Efficient Synthesis of Two Sialylated Tetrasaccharides Found in Goat Milk

Abstract

Two regioisomeric sialylated tetrasaccharides found in goat milk have been obtained in excellent yield by a concise synthesis using a common disaccharide intermediate. Regioselective glycosylations and a minimal number of protecting-group manipulations are the key features of this synthetic strategy.

Mammalian milk is an excellent source of complex oligosaccharide antigens and bifidus factors for breastfed newborns. To date, more than 130 different oligosaccharide structures have been isolated and characterized from different mammalian milks. [¹] For a long time, the biological significance of a large number of complex oligosaccharides found in milk were not properly studied because it was thought that they are not directly related to nutrition and were treated as byproducts formed during the synthesis of milk. Recently, it has been demonstrated that milk oligosaccharides have strong potential to inhibit pathogenic microbial adhesion to the host cell surface, which is the initial step of bacterial infections. [²] Oligosaccharides found in human milk can inhibit the adhesion of Streptococcus pneumoniae and Haemophilus influenzae to human pharyngeal or buccal epithelial cells and Escherichia coli adhesion to uroepithelial cells. [²] [³] A number of glycoproteins or glycolipids that function as tumor-associated antigens are found in human milk. [4] In addition, milk oligosaccharides may function as prebiotics, promoting the growth of benign microorganisms, such as Bifidobacterium bifidus, within the lower gastrointestinal tract, and inhibiting the proliferation of pathogenic organisms. [5] In spite of several investigations, the physiological role of the milk-derived oligosaccharides is not yet completely established. Different types of milk have been analyzed for their oligosaccharide structures that have immunological properties. [6] Recently, it has been reported that goat milk contains two regioisomeric sialylated tetrasaccharides. [7] Although these oligosaccharides can be isolated from the natural sources, it is essential to synthesize them chemically to provide larger quantities for their proper bioevaluations. We report herein the concise chemical synthesis of two sialylated tetrasaccharides found in goat milk (Figure  [¹] ).

Two regioisomeric sialylated tetrasaccharides 1 and 2 (Figure  [¹] ) were synthesized from a common disaccharide derivative 8 in a very concise manner (Scheme  [¹] and Scheme  [²] ). For this purpose, a number of suitably protected monosaccharide synthons 3, [8] 4, [9] 5, [¹0] 6, [¹¹] and 7 [¹²] (Figure  [²] ) were prepared from naturally available monosaccharides according to methodologies reported in the literature.

Condensation of monosaccharide acceptor 3 with thioglycoside donor 4 in the presence of a combination of N-iodosuccinimide­ and trimethylsilyl trifluoromethane­sulfonate [¹³] furnished the disaccharide derivative 8 in 92% yield; this was the common starting point for the synthesis of both 1 (Scheme  [¹] ) and 2 (Scheme  [²] ). The presence of signals at δ = 5.46 (s, PhCH), 4.86 (d, J = 7.4 Hz, H-1_A), and 4.70 (d, J = 8.1 Hz, H-1_B) in the ^¹H NMR and at δ = 102.8 (C-1_A), 101.0 (PhCH), and 100.8 (C-1_B) in the ^¹³C NMR spectra confirmed its formation.

Removal of the benzylidene acetal from compound 8 by use of perchloric acid supported on silica [¹4] at room temperature resulted in the formation of disaccharide diol derivative 9 in 95% yield (Scheme  [¹] ). Regioselective glycosylation of compound 9 with d-galactose-derived trichloroacetimidate derivative 5 in the presence of tri­methylsilyl trifluoromethanesulfonate [¹5] afforded expected trisaccharide derivative 10 in 82% yield. Presence of ^¹H NMR signals at δ = 4.86 (d, J = 8.0 Hz, H-1_A), 4.61 (d, J = 3.9 Hz, H-1_B), and 4.30 (d, J = 8.0 Hz, H-1_C) and ^¹³C NMR signals at δ = 102.9 (C-1_A), 101.3 (C-1_C), and 100.2 (C-1_B) supported its formation. Compound 10 was deacetylated with sodium methoxide, to furnish trisaccharide triol acceptor 11 in quantitative yield, to be used for glycosylation with the sialic acid derivative. Stereo- and regioselective glycosylation [¹6] of sialic acid derived thioglycoside donor 7 with compound 11 in the presence of the N-iodosuccinimide-trifluoromethanesulfonic acid combination [¹³] in a mixed solvent (MeCN-CH₂Cl₂, 5:1) furnished sialylated tetrasaccharide derivative 12 in 50% yield. Hydrogenolysis of tetrasaccharide derivative 12 over Pearlman’s catalyst [¹7] followed by conventional saponification furnished tetrasaccharide 1 as its sodium salt and 4-methoxyphenyl glycoside in 80% overall yield; it was purified over Sephadex LH-20 gel (MeOH-H₂O, 4:1) (Scheme  [¹] ). The presence of signals at δ = 4.79 (d, J = 7.3 Hz, H-1_A), 4.42 (d, J = 7.9 Hz, H-1_B), 4.28 (d, J = 7.0 Hz, H-1_C), 2.74 (dd, J = 12.1, 3.7 Hz, 1 H, H-3e_D), and 1.87 (t, J = 11.8 Hz, 1 H, H-3a_D) in the ^¹H NMR and at δ = 104.3 (C-2_D), 104.2 (2 C, C-1_C, C-1_B), and 101.9 (C-1_A) in the ^¹³C NMR spectra confirmed the formation of compound 1.

