-d-glucosyl bromide was prepared by following the general procedure D. Powdered molecular sieves (4 Å; 3.0 g) was added to a solution of the above bromide donor (4.80 g, 5.44 mmol) and 13 (800 mg, 0.907 mmol) in anhydrous dichloromethane (20 mL). The suspension was stirred under nitrogen for 1.5 h at room temperature and then cooled to −30 °C. Then 2,4,6-collidine (0.72 mL, 5.44 mmol), and freshly dried AgOTf (1.40 g, 5.44 mmol) were sequentially added to the reaction mixture. After it was stirred for 2 h at −30 °C, the mixture was warmed to room temperature overnight, diluted with CH₂Cl₂, and filtered through Celite. The filtrate was diluted with CH₂Cl₂, washed with aqueous NaHCO₃ and brine, dried over Na₂SO₄, and concentrated. The residue was purified on a silica gel column (hexanes/EtOAc 5/1) to afford the product 14 (1.33 g, 85%) as a white powder. [α]_D²⁰ = +6.4 (c 1.0, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 7.85 (t, J = 4.01 Hz, 1 H), 7.72–7.74 (m, 2 H), 7.43–7.56 (m, 2 H), 7.18–7.40 (m, 27 H), 7.08–7.16 (m, 3 H), 6.97 (m, 2 H), 5.76–5.87 (m, 2 H), 5.62 (d, J = 8.24 Hz, 1 H), 5.39 (d, J = 8.43, 1H), 5.16–5.24 (m, 2 H), 4.85–4.93 (m, 4 H), 4.75 (d, J = 10.6 Hz, 1 H), 4.55–4.66 (m, 3 H), 4.35–4.44 (m, 5 H), 4.19–4.33 (m, 5 H), 4.12 (d, J = 12.7 Hz, 1 H), 3.91 (t, J = 10.1 Hz, 1 H), 3.71–3.86 (m, 5 H), 3.47–3.59 (m, 5 H), 3.33–3.47 (m, 2 H), 3.21 (t, J = 5.9 Hz, 1 H), 3.05(dd, J = 3.2, 9.7 Hz, 1 H), 2.09 (s, 3 H), 2.09 (s, 3 H), 2.02 (s, 3 H), 1.98 (s, 3 H), 1.87(d, 6 H). ¹³C NMR (CDCl₃, 100 MHz): δ 170.7, 170.6, 170.1, 170.0, 169.5, 169.5, 139.1, 139.0, 138.6, 138.5, 138.4, 137.7, 134.4, 134.0, 128.4, 128.3, 128.2, 128.2, 128.1, 128.0, 127.8, 127.6, 127.5, 127.4, 127.4, 127.1, 127.0, 126.9, 126.5, 102.4, 102.3, 99.0, 97.5, 83.1, 81.7, 81.7, 78.6, 76.2, 76.1, 75.6, 75.2, 75.0, 74.9, 74.1, 73.1, 72.8 (2 C), 71.7, 71.6, 70.8, 70.8 (2 C), 70.5, 68.9, 68.8, 68.0, 66.8, 61.7, 61.6, 55.1, 54.7, 20.8, 20.7, 20.7, 20.6, 20.5, 20.4. HRMS (ESI-Orbitrap): [M + Na]⁺ calcd for C₉₄H₉₆N₂NaO₂₉ 1739.5996, found 1739.5980.

Benzyl O-(3,4,6-Tri-O-acetyl-2-deoxyacetamido-β-d-glucopyranosyl)-(1→3)-[3,4,6-tri-O-acetyl-2-deoxyacetamido-β-d-glucopyranosyl]-(1→6)-(2,4-di-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (15)

Following the general procedure A compound 14 (1.16 g, 0.66 mmol) yielded the compound 15 (762 mg, 75% over two steps). [α]_D²⁰ = −1.9 (c 0.4, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 7.25–7.48 (m, 27 H), 7.10– 7.18 (m, 3 H), 5.78 (d, J = 9.5 Hz, 1 H), 5.01–5.16 (m, 6 H), 4.90–5.00 (m, 3 H), 4.75–4.91 (m, 4 H), 4.60–4.71 (m, 3 H), 4.41–4.55 (m, 4 H), 4.29 (d, J = 3.5 Hz, 1 H), 3.96–4.15 (m, 5 H), 3.64–3.86 (m, 8 H), 3.52–3.64 (m, 4 H), 3.45–3.51 (m, 1 H), 3.23 (d, J = 9.7 Hz, 1 H), 2.07 (s, 3 H), 2.06 (s, 3 H), 2.05 (s, 3 H), 2.00 (s, 3 H), 1.99 (s, 3 H), 1.98 (s, 3 H), 1.51 (s, 3 H). ¹³C NMR (CDCl₃, 100 MHz): δ 170.9, 170.8, 170.6, 170.5, 170.3, 169.8, 169.3, 169.2, 139.4, 138.9, 138.8, 138.6, 137.9, 137.4, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 126.2, 102.6, 102.5, 102.4, 101.4, 84.3, 82.4, 82.1, 79.5, 76.7, 76.4, 76.2, 75.2, 74.9, 74.7, 74.5, 74.4, 73.3, 73.2, 72.7, 72.0, 70.9, 70.6, 68.8, 68.5, 67.9, 67.5, 62.0, 61.6, 54.4, 53.7, 23.6, 22.8, 20.8, 20.8, 20.7, 20.6 (3 C). HRMS: [M + Na]⁺ calcd for C₈₂H₉₆N₂NaO₂₇ 1563.6098, found 1563.6126.

2-Deoxyacetamido-β-d-glucopyranosyl-(1→3)-[2-deoxyacetamido-β-d-glucopyranos-yl]-(1→6)-β-d-galactopyranosyl-(1→ 4)-α,β-d-glucopyranose (HMO1)

Following the general procedures B and C, compound 15 (400 mg, 0.26 mmol) yielded the compound HMO1 (152 mg, 80% over two steps). ¹H NMR (D₂O, 400 MHz): δ 5.15 (d, J = 3.6 Hz, 0.55 H, Glc-1 H-1 of α form), 4.53–4.62 (m, overlap with D₂O, 2.45 H, GlcNAc-1 H-1, GlcNAc-2 H-1 and Glc-1 H-1 of β form), 4.36 (d, J = 7.7 Hz, 1 H, Gal-1 H-1), 4.07(d, J = 2.4 Hz, 1 H), 3.58–3.95 (m, 13 H), 3.45–3.58 (m, 5 H), 3.32–3.45 (m, 4 H), 3.16–3.26 (m, 1 H), 1.99 (s, 3 H), 1.96 (s, 3 H). ¹³CNMR(D₂O, 100 MHz): δ 174.9, 174.6, 102.9, 102.8 (GlcNAc-1, GlcNAc-2, C-1),101.1 (Gal-1, C-1), 95.7 (Glc-1, C-1 of β form), 91.8 (Glc-1, C-1 of α form), 81.7, 78.8, 75.8 (2 C), 75.6, 73.8, 74.7, 74.3, 73.8, 73.5, 73.4,69.9, 69.8, 69.6, 68.7, 68.4,60.7, 60.5, 55.6, 55.5, 22.4, 22.2. HRMS (ESI-Orbitrap): [M + Na]⁺ calcd for C₂₈H₄₈N₂NaO₂₁ 771.2647, found 771.2665.