For the synthesis of compound 2 (Scheme  [²] ), disaccharide derivative 8 was deacetylated with sodium methoxide to afford disaccharide diol acceptor 13 in quantitative yield. Compound 13 was regioselectively glycosylated with thioglycoside 6 under N-iodosuccinimide-trimethylsilyl trifluoromethanesulfonate catalysis; conventional acetylation followed, to furnish trisaccharide derivative 14 in 74% overall yield. Presence of signals at δ = 4.80 (d, J = 7.5 Hz, H-1_A), 4.62 (d, J = 7.9 Hz, H-1_B), and 4.58 (d, J = 8.0 Hz, H-1_C) in the ^¹H NMR and at δ = 102.7 (C-1_A), 101.6 (C-1_B), and 100.9 (C-1_C) in the ^¹³C NMR spectra confirmed its formation. Removal of the benzylidene acetal from compound 14 was achieved by use of perchloric acid supported on silica at room temperature; this gave trisaccharide diol derivative 15 in 92% yield. Stereo- and regioselective glycosylation of trisaccharide acceptor 15 with sialic acid thioglycoside derivative 7 in the presence of the N-iodosuccinimide-trifluoromethanesulfonic acid combination in a mixed solvent (MeCN-CH₂Cl₂, 5:1) furnished 6-O-α-sialylated tetrasaccharide derivative 16 in 67% yield. Hydrogenolysis of tetrasaccharide derivative 16 over Pearlman’s catalyst followed by conventional saponification furnished tetrasaccharide 2 as its sodium salt and 4-methoxyphenyl glycoside in 78% overall yield; it was purified over Sephadex LH-20 gel (MeOH-H₂O, 4:1) (Scheme  [²] ). The presence of signals at δ = 4.79 (d, J = 7.3 Hz, H-1_A), 4.47 (d, J = 7.3 Hz, H-1_B), 4.41 (d, J = 7.5 Hz, H-1_C), 2.73 (dd, J = 12.3, 4.2 Hz, 1 H, H-3e_D), and 1.75 (t, J = 11.9 Hz, 1 H, H-3a_D) in the ^¹H NMR and at δ = 105.3 (C-1_B), 103.7 (C-1_C), 101.9 (C-1_A), 99.5 (C-2_D) in the ^¹³C NMR spectra supported the formation of compound 2.

In summary, efficient syntheses of two regioisomers of sialylated tetrasaccharides 1 and 2 found in goat milk were achieved in excellent yield. A common disaccharide derivative has been used as the starting point for the preparation of both tetrasaccharides in a minimal number of steps. Both tetrasaccharides contain the 4-methoxyphenyl group as a temporary anomeric protecting group, which can be removed by standard procedures for the preparation of glycoconjugates.

All the reactions were monitored by TLC on plates coated with silica gel. The TLC spots were visualized by warming the TLC plates sprayed with 2% Ce(SO₄)₂ in 2 N H₂SO₄ on a hot plate. Silica gel (230-400 mesh) was used for column chromatography. ^¹H and ^¹³C NMR, 2D COSY, and HSQC spectra were recorded on a Bruker Avance DPX 300 MHz spectrometer; CDCl₃ and D₂O were used as solvents and TMS as internal reference, unless stated otherwise. ESI-MS was carried out on a MICROMASS QUATTRO II triple quadrupole mass spectrometer. Elementary analysis was carried out on a Carlo ERBA-1108 analyzer. Optical rotations were measured at 25 ˚C on a Rudolf Autopol III polarimeter. Commercially available grades of organic solvents of adequate purity were used in many reactions.