Benzyl O-(2,3,4,6-Tetra-O-acetyl-β-d-galactopyranosyl)-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl-(1→3)-(2-O-benzyl-4,6-O-benzylidene-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (16)

Powdered molecular sieves (4 Å; 3.0 g) was added to a solution of compounds 3 (871 mg, 0.96 mmol) and 1 (652 mg, 0.74 mmol) in anhydrous dichloromethane (20 mL). The suspension was stirred under nitrogen for 1.5 h at room temperature and then cooled to −30 °C. Then NIS (260 mg, 1.15 mmol) and TMSOTf (35 µL, 0.19 mmol) were sequentially added to the reaction mixture. After it was stirred for 2 h at −30 °C, the mixture was warmed to room temperature overnight, diluted with CH₂Cl₂, and filtered through Celite. The filtrate was diluted with CH₂Cl₂, washed with aqueous NaHCO₃ and brine, dried over Na₂SO₄, and concentrated. The residue was purified on a silica gel column (hexanes/EtOAc 5/1) to afford the product 16 (1.04 g, 86%) as a white powder. [α]_D²⁰ = +12.9 (c 1.0, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 7.53–7.55 (m, 2 H), 7.45–7.50 (m, 2 H), 7.27–7.45 (m, 27 H), 7.19 (t, J = 3.6 Hz, 2 H), 7.09–7.16 (m, 3 H), 7.02–7.06 (m, 2 H), 6.84–6.94 (m, 5 H), 5.49 (s, 1 H), 5.47 (d, J = 7.4 Hz, 1 H), 5.35 (d, J = 3.2 Hz, 1 H), 5.25 (dd, J = 7.9, 10.4 Hz, 1 H), 5.09 (d, J = 10.7 Hz, 1 H), 4.88–4.99 (m, 3 H), 4.85 (d, J = 12.0 Hz, 1 H), 4.65–4.78 (m, 4 H), 4.61 (d, J = 12.0 Hz, 1 H), 4.44–4.55 (m, 3 H), 4.30–4.39 (m, 4 H), 4.18–4.28 (m, 5 H), 4.09 (t, J = 9.3 Hz, 1 H), 3.98–4.04 (m, 2 H), 3.89–3.96 (m, 1 H), 3.80–3.89 (m 2 H), 3.66–3.79 (m, 3 H), 3.53–3.64 (m, 2 H), 3.44–3.51 (m, 3 H), 3.38 (d, J = 10.1 Hz, 1 H), 2.93–3.02 (m, 2 H), 2.12 (s, 3 H), 2.10 (s, 3 H), 2.05 (s, 3 H), 2.03 (s, 3 H). ¹³C NMR (CDCl₃, 100 MHz): δ 170.3, 170.2, 170.1, 169.2, 139.0, 138.6 (2 C), 138.5, 138.3, 137.8, 137.6, 133.5, 131.3, 128.7, 128.6 (2 C), 128.4, 128.3 (2 C), 128.2, 128.1 (2 C), 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.2, 127.1, 126.7, 126.5, 126.3, 123.1, 102.4 (2 C), 100.7, 100.6, 99.6, 83.1, 81.8, 80.9, 78.4, 77.7, 76.0, 75.7, 75.0, 74.8, 74.7, 74.5, 74.3, 73.8 (2 C), 73.0 (2 C), 71.1, 70.9 (2 C), 70.6, 69.7, 68.8, 68.7, 67.9, 67.0, 66.4,60.8, 55.8, 20.9, 20.7 (2 C), 20.6. HRMS (ESI-Orbitrap): [M + Na]⁺ calcd for C₉₆H₉₉NNaO₂₆ 1704.6353, found 1704.6383.

Benzyl O-(2,3,4,6-Tetra-O-acetyl-β-d-galactopyranosyl)-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl-(1→3)-(2-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (19)

To a solution of compound 16 (800 mg, 0.49 mmol) in anhydrous MeOH (10 mL) were added TsOH (8.4 mg) and EtSH (0.21 mL, 2.93 mmol). The reaction mixture was stirred at room temperature for 6 h and then quenched with triethylamine and evaporated under reduced pressure. The mixture was purified with silica column (hexanes/acetone 5/1) to afford compound 19 as a white powder (702 mg, 90%). [α]_D²⁰ = +18.4 (c 0.5, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 7.38–7.52 (m, 11 H), 7.25–7.36 (m, 16 H), 7.22–7.26 (m, 2 H), 7.03–7.14 (m, 3 H), 6.99–7.03 (m, 2 H), 6.85–6.92 (m, 2 H), 6.78 (d, J = 7.1 Hz, 2 H), 5.29–5.31 (m, 2 H), 5.19–5.25 (m, 1 H), 4.86–4.98 (m, 4 H), 4.83 (d, J = 12.2 Hz, 1 H), 4.68–4.77 (m, 3 H), 4.65 (d, J = 8.3 Hz, 1 H), 4.57–4.62 (m, 2 H), 4.46 (t, J = 12.4 Hz, 2 H), 4.34–4.40 (m, 1 H), 4.26–4.34 (m, 3 H), 4.17–4.26 (m, 3 H), 3.95–4.08 (m, 3 H), 3.81–3.88 (m, 1 H), 3.72–3.79 (m, 3 H), 3.64–3.72 (m, 2 H), 3.47–3.55 (m, 2 H), 3.34–3.46 (m, 5 H), 3.17–3.23 (m, 1 H), 3.00–3.06 (m, 1 H), 2.87 (s, 1 H), 2.11 (s, 3 H), 2.08 (s, 3 H), 2.05 (s, 3 H), 2.02 (s, 3 H). ¹³C NMR (CDCl₃, 100 MHz): δ 170.3, 170.2, 170.1, 169.2, 138.9, 138.4, 138.3 (2 C), 137.6, 137.5, 133.6, 131.1, 128.8, 128.4, 128.3 (3 C), 128.2 (2 C), 128.1, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.2, 126.7, 126.4, 123.2, 102.4, 102.1, 100.6, 99.0, 83.8, 82.8, 81.5, 78.3, 77.9, 76.6, 76.4, 75.6, 74.9, 74.6, 74.3, 73.8, 73.7, 73.1, 71.0, 70.9, 70.7, 69.6, 68.3,67.9, 67.7, 66.9, 62.2, 60.8, 55.6, 20.8, 20.68, 20.6, 20.6. HRMS (ESI-Orbitrap): [M + Na]⁺ calcd for C₈₉H₉₅NNaO₂₆ 1616.6040, found 1616.6065.

Benzyl O-(2,3,4,6-Tetra-O-acetyl-β-d-galactopyranosyl)-(1→4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl-(1→3))-[3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl]-(1→6)-(2-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (20)