4-Methoxyphenyl (2,3-Di- O -acetyl-4,6- O -benzylidene-β- d -galactopyranosyl)-(1→4)-2,3,6-tri- O -benzyl-β- d -glucopyranoside (8)

To a soln of 3 (5 g, 9 mmol) and thioglycoside donor 4 (4.3 g, 10.8 mmol) in CH₂Cl₂ (50 mL) was added 4 Å MS (5 g), and the mixture was allowed to stir at r.t. under argon for 30 min. The mixture was cooled to -30 ˚C and NIS (3.2 g, 14 mmol) and TMSOTf (30 µL) were added. After stirring of the mixture at the same temperature for 45 min, it was filtered through a Celite bed and washed with CH₂Cl₂ (100 mL). The organic layer was washed with 5% Na₂S₂O₃ (100 mL), sat. NaHCO₃ (100 mL), and H₂O (100 mL) in succession, dried (Na₂SO₄), and evaporated to dryness. The crude mass was purified by chromatography (silica gel, hexane-EtOAc, 5:1); this gave pure 8.

Yield: 7.4 g (92%); R _f = 0.3 (hexane-EtOAc, 3:1); white solid; mp 139-41 ˚C; [α]_D ^²5 -4.2 (c 1.5, CHCl₃).

IR (KBr): 2915, 2871, 1740, 1506, 1454, 1374, 1230, 1061, 736, 698 cm^-¹.

^¹H NMR (300 MHz, CDCl₃): δ = 7.49-7.19 (m, 20 H, Ar-H), 7.02 (d, J = 9.0 Hz, 2 H, Ar-H), 6.81 (d, J = 9.0 Hz, 2 H, Ar-H), 5.46 (s, 1 H, PhCH), 5.33 (dd, J = 10.3, 8.1 Hz, 1 H, H-2_B), 5.12 (d, J = 11.0 Hz, 1 H, PhCH ₂), 5.02 (d, J = 11.0 Hz, 1 H, PhCH ₂), 4.86 (d, J = 7.4 Hz, 1 H, H-1_A), 4.84-4.73 (m, 4 H, H-3_B, 3 PhCH ₂), 4.70 (d, J = 8.1 Hz, 1 H, H-1_B), 4.53 (d, J = 11.9 Hz, 1 H, PhCH ₂), 4.25 (d, J = 3.4 Hz, 1 H, H-4_B), 4.18 (d, J = 12.4 Hz, 1 H, H-6a_B), 4.00 (t, J = 9.0 Hz, 1 H, H-3_A), 3.87-3.75 (m, 3 H, H-6b_B, H-6ab_A), 3.79 (s, 3 H, OCH ₃), 3.72-3.66 (m, 3 H, H-2_A, H-5_A, H-5_B), 3.52-3.46 (m, 1 H, H-4_A), 2.09, 2.00 (2 s, 6 H, 2 COCH ₃).

^¹³C NMR (75 MHz, CDCl₃): δ = 170.6, 168.9 (2 COCH₃), 155.3-114.5 (Ar-C), 102.8 (C-1_A), 101.0 (PhCH), 100.8 (C-1_B), 82.9 (C-2_A), 81.7 (C-5_A), 77.4 (C-3_A), 75.6 (PhCH₂), 75.0 (3 C, PHCH₂, C-4_A, C-5_B), 73.6 (PhCH₂), 73.3 (C-4_B), 72.2 (C-3_B), 69.4 (C-2_B), 68.6 (C-6_B), 68.0 (C-6_A), 55.6 (OCH₃), 20.8 (2 C, COCH₃).

ESI-MS: m/z = 913.2 [M + Na]⁺.

Anal. Calcd for C₅₁H₅₄O₁₄ (890.35): C, 68.75; H, 6.11. Found: C, 68.58; H, 6.30.

4-Methoxyphenyl (2,3-Di- O -acetyl-β- d -galactopyranosyl)-(1→4)-2,3,6-tri- O -benzyl-β- d -glucopyranoside (9)

HClO₄˙SiO₂ (500 mg) was added to a soln of 8 (2.5 g, 2.8 mmol) in MeCN-H₂O (9:1; 50 mL), and the mixture was allowed to stir at r.t. for 30 min. It was then filtered through a Celite bed and evaporated to dryness. The crude product was purified through a short pad of silica gel (hexane-EtOAc, 1:1); this gave pure 9.

Yield: 2.2 g (95%); R _f = 0.2 (hexane-EtOAc, 1:2); white solid; mp 156-58 ˚C; [α]_D ^²5 -31.4 (c 1.5, CHCl₃).

IR (KBr): 3432, 2870, 1743, 1508, 1455, 1373, 1229, 1059, 751, 698 cm^-¹.