3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-d-glucosyl bromide was prepared by following the general procedure D. Powdered molecular sieves (4 Å; 3.0 g) was added to a solution of 19 (460 mg, 0.29 mmol), 2,4,6-collidine (76 µL, 0.58 mmol), and freshly dried AgOTf (150 mg, 0.58 mmol) in anhydrous dichloromethane (20 mL). The suspension was stirred under nitrogen for 1.5 h at room temperature and then cooled to −30 °C. Then a solution of the above bromide donor (460 mg, 0.29 mmol) in dichloromethane (5.0 mL) was added dropwise during 30 min to the reaction mixture. After it was stirred for 2 h at −30 °C, the mixture was warmed to room temperature overnight, diluted with CH₂Cl₂, and filtered through Celite. The filtrate was diluted with CH₂Cl₂, washed with aqueous NaHCO₃ and brine, dried over Na₂SO₄, and concentrated. The residue was purified on a silica gel column (hexanes/EtOAc 5/1) to afford the product 20 (495 mg, 85%) as a white powder. [α]_D²⁰ = +11.7 (c 1.0, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 7.76–7.91 (m, 4 H), 7.49–7.56 (m, 2 H), 7.41–7.49 (m, 8 H), 7.14–7.20 (m, 2 H), 7.05–7.11 (m, 1 H), 6.97–7.04 (m, 4 H), 6.85–6.94 (m, 3 H), 6.75 (d, J = 7.4 Hz, 2 H), 5.73 (dd, J = 9.1, 10.61 Hz, 1 H), 5.43 (d, J = 8.3 Hz, 1 H), 5.33 (d, J = 3.5 Hz, 1 H), 5.12–5.25 (m, 2 H), 4.99 (d, J = 8.9 Hz, 1 H), 4.77– 4.95 (m, 3 H), 4.72–4.84 (m, 3 H), 4.61–4.71 (m, 2 H), 4.48–4.58 (m, 2 H), 4.33–4.47 (m, 3 H), 4.09–4.31 (m, 8 H), 3.80–4.06 (m, 6 H), 3.65–3.80 (m, 5 H), 3.62 (d, J = 10.0 Hz, 1 H), 3.50–3.56 (m, 2 H), 3.36–3.45 (m, 3 H), 3.20–3.27 (m, 2 H), 3.09–3.17 (m, 2 H), 2.87 (s, 1 H), 2.14 (s, 3 H), 2.11 (s, 3 H), 2.09 (s, 3 H), 2.04 (s, 3 H), 2.02 (s, 3 H), 1.99 (s, 3 H), 1.87 (s, 3 H). ¹³C NMR (CDCl₃, 100 MHz): δ 170.7, 170.3, 170.2, 170.0 (2 C), 169.5, 169.5, 139.1, 138.7, 138.4, 138.3 (2 C), 137.7, 137.6, 131.2, 128.8, 128.4, 128.3 (3 C), 128.2, 128.1, 127.9 (2 C), 127.8, 127.7, 127.6 (2 C), 127.4, 127.2, 126.5, 123.2, 102.4, 101.9, 100.7, 98.5, 97.6, 83.8, 83.1, 81.9, 78.4, 77.8, 76.5, 76.3, 75.7, 75.1, 74.9, 74.5, 74.4, 74.4, 73.9, 72.9 (2 C), 72.0, 71.7, 71.1, 71.0, 70.8, 70.6, 69.5, 68.8, 68.1, 67.6, 66.9, 66.6, 61.7, 60.8, 55.4, 54.7, 20.9, 20.8, 20.67, 20.6 (3 C), 20.5. HRMS (ESI-Orbitrap): [M + Na]⁺ calcd for C₁₀₉H₁₁₄N₂NaO₃₅ 2033.7100, found 2033.7089.

Benzyl O-(2,3,4,6-Tetra-O-acetyl-β-d-galactopyranosyl)-(1→4)-3,6-di-O-benzyl-2-deoxyacetamido-β-d-glucopyranosyl-(1→3)-[3,4,6-tri-O-acetyl-2-deoxyacetamido-β-d-glucopyranosyl]-(1→6)-(4-O-acetyl-2-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (21)

Following the general procedure A, compound 20 (400 mg, 0.20 mmol) yielded the compound 21 (232 mg, 62% over two steps). [α]_D²⁰ = −1.0 (c 1.2, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 7.51 (m, 2H), 7.21–7.43 (m, 33 H), 5.89 (d, J = 9.5 Hz, 1 H), 5.43 (d, J = 8.4 Hz, 1 H), 5.33 (d, J = 2.8 Hz, 1 H), 4.33–5.24 (m, 23 H), 4.24 (d, J = 8.2 Hz, 1 H), 3.94–4.10 (m, 6 H), 3.45–3.86 (m, 15 H), 3.23 (d, J = 9.7 Hz, 1 H), 2.12 (s, 3 H), 2.11 (s, 3 H), 2.05 (s, 3 H), 2.04 (s, 3 H), 2.01 (s, 3 H), 2.00 (s, 3 H), 1.99 (s, 3 H), 1.97 (s, 3 H), 1.94 (s, 3 H), 1.54 (s, 3 H). ¹³C NMR (CDCl₃, 100 MHz): δ 170.8, 170.7, 170.4, 170.3, 170.2, 170.0, 170.0 (2 C), 169.7, 169.2, 139.4, 138.7, 138.6 (2 C), 138.0, 137.7, 137.4, 128.7, 128.6, 128.4, 128.4 (2 C), 128.3, 128.2, 128.1, 128.0, 127.9 (2 C), 127.8, 127.7, 127.6, 127.0, 102.6, 102.3, 101.1, 100.7, 100.0, 83.8, 82.4, 79.5, 78.3, 77.9, 76.2 (2 C), 75.6, 75.0, 74.9, 74.6, 74.4, 73.9, 73.5, 73.3, 73.2 (2 C), 73.2, 70.9, 70.8, 70.7, 70.6, 70.0, 69.4,68.3,68.0, 67.9, 67.9, 67.3,66.9, 61.5, 60.8, 54.5, 53.6, 20.9, 20.8, 20.7 (3 C), 20.6 (3 C), 20.5 (2 C). HRMS: [M + Na]⁺ calcd for C₉₉H₁₁₆N₂NaO₃₄ 1899.7307, found 1899.7287.

β-d-Galactopyranosyl-(1→4)-2-deoxyacetamido-β-d-glucopyranosyl-(1→3)-[2-deoxyacetamido-β-d-glucopyranosyl]-(1→ 6)-β-d-galactopyranosyl-(1→4)-α,β-d-glucopyranose (HMO2)

Following the general procedures B and C, compound 21 (200 mg, 0.11 mmol) yielded the compound HMO2 (87 mg, 90%). ¹H NMR (D₂O, 400 MHz): δ 5.11 (d, J = 3.6 Hz, 0.42 H, Glc-1 H-1 of α form), 4.53–4.62 (m, 1.58 H, GlcNAc-1 H-1, Glc-1 H-1 of β form), 4.50 (d, J = 8.4 Hz, 1 H, GlcNHAC-2 H-1), 4.36 (d, J = 7.9 Hz, 1 H, Gal-1 H-1), 4.31 (d, J = 8.0 Hz, 1 H, Gal-2 H-1), 4.03 (d, J = 3.0 Hz, 1 H), 3.77–3.92 (m, 5 H), 3.40–3.77 (m, 21 H), 3.30–3.38 (m, 2 H), 3.17 (t, J = 8.9 Hz, 1 H), 1.95 (s, 3 H), 1.92 (s, 3 H). ¹³C NMR (D₂O, 100 MHz): δ 174.9, 174.5, 103.0, 102.8, 102.7 (GlcNAc-1, GlcNAc-2, Gal-2, C-1), 101.0 (Gal-1, C-1), 95.7 (Glc-1, C-1 of β form), 91.8 (Glc-1, C-1 of α form), 81.8, 78.9, 78.8, 78.1, 75.8, 75.3, 74.7, 74.5, 74.3, 73.8, 73.4, 72.5, 72.1, 71.4, 71.2, 70.9, 69.6, 68.5, 68.4,61.0, 60.1, 60.0, 59.8, 55.5, 55.2, 22.4, 22.2. HRMS (ESI-Orbitrap): [M + Na]⁺ calcd for C₃₄H₅₈N₂NaO₂₆ 933.3175, found 933.3195.

Benzyl O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)-(1→3)-(2-O-benzyl-4,6-O-benzylidene-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (22)

3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-d-glucosyl bromide was prepared by following the general procedure D. Powdered molecular sieves (4 Å; 2.0 g) was added to a solution of the above bromide donor 2 (1.2 g, 2.43 mmol) and 1 (500 mg, 0.61 mmol) in anhydrous dichloromethane (20 mL). The suspension was stirred under nitrogen for 1.5 h at room temperature and then cooled to −30 °C. Then 2,4,6-collidine (0.32 mL, 2.43 mmol), and freshly dried AgOTf (624 mg, 2.43 mmol) were sequentially added to the reaction mixture. After it was stirred for 2 h at −30 °C, the mixture was warmed to room temperature overnight, diluted with CH₂Cl₂, and filtered through Celite. The filtrate was diluted with CH₂Cl₂, washed with aqueous NaHCO₃ and brine, dried over Na₂SO₄, and concentrated. The residue was purified on a silica gel column (hexanes/acetone 6/1) to afford the product 22 (626 mg, 85%) as a white powder. [α]_D²⁰ = +12.6 (c 1.0, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 7.30–7.63 (m, 26 H), 7.22–7.27 (m, 3 H), 7.16–7.21 (m, 3 H), 6.89–6.96 (m, 2 H), 5.90 (dd, J = 9.4, 10.65 Hz, 1 H), 5.76 (d, J = 8.3 Hz, 1 H), 5.59 (s, 1 H), 5.29 (t, J = 9.7 Hz, 1 H), 5.17 (d, J = 10.6 Hz, 1 H), 4.92–4.99 (m, 2 H), 4.72–4.82 (m, 2 H), 4.63 (t, J = 11.6 Hz, 1 H), 4.54 (dd, J = 8.3, 10.8 Hz, 1 H), 4.25–4.47 (m, 9 H), 3.93–4.05 (m, 3 H), 3.61–3.73 (m, 2 H), 3.49–3.60 (m, 3 H), 3.44 (d, J = 11.3 Hz, 1 H), 3.14 (s, 1 H), 2.98–3.04 (m, 1 H), 2.13 (s, 3 H), 2.12 (s, 3 H), 1.90 (s, 3 H). ¹³C NMR (D₂O, 100 MHz): δ 170.5, 170.2, 169.6, 139.0, 138.7, 138.6, 138.5, 138.3, 137.6, 134.2, 128.7, 128.4, 128.4, 128.3, 128.2, 128.2, 128.1, 128.0, 127.8, 127.7, 127.6, 127.3, 126.9, 126.4, 123.4, 102.4 (2 C), 100.9, 99.3, 83.1, 81.8, 81.3, 76.9, 75.9, 75.8, 75.1, 74.8, 74.3, 73.1, 71.9, 71.0, 70.8, 69.0, 68.9, 67.9, 66.4,62.2, 54.8, 53.7, 20.9, 20.8, 20.5. HRMS (ESI-Orbitrap): [M + Na]⁺ calcd for C₇₄H₇₅NNaO₂₀ 1320.4780, found 1320.4806.

Benzyl O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)-(1→3)-(2-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (23)

To a solution of compound 22 (602 mg, 0.46 mmol) in anhydrous MeOH (10 mL) were added TsOH (16.0 mg) and EtSH (0.21 mL, 2.93 mmol). The reaction mixture was stirred at room temperature for 6 h and then quenched with triethylamine and evaporated under reduced pressure. The mixture was purified with a silica column (hexanes/acetone 5/1) to give compound 23 as a white powder (445 mg, 80%). [α]_D²⁰ = +17.7 (c 1.0, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 7.51–7.66 (m, 4 H), 7.41–7.45 (m, 2 H), 7.25–7.40 (m, 18 H), 7.06–7.17 (m, 3 H), 6.83 (d, J = 6.7 Hz, 2 H), 5.86 (dd, J = 9.1, 10.6 Hz, 1 H), 5.65 (d, J = 8.5 Hz, 1 H), 5.18 (t, J = 9.4 Hz, 1 H), 4.88–4.96 (m, 3 H), 4.69–4.76 (m, 2 H), 4.62 (d, J = 12.0 Hz, 1 H), 4.43–4.56 (m, 2 H), 4.37–4.41 (m, 1 H), 4.23–4.32 (m, 9 H), 3.95–4.03 (m, 3 H), 3.87–3.93 (m, 1 H), 3.68–3.75 (m, 1 H), 3.52–3.66 (m, 2 H), 3.38–3.50 (m, 5 H), 3.23–3.29 (m, 1 H), 3.03–3.08 (m, 1 H), 2.83 (d, J = 1.5 Hz, 1 H), 2.15 (s, 3 H), 2.09 (s, 3 H), 1.88 (s, 3 H). ¹³C NMR (D₂O, 100 MHz): δ 170.8, 170.1, 169.5, 138.8, 138.6, 138.4, 138.3, 134.3, 128.4 (2 C), 128.3, 128.2, 128.2, 128.0, 127.9, 127.8, 127.7 (2 C), 127.6, 127.5, 126.8, 126.3, 123.5, 102.4, 102.1, 98.7, 84.3, 82.8, 81.7, 77.8, 76.3, 75.7, 75.0, 74.7, 74.3, 73.9, 73.1, 72.1, 70.9, 70.5, 69.0, 68.0, 67.7, 62.2, 61.9, 54.6, 20.8, 20.7, 20.4. HRMS (ESI-Orbitrap): [M + Na]⁺ calcd for C₆₇H₇₁NNaO₂₀ 1232.4467, found 1232.4490.

Benzyl O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)-(1→3)-[2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-(1→4)-3,6-di-O-benzyl-2-deoxyacetamido-β-d-glucopyranosyl]-(1→6)-(2-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (24)

Powdered molecular sieves (4 Å; 1.0 g) was added to a solution of compound 3 (230 mg, 0.27 mmol) and 23 (261 mg, 0.21 mmol) in anhydrous dichloromethane (10 mL). The suspension was stirred under nitrogen for 1.5 h at room temperature and then cooled to −30 °C. Then NIS (61 mg, 0.27 mmol), and AgOTf (35 mg, 0.105 mmol) were sequentially added to the reaction mixture. After it was stirred for 2 h at −30 °C, the mixture was warmed to room temperature overnight, diluted with CH₂Cl₂, and filtered through Celite. The filtrate was diluted with CH₂Cl, washed with aqueous NaHCO₃ and brine, dried over Na₂SO₄, and concentrated. The residue was purified on a silica gel column (hexanes/acetone 5/1) to afford the product 24 (295 mg, 70%) as a white powder. [α]_D²⁰ = +13.0 (c 1.0, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 7.76–8.08 (m, 5 H), 7.49–7.72 (m, 3 H), 7.17–7.48 (m, 25 H), 6.96–7.11 (m, 5 H), 6.87–6.95 (m, 3 H), 6.74 (d, J = 7.3 Hz, 2 H), 5.55–5.61 (dd, J = 9.3, 10.37 Hz, 1 H), 5.30 (d, J = 3.3 Hz, 1 H), 5.11–5.20 (m, 2 H), 5.04 (d, J = 8.5 Hz, 1 H), 4.69–4.95 (m, 9 H), 4.42–4.66 (m, 6 H), 4.21–4.39 (m, 6 H), 4.03–4.15 (m, 5 H), 3.91–4.02 (m, 3 H), 3.70–3.88 (m, 4 H), 3.50–3.68 (m, 4 H), 3.23–3.46 (m, 8 H), 3.00–3.07 (m, 1 H), 2.60–2.69 (m, 1 H), 2.11 (s, 3 H), 2.09 (s, 3 H), 2.08 (s, 3 H), 2.07 (s, 3 H), 2.03 (s, 3 H), 2.00 (s, 3 H), 1.85 (s, 3 H). ¹³C NMR (D₂O, 100 MHz): δ 171.2, 170.4, 170.3, 170.1, 170.0, 169.2, 169.2, 138.8, 138.7, 138.5, 138.3, 138.2, 137.7, 137.6, 128.7, 128.5, 128.4, 128.3, 128.2 (2 C), 128.0 (2 C), 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.3, 126.6, 126.2, 102.5, 101.8, 100.3, 97.8, 97.4, 84.6, 82.9, 81.8, 77.9 (2 C), 77.3, 76.5, 76.1, 75.7, 75.0, 74.8, 74.7, 74.5, 74.4, 73.7, 73.1, 71.4, 71.0, 70.9, 70.4, 70.1, 70.0, 69.5, 69.4,69.2, 67.8, 67.2, 66.9, 65.5, 64.5, 62.2, 60.7, 55.6, 54.1, 20.9, 20.7, 20.7, 20.6 (3 C), 20.4. HRMS (ESI-Orbitrap): [M + Na]⁺ calcd for C₁₀₉H₁₁₄N₂NaO₃₅ 2033.7100, found 2033.7129.

Benzyl O-(3,4,6-Tri-O-acetyl-2-deoxyacetamido-β-d-glucopyranosyl)-(1→3)-[2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-(1→4)-3,6-di-O-benzyl-2-deoxyacetamido-β-d-glucopyranosyl]-(1→6)-(4-O-acetyl-2-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-benzyl-β-d-glucopyranoside (25)

Following the general procedure A, compound 24 (400 mg, 0.20 mmol) yielded compound 25 (183 mg, 70% over two steps). [α]_D²⁰ = −2.0 (c 1.0, CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz): δ 7.16–7.51 (m, 35 H), 5.20–5.36 (m, 2 H), 4.97–5.20 (m, 6 H), 4.89–4.97 (m, 2 H), 4.69–4.89 (m, 8 H), 4.56–4.69 (m, 4 H), 4.29–4.56 (m, 7 H), 4.08–4.26 (m, 2 H), 3.41–4.04 (m, 20 H), 3.32–3.40 (m, 1 H), 3.23–3.31 (m, 1 H), 3.08–3.19 (d, J = 8.6 Hz, 1 H), 2.10 (s, 3 H), 2.09 (s, 3 H), 2.08 (s, 3 H), 2.04 (s, 3 H), 2.01 (s, 3 H), 1.98 (s, 9 H), 1.92 (s, 3 H), 1.57 (s, 3 H). ¹³C NMR(D₂O, 100 MHz): δ 171.0, 170.9, 170.6 (2 C), 170.2, 170.0, 169.8, 169.7, 169.3 (2 C), 139.1, 139.0, 138.7, 138.5, 138.2, 137.9, 137.4, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7 (2 C), 127.6, 127.5, 126.6, 102.4, 102.0, 101.1, 100.0, 99.8, 82.9, 82.0, 79.9, 78.4, 78.2, 77.4, 76.4, 75.5, 75.0, 74.8, 74.1, 73.7, 73.6, 73.3, 72.7, 72.3, 71.7, 71.0, 70.9, 70.4,69.5, 68.3,67.9, 66.9, 66.7, 61.5, 60.8, 55.2, 54.3, 53.9, 20.9, 20.8 (2 C), 20.7 (2 C), 20.6 (3 C), 20.50 (2 C). HRMS (ESI-Orbitrap): [M + Na]⁺ calcd for C₉₉H₁₁₆N₂NaO₃₄ 1899.7307, found 1899.7357.

2-Deoxyacetamido-β-d-glucopyranosyl-(1→3)-[β-d-galactopyranosyl-(1→4)-2-deoxyacetamido-β-d-glucopyranosyl]-(1→6)-β-d-galactopyranosyl-(1→4)-α, β-d-glucopyranose (HMO3)

Following the general procedures B and C, compound 25 (183 mg, 0.098 mmol) yielded the compound HMO3 (85 mg, 95%). ¹H NMR (D₂O, 400 MHz): δ 5.14 (d, J = 3.8 Hz, 0.54 H, Glc-1 H-1 of α form), 4.54–4.63 (m, overlap with D₂O, 2.46 H, GlcNAc-1, GlcNAc-2, H-1, Glc-1 H-1 of β form), 4.39 (d, J = 7.8 Hz, Gal-1, 1 H), 4.35 (d, J = 7.8 Hz, Gal-1, 1 H), 4.06 (d, J = 3.1 Hz, 1 H), 3.82–3.95 (m, 4 H), 3.42–3.82(m, 22 H), 3.33–3.42 (m, 2 H), 3.21 (t, J = 8.39 Hz, 1 H), 1.98 (s, 3 H), 1.96 (s, 3 H). ¹³C NMR (D₂O, 100 MHz): δ 174.9, 174.5, 103.0, 102.9, 102.8 (GlcNAc-1, GlcNAc-2, Gal-2, C-1), 101.0 (Gal-1, C-1), 95.7 (Glc-1, C-1 of β form), 91.8 (Glc-1, C-1 of α form), 81.7, 79.0, 78.9, 78.4, 75.7, 75.3, 74.7, 74.7, 74.4, 73.9, 73.6, 73.5, 72.5, 72.4, 71.0, 69.9, 69.7, 68.7, 68.6, 68.4,61.0, 60.5, 60.0, 55.7, 55.0, 22.4, 22.2. HRMS (ESI-Orbitrap): [M + Na]⁺ calcd for C₃₄H₅₈N₂NaO₂₆ 933.3175, found 933.3161.

HMO11

Yield: 40.0 mg, 92%. ¹H NMR (D₂O, 500 MHz): δ 5.10 (d, J = 3.5 Hz, 0.61 H, Glc-1 H-1 of α form), 4.50–4.60 (m, 2.39 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.33–4.37 (m, 2 H, Gal-2 H-1, Gal-3 H-1), 4.31 (d, J = 7.9 Hz, 1 H, Gal-1 H-1), 4.02 (d, J = 3.1 Hz, 1 H), 3.78–3.79 (m, 6 H), 3.57–3.76 (m, 18 H), 3.53–3.57 (m, 2 H), 3.39–3.51 (m, 7 H), 3.17 (t, J = 8.3 Hz, 1 H), 1.94 (s, 3 H), 1.91 (s, 3 H). MS (MALDI-TOF): [M + Na]⁺ calcd for C₄₀H₆₈N₂NaO₃₁ 1095.370, found 1095.368.

HMO12

Yield: 2.0 mg, 90%. ¹H NMR (D₂O, 500 MHz): δ 5.10 (d, J = 3.5 Hz, 0.51 H, Glc-1 H-1 of α form), 4.50–4.58 (m, 2.49 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.28–4.36 (m, 3 H, Gal-1 H-1, Gal-2 H-1, Gal-3 H-1), 4.03 (d, J = 3.0 Hz, 1 H), 3.38–3.91 (m, 47H), 3.17 (t, J = 8.3 Hz, 1 H), 2.52–2.57 (m, 2 H), 1.97 (s, 3 H), 1.93 (s, 3 H), 1.91 (s, 6 H), 1.55–1.63 (m, 2 H). HRMS (ESI-Orbitrap): [M − 2H]²⁻ calcd for C₆₂H₁₀₀N₄O₄₇ 826.2784, found 826.2760.

HMO13

Yield: 2.0 mg, 90%. ¹H NMR (D₂O, 500 MHz): δ 5.09 (d, J = 3.7 Hz, 0.29 H, Glc-1 H-1 of α form), 4.50–4.63 (m, 2.71 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.28–4.36 (m, 3 H, Gal-1 H-1, Gal-2 H-1, Gal-3 H-1), 4.02 (d, J = 3.0 Hz, 1 H), 3.99 (s, 4 H), 3.38–3.91 (m, 48 H), 3.18 (t, J = 8.3 Hz, 1 H), 2.52–2.60 (m, 2 H), 1.97 (s, 3 H), 1.93 (s, 3 H), 1.55–1.65 (m, 2 H). HRMS (ESI-Orbitrap): [M − 2H]²⁻ calcd for C₆₂H₁₀₀N₄O₄₉ 842.2734, found 842.2760.

HMO14

Yield: 2.3 mg, 95%. ¹H NMR (D₂O, 500 MHz): δ 5.10 (d, J = 3.5 Hz, 0.33 H, Glc-1 H-1 of α form), 4.96–5.02 (m, 2 H, Fuc-1 H-1, Fuc-2 H-1), 4.50–4.61 (m, 2.67 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.28–4.36 (m, 3H, Gal-1 H-1, Gal-2 H-1, Gal-3 H-1), 4.01 (d, J = 3.1 Hz, 1 H), 3.63–3.91 (m, 25 H), 3.40–3.63 (m, 21 H), 3.35–3.39 (m, 2 H), 3.17 (t, J = 8.4 Hz, 1 H), 1.90–1.93 (d, J = 6.5 Hz, 6 H). MS (MALDI-TOF): [M + Na]⁺ calcd for C₅₂H₈₈N₂NaO₃₉ 1387.487, found 1387.486.

HMO15

Yield: 2.0 mg, 93%. ¹H NMR (D₂O, 500 MHz): δ 5.15–5.22 (m, 2 H, Fuc-1 H-1, Fuc-2 H-1), 5.10(d, J = 3.5 Hz, 0.33 H, Glc-1 H-1 of α form), 4.45–4.61 (m, 2.67 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.39–4.45 (m, 2 H, Gal-2 H-1, Gal-3 H-1), 4.30 (d, J = 7.9 Hz, 1 H, Gal-1 H-1), 4.06–4.13 (m, 2 H), 4.01 (d, J = 3.1 Hz, 1 H), 3.41–3.91 (m, 44 H), 3.30–3.39 (m, 2 H), 3.16 (t, J = 8.4 Hz, 1 H), 1.93 (s, 3 H), 1.91 (s, 3 H), 1.09–1.11 (d, J = 6.7 Hz, 6 H). MS (MALDI-TOF): [M + Na]⁺ calcd for C₅₂H₈₈N₂NaO₃₉ 1387.487, found 1387.490.

HMO16

Yield: 1.0 mg, 95%. ¹H NMR (D₂O, 500 MHz): δ 5.12–5.18(m, 2 H, Fuc-2 H-1, Fuc-4 H-1), 5.10 (d, J = 3.5 Hz, 0.40 H, Glc-1 H-1 of α form), 4.93–5.01 (m, 2 H, Fuc-1 H-1, Fuc-3 H-1), 4.70–4.78 (m, 2 H), 4.45–4.61 (m, 2.60 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.35–4.41 (m, 2 H, Gal-2 H-1, Gal-3 H-1), 4.31 (d, J = 7.9 Hz, 1 H, Gal-1 H-1), 4.09–4.16 (m, 2 H), 4.00 (d, J = 3.1 Hz, 1 H), 3.41–3.91 (m, 44 H), 3.28–3.38 (m, 2 H), 3.16 (t, J = 8.4 Hz, 1 H), 1.93 (s, 3 H), 1.90 (s, 3 H), 1.10–1.15 (m, 12 H). MS (MALDI-TOF): [M + Na]⁺ calcd for C₆₄H₁₀₈N₂NaO₄₇ 1679.602, found 1679.607.

HMO21

Yield: 2 mg, 90%. ¹H NMR (D₂O, 500 MHz): δ 5.09 (d, J = 3.6 Hz, 0.22 H, Glc-1 H-1 of α form), 4.45–4.61 (m, 2.78 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.26–4.36 (m, 2 H, Gal-1 H-1, Gal-2 H-1), 4.17–4.22 (m, 1 H), 4.09–4.16 (m, 2 H), 4.00 (d, J = 3.1 Hz, 1 H), 3.36–3.90 (m, 32 H), 3.26–3.36 (m, 2 H), 3.16 (t, J = 8.4 Hz, 1 H), 2.50–2.56 (m, 1 H), 1.92 (s, 3 H), 1.91 (s, 3 H), 1.89 (s, 3 H), 1.58 (t, J = 12.3 Hz, 1 H). MS (ESI-Orbitrap): [M − H]⁻ calcd for C₄₅H₇₄N₃O₃₄ 1200.4159, found 1200.4190.

HMO22

Yield: 2.0 mg, 90%. ¹H NMR (D₂O, 500 MHz): δ 5.09 (d, J = 3.6 Hz, 0.27 H, Glc-1 H-1 of α form), 4.47–4.62 (m, 2.73 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.28–4.35 (m, 2 H, Gal-1 H-1, Gal-2 H-1), 4.06–4.16 (m, 1 H), 4.01 (d, J = 3.1 Hz, 1 H), 3.99 (s, 2 H), 3.38–3.90 (m, 33 H), 3.29–3.37 (m, 2 H), 3.16 (t, J = 8.4 Hz, 1 H), 2.53–2.58 (m, 1 H), 1.93 (s, 3 H), 1.92 (s, 3 H), 1.60 (t, J = 12.3 Hz, 1 H). HRMS (ESI-Orbitrap): [M − H]⁻ calcd for C₄₅H₇₄N₃O₃₅ 1216.4108, found 1216.4129.

HMO23

Yield: 2.0 mg, 95%. ¹H NMR (D₂O, 500 MHz): δ 5.09 (d, J = 3.6 Hz, 0.36 H, Glc-1 H-1 of α form), 5.00 (d, J = 3.9 Hz, 1H, Fuc-1 H-1), 4.46–4.60 (m, 2.64 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.28–4.36 (m, 2 H, Gal-1 H-1, Gal-2 H-1), 4.02 (d, J = 3.1 Hz, 1 H), 3.40–3.90 (m, 31 H), 3.29–3.40 (m, 2 H), 3.16 (t, J = 8.4 Hz, 1 H), 2.50–2.56 (m, 1 H), 1.94 (s, 3 H), 1.90 (s, 3 H), 1.05 (d, J = 6.7 Hz, 3 H). MS (MALDI-TOF): [M + Na]⁺ calcd for C₄₀H₆₈N₂NaO₃₀ 1079.376, found 1079.371.

HMO24

Yield: 2.0 mg, 92%. ¹H NMR (D₂O, 500 MHz): δ 5.19 (d, J = 2.6 Hz, 1 H, Fuc-1 H-1), 5.09 (d, J = 3.6 Hz, 0.50 H, Glc-1 H-1 of α form), 4.46–4.60 (m, 2.50 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.42 (d, J = 7.8 Hz, 1 H, Gal-2 H-1), 4.31 (d, J = 8.0 Hz, 1 H, Gal-1 H-1), 4.07–4.12 (m, 1 H), 4.01 (d, J = 3.1 Hz, 1 H), 3.41–3.90 (m, 28 H), 3.29–3.38 (m, 3 H), 3.16 (t, J = 8.4 Hz, 1 H), 1.94 (s, 3 H), 1.91 (s, 3 H), 1.10 (d, J = 6.5 Hz, 3 H). MS (MALDI-TOF): [M + Na]⁺ calcd for C₄₀H₆₈N₂NaO₃₀ 1079.376, found 1079.379.

HMO25

Yield: 1.0 mg, 90%. ¹H NMR (D₂O, 500 MHz): δ 5.09(d, J = 3.9 Hz, 0.28 H, Glc-1 H-1 of α form), 4.48–4.62 (m, 2.72 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.28–4.37 (m, 3 H, Gal-1 H-1, Gal-2 H-1, Gal-3 H-1), 4.01 (d, J = 3.1 Hz, 1 H), 3.38–3.90 (m, 41 H), 3.17 (t, J = 8.4 Hz, 1 H), 2.50–2.57 (m, 1 H), 1.93 (s, 3 H), 1.92 (s, 3 H), 1.90 (s, 3 H), 1.59 (t, J = 11.9 Hz, 1 H). MS (ESI-Orbitrap): [M – H]⁻ calcd for C₅₁H₈₄N₃O₃₉ 1362.4687, found 1362.4759.

HMO26

Yield: 1.0 mg, 90%. ¹H NMR (D₂O, 500 MHz): δ 5.10 (d, J = 3.7 Hz, 0.51 H, Glc-1 H-1 of α form), 4.50–4.63 (m, 2.49 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.29–4.37 (m, 3 H, Gal-1 H-1, Gal-2 H-1, Gal-3 H-1), 4.02 (d, J = 2.9 Hz, 1 H), 3.99 (s, 2 H), 3.39–3.90 (m, 41 H), 3.17 (t, J = 8.3 Hz, 1 H), 2.54–2.59 (m, 1 H), 1.94 (s, 3 H), 1.93 (s, 3 H), 1.61 (t, J = 12.2 Hz, 1 H). HRMS (ESI-Orbitrap): [M – H]⁻ calcd for C₅₁H₈₄N₃O₄₀ 1378.4637, found 1378.4660.

HMO27

Yield: 1.0 mg, 90%. ¹H NMR (D₂O, 500 MHz): δ 5.10 (d, J = 3.7 Hz, 0.55 H, Glc-1 H-1 of α form), 5.00 (d, J = 4.0 Hz, 1H, Fuc-1 H-1), 4.69–4.74 (m, 1 H), 4.50–4.61 (m, 2.45 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.28–4.38 (m, 3 H, Gal-1 H-1, Gal-2 H-1, Gal-3 H-1), 4.02 (d, J = 2.9 Hz, 1 H), 3.34–3.91 (m, 32 H), 3.17 (t, J = 8.3 Hz, 1 H), 2.54–2.59 (m, 1 H), 1.94 (s, 3 H), 1.90 (s, 3 H), 1.05 (d, J = 6.6 Hz, 3 H). MS (MALDI-TOF): [M + Na]⁺ calcd for C₄₆H₇₈N₂NaO₃₅ 1241.428, found 1241.432.

HMO28

Yield: 1.0 mg, 90%. ¹H NMR (D₂O, 500 MHz): δ 5.19 (d, J = 2.4 Hz, 1H, Fuc-1 H-1), 5.10 (d, J = 3.7 Hz, 0.42 H, Glc-1 H-1 of α form), 4.48–4.60 (m, 2.58 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.28–4.45 (m, 3 H, Gal-1 H-1, Gal-2 H-1, Gal-3 H-1), 4.06–4.15 (m, 1 H), 4.01 (d, J = 2.9 Hz, 1 H), 3.38–3.90 (m, 31 H), 3.31–3.37 (m, 1 H), 3.17(t, J = 8.8 Hz, 1 H), 1.93 (s, 3 H), 1.91 (s, 3 H), 1.10 (d, J = 6.5 Hz, 3 H). MS (MALDI-TOF): [M + Na]⁺ calcd for C₄₆H₇₈N₂NaO₃₅ 1241.428, found 1241.423.

HMO29

Yield: 1.0 mg, 93%. ¹H NMR (D₂O, 500 MHz): δ 5.15 (d, J = 2.6 Hz, 1 H, Fuc-2 H-1), 5.09(d, J = 3.7 Hz, 0.36 H, Glc-1 H-1 of α form), 4.99 (d, J = 3.7 Hz, 1 H, Fuc-1 H-1), 4.72–4.78 (m, 1 H), 4.46–4.61 (m, 2.64 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.42 (d, J = 7.8 Hz, 1 H, Gal-2 H-1), 4.31 (d, J = 8.0 Hz, 1 H, Gal-1 H-1), 4.10–4.16 (m, 1 H), 4.01 (d, J = 2.9 Hz, 1 H), 3.40–3.92 (m, 33 H), 3.29–3.37 (m, 3 H), 3.16 (t, J = 8.4 Hz, 1 H), 1.94 (s, 3 H), 1.90 (s, 3 H), 1.09–1.16 (m, 6 H). MS (MALDI-TOF): [M + Na]⁺ calcd for C₄₆H₇₈N₂NaO₃₄ 1225.433, found 1225.438.

HMO210

Yield: 0.5 mg, 95%. ¹H NMR (D₂O, 500 MHz): δ 5.16 (d, J = 2.6 Hz, 1 H, Fuc-2 H-1), 5.10 (d, J = 3.5 Hz, 0.40 H, Glc-1 H-1 of α form), 4.93–5.01 (m, 2 H, Fuc-1 H-1, Fuc-3 H-1), 4.67–4.79 (m, 2 H), 4.45–4.61 (m, 2.60 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.26–4.41 (m, 3 H, Gal-1 H-1, Gal-2 H-1, Gal-3 H-1), 4.09–4.16 (m, 1 H), 4.00 (d, J = 3.1 Hz, 1 H), 3.41–3.91 (m, 44 H), 3.28–3.40 (m, 3 H), 3.16 (t, J = 8.4 Hz, 1 H), 1.93 (s, 3 H), 1.90 (s, 3 H), 1.02–1.17 (m, 9 H). MS (MALDI-TOF): [M + Na]⁺ calcd for C₅₈H₉₈N₂NaO₄₃ 1533.544, found 1533.538.

HMO211

Yield: 0.5 mg, 90%. ¹H NMR (D₂O, 500 MHz): δ 5.16 (d, J = 2.7 Hz, 1 H, Fuc-2 H-1), 5.10 (d, J = 3.7 Hz, 0.47 H, Glc-1 H-1 of α form), 4.99 (d, J = 3.7 Hz, 1 H, Fuc-1 H-1), 4.72–4.78 (m, 1 H), 4.49–4.62 (m, 2.53 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.29–4.41 (m, 3 H, Gal-1 H-1, Gal-2 H-1, Gal-3 H-1), 4.10–4.16 (m, 1 H), 4.01 (d, J = 3.2 Hz, 1 H), 3.77–3.91 (m, 8 H), 3.39–3.77 (m, 35 H), 3.30–3.36 (m, 1 H), 3.17 (t, J = 8.4 Hz, 1 H), 1.94 (s, 3 H), 1.90 (s, 3 H), 1.09–1.16 (m, 6 H). MS (MALDI-TOF): [M + Na]⁺ calcd for C₅₂H₈₈N₂NaO₃₉ 1387.486, found 1387.490.

HMO31

Yield: 2.0 mg, 90%. ¹H NMR (D₂O, 500 MHz): δ 5.09(d, J = 3.7 Hz, 0.31 H, Glc-1 H-1 of α form), 4.47–4.58 (m, 2.69 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.28–4.34 (m, 2 H, Gal-1 H-1, Gal-2 H-1), 4.06–4.16 (m, 1 H), 4.02 (d, J = 3.1 Hz, 1 H), 3.28–3.91 (m, 35 H), 3.26–3.36 (m, 2 H), 3.18 (t, J = 8.4 Hz, 1 H), 2.51–2.57 (m, 1 H), 1.96 (s, 3 H), 1.92 (s, 3 H), 1.91 (s, 3 H), 1.59 (t, J = 12.2 Hz, 1 H). MS(ESI-Orbitrap): [M–H]⁻ calcd for C₄₅H₇₄N₃O₃₄ 1200.4159, found 1200.4122.

HMO32

Yield: 2.0 mg, 90%. ¹H NMR (D₂O, 500 MHz): δ 5.09 (d, J = 3.9 Hz, 0.28 H, Glc-1 H-1 of α form), 4.50–4.58 (m, 2.72 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.28–4.34 (m, 2 H, Gal-1 H-1, Gal-2 H-1), 4.02 (d, J = 3.2 Hz, 1 H), 3.28–3.91 (m, 35 H), 3.18 (t, J = 8.3 Hz, 1 H), 2.53–2.58 (m, 1 H), 1.96 (s, 3 H), 1.91 (s, 3 H), 1.59 (t, J = 12.2 Hz, 1 H). MS (ESI-Orbitrap): [M – H]⁻ calcd for C₄₅H₇₄N₃O₃₅ 1216.4108, found 1216.4139.

HMO33

Yield: 2.2 mg, 95%. ¹H NMR (D₂O, 500 MHz): δ 5.09 (d, J = 3.7 Hz, 0.19 H, Glc-1 H-1 of α form), 4.98 (d, J = 3.9 Hz, 1H, Fuc-1 H-1), 4.66–4.73 (m, 1 H), 4.48–4.58 (m, 2.81 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.27–4.35 (m, 2 H, Gal-1 H-1, Gal-2 H-1), 4.02 (d, J = 3.1 Hz, 1 H), 3.28–3.90 (m, 38 H), 3.16 (t, J = 8.4 Hz, 1 H), 1.92 (s, 3 H), 1.90 (s, 3 H), 1.04 (d, J = 6.6 Hz, 3 H). MS (MALDI-TOF): [M + Na]⁺ calcd for C₄₀H₆₈N₂NaO₃₀ 1079.376, found 1079.380.

HMO34

Yield: 2.0 mg, 92%. ¹H NMR (D₂O, 500 MHz): δ 5.18 (d, J = 2.6 Hz, 1 H, Fuc-1 H-1), 5.09(d, J = 3.9 Hz, 0.41 H, Glc-1 H-1 of α form), 4.66–4.73 (m, 1 H), 4.45–4.58 (m, 2.59 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.39–4.44 (m, 2 H, Gal-1 H-1, Gal-2 H-1), 4.31 (d, J = 7.5 Hz, 1 H), 4.06–4.12 (m, 2 H), 4.02 (d, J = 3.1 Hz, 1 H), 3.39–3.90 (m, 28 H), 3.28–3.38 (m, 3 H), 3.16 (t, J = 8.4 Hz, 1 H), 1.94 (s, 3 H), 1.91 (s, 3 H), 1.11 (d, J = 6.7 Hz, 3 H). MS (MALDI-TOF): [M + Na]⁺ calcd for C₄₀H₆₈N₂NaO₃₀ 1079.376, found 1079.372.

HMO35

Yield: 1 mg, 90%. ¹H NMR (D₂O, 500 MHz): δ 5.09 (d, J = 3.8 Hz, 0.53 H, Glc-1 H-1 of α form), 4.51–4.60 (m, 2.47 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.28–4.37 (m, 3 H, Gal-1 H-1, Gal-2 H-1, Gal-3 H-1), 4.02 (d, J = 3.0 Hz, 1 H), 3.38–3.91 (m, 41 H), 3.18 (t, J = 8.4 Hz, 1 H), 2.50–2.57 (m, 1 H), 1.96 (s, 3 H), 1.91 (s, 6 H), 1.59 (t, J = 12.0 Hz, 1 H). MS (ESI-Orbitrap): [M – H]⁻ calcd for C₅₁H₈₄N₃O₃₉ 1362.4687, found 1362.4799.

HMO36

Yield: 1 mg, 90%. ¹H NMR (D₂O, 500 MHz): δ 5.09 (d, J = 3.8 Hz, 0.52 H, Glc-1 H-1 of α form), 4.50–4.60 (m, 2.48 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.28–4.38 (m, 3 H, Gal-1 H-1, Gal-2 H-1, Gal-3 H-1), 4.02 (d, J = 3.0 Hz, 1 H), 3.99 (s, 2 H), 3.38–3.91 (m, 41 H), 3.18 (t, J = 8.4 Hz, 1 H), 2.53–2.59 (m, 1 H), 1.96 (s, 3 H), 1.91 (s, 3 H), 1.59 (t, J = 12.0 Hz, 1 H). MS (ESI-Orbitrap): [M – H]⁻ calcd for C₅₁H₈₄N₃O₄₀ 1378.4637, found 1378.4609.

HMO37

Yield: 1.1 mg, 90%. ¹H NMR (D₂O, 500 MHz): δ 5.09 (d, J = 3.8 Hz, 0.34 H, Glc-1 H-1 of α form), 4.98 (d, J = 4.0 Hz, 1H, Fuc-1 H-1), 4.67–4.74 (m, 1 H), 4.50–4.61 (m, 2.66 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.27–4.38 (m, 3 H, Gal-1 H-1, Gal-2 H-1, Gal-3 H-1), 4.01 (d, J = 3.1 Hz, 1 H), 3.30–3.90 (m, 37 H), 3.16 (t, J = 8.6 Hz, 1 H), 1.92 (s, 3 H), 1.91 (s, 3 H), 1.05 (d, J = 6.6 Hz, 3 H). MS (MALDITOF): [M + Na]⁺ calcd for C₄₆H₇₈N₂NaO₃₅ 1241.428, found 1241.436.

HMO38

Yield: 1 mg, 90%. ¹H NMR (D₂O, 500 MHz): δ 5.18 (d, J = 2.6 Hz, 1 H, Fuc-1 H-1), 5.09 (d, J = 3.8 Hz, 0.69 H, Glc-1 H-1 of α form), 4.46–4.60 (m, 2.31 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.28–4.43 (m, 3 H, Gal-1 H-1, Gal-2 H-1, Gal-3 H-1), 4.07–4.13 (m, 1 H), 4.02 (d, J = 3.2 Hz, 1 H), 3.39–3.89 (m, 36 H), 3.33–3.38 (m, 1 H), 3.17 (t, J = 8.4 Hz, 1 H), 1.94 (s, 3 H), 1.91 (s, 3 H), 1.11 (d, J = 6.5 Hz, 3 H). MS (MALDI-TOF): [M + Na]⁺ calcd for C₄₆H₇₈N₂NaO₃₅ 1241.428, found 1241.435.

HMO39

Yield: 1.1 mg, 93%. ¹H NMR (D₂O, 500 MHz): δ 5.16 (d, J = 2.9 Hz, 1 H, Fuc-2 H-1), 5.10(d, J = 3.7 Hz, 0.39 H, Glc-1 H-1 of α form), 4.97 (d, J = 4.0 Hz, 1 H, Fuc-1 H-1), 4.72–4.78 (m, 1 H), 4.45–4.58 (m, 2.61 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.28–4.40 (m, 2 H, Gal-1 H-1, Gal-2 H-1), 4.10–4.16 (m, 1 H), 4.02 (d, J = 3.06 Hz, 1 H), 3.40–3.93 (m, 33 H), 3.29–3.38 (m, 3 H), 3.16(t, J = 8.4 Hz, 1 H), 1.93 (s, 3 H), 1.91 (s, 3 H), 1.08–1.17 (m, 6 H). MS (MALDI-TOF): [M + Na]⁺ calcd for C₄₆H₇₈N₂NaO₃₄ 1225.433, found 1225.430.

HMO310

Yield: 0.4 mg, 93%. ¹H NMR (D₂O, 500 MHz): δ 5.16 (d, J = 3.0 Hz, 1 H, Fuc-3 H-1), 5.09 (d, J = 3.7 Hz, 0.62 H, Glc-1 H-1 of α form), 4.96–5.01 (m, 2 H), 4.69–4.78 (m, overlap with D₂O, 2 H), 4.47–4.61 (m, 2.38 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.28–4.40 (m, 3H, Gal-1 H-1, Gal-2 H-1, Gal-3 H-1), 4.10–4.16 (m, 1 H), 4.02 (d, J = 3.2 Hz, 1 H), 3.32–3.93 (m, 43 H), 3.16 (t, J = 8.4 Hz, 1 H), 1.93 (s, 3 H), 1.90 (s, 3 H), 1.02–1.17 (m, 9 H). MS (MALDI-TOF): [M + Na]⁺ calcd for C₅₈H₉₈N₂NaO₄₃ 1533.544, found 1533.550.

HMO311

Yield: 0.5 mg, 90%. ¹H NMR (D₂O, 500 MHz): δ 5.16 (d, J = 3.2 Hz, 1 H, Fuc-2 H-1), 5.10 (d, J = 3.6 Hz, 0.67 H, Glc-1 H-1 of α form), 4.97 (d, J = 3.9 Hz, 1 H, Fuc-1 H-1), 4.72–4.77 (m, 1 H), 4.47–4.60 (m, 2.37 H, GlcNAc-1 H-1, GlcNAc-2 H-1, Glc-1 H-1 of β form), 4.29–4.39 (m, 3 H, Gal-1 H-1, Gal-2 H-1, Gal-3 H-1), 4.10–4.16 (m, 1 H), 4.02 (d, J = 3.1 Hz, 1 H), 3.39–3.93 (m, 41 H), 3.30–3.38 (m, 1 H), 3.16 (t, J = 8.4 Hz, 1 H), 1.94 (s, 3 H), 1.91 (s, 3 H), 1.09–1.17 (m, 6 H). MS (MALDITOF): [M + Na]⁺ calcd for C₅₂H₈₈N₂NaO₃₉ 1387.486, found 1387.478.