^¹H NMR (300 MHz, CDCl₃): δ = 7.43-7.29 (m, 15 H, Ar-H), 7.05 (d, J = 9.1 Hz, 2 H, Ar-H), 6.81 (d, J = 9.2 Hz, 2 H, Ar-H), 5.26 (dd, J = 10.1, 8.0 Hz, 1 H, H-2_B), 5.02 (d, J = 11.0 Hz, 1 H, PhCH ₂), 4.98 (d, J = 11.2 Hz, 1 H, PhCH ₂), 4.85-4.79 (m, 3 H, H-1_A, PhCH ₂), 4.75 (d, J = 10.4, 3.6 Hz, 1 H, H-3_B), 4.70 (d, J = 11.9 Hz, 1 H, PhCH ₂), 4.58 (d, J = 8.0 Hz, 1 H, H-1_B), 4.52 (d, J = 11.9 Hz, 1 H, PhCH ₂), 4.03-3.97 (m, 2 H, H-4_B, H-3_A), 3.73 (s, 3 H, OCH ₃), 3.75-3.65 (m, 2 H, H-6ab_A), 3.63-3.60 (m, 2 H, H-2_A, H-5_A), 3.57-3.54 (m, 2 H, H-6ab_B), 3.52-3.48 (m, 1 H, H-4_A), 3.25-3.22 (m, 1 H, H-5_B), 2.09, 1.99 (2 s, 6 H, 2 COCH ₃).

^¹³C NMR (75 MHz, CDCl₃): δ = 170.1, 169.3 (2 COCH₃), 155.3-114.5 (Ar-C), 102.7 (C-1_A), 100.3 (C-1_B), 82.5 (C-2_A), 81.3 (C-5_A), 76.4 (C-3_A), 75.3 (C-3_B), 74.9 (PhCH₂), 74.8 (PhCH₂), 73.7 (C-4_A), 73.5 (2 C, PhCH₂, C-5_B), 69.4 (C-2_B), 68.1 (C-4_B), 67.8 (C-6_A), 62.1 (C-6_B), 55.5 (OCH₃), 20.7 (2 C, COCH₃).

ESI-MS: m/z = 825.1 [M + Na]⁺.

Anal. Calcd for C₄₄H₅₀O₁₄ (802.32): C, 65.82; H, 6.28. Found: C, 65.60; H, 6.50.

4-Methoxyphenyl (2- O -Acetyl-3,4,6-tri- O -benzyl-β- d -galactopyranosyl)-(1→6)-(2,3-di- O -acetyl-β- d -galactopyranosyl)-(1→4)-2,3,6-tri- O -benzyl-β- d -glucopyranoside (10)

A soln of 9 (2 g, 2.5 mmol) and 5 (2 g, 3.2 mmol) in anhyd CH₂Cl₂ (25 mL) was cooled to -50 ˚C. TMSOTf (100 µL) was added, and the mixture was stirred at -40 ˚C for 1 h. It was diluted with CH₂Cl₂ (50 mL), and the organic layer was washed with sat. aq NaHCO₃ (100 mL) and H₂O (100 mL) in succession, dried (Na₂SO₄), and evaporated to dryness. The crude product was purified by chromatography (silica gel, hexane-EtOAc, 2:1); this gave pure 10.

Yield: 2.6 g (82%); R _f = 0.4 (hexane-EtOAc, 1:2); white solid; mp 176-78 ˚C; [α]_D ^²5 -17.3 (c 1.5, CHCl₃).

IR (KBr): 3465, 2874, 1747, 1506, 1371, 1230, 1063, 747, 698 cm^-¹.

^¹H NMR (300 MHz, CDCl₃): δ = 7.43-7.17 (m, 30 H, Ar-H), 7.00 (d, J = 9.0 Hz, 2 H, Ar-H), 6.80 (d, J = 9.0 Hz, 2 H, Ar-H), 5.28 (t, J = 9.0 Hz, 1 H, H-2_C), 5.21 (t, 9.1 Hz, 1 H, H-2_B), 4.97 (d, J = 11.1 Hz, 2 H, PhCH ₂), 4.90 (d, J = 12.1 Hz, 1 H, PhCH ₂), 4.86 (d, J = 8.0 Hz, 1 H, H-1_A), 4.84-4.80 (m, 1 H, H-3_B), 4.79 (d, J = 11.8 Hz, 1 H, PhCH ₂), 4.78 (d, J = 12.0 Hz, 1 H, PhCH ₂), 4.71 (d, J = 12.0 Hz, 1 H, PhCH ₂), 4.61 (d, J = 3.9 Hz, 1 H, H-1_B), 4.58-4.54 (d, J = 12.0 Hz, 3 H, PhCH ₂), 4.49-4.39 (m, 3 H, PhCH ₂), 4.30 (d, J = 8.0 Hz, 1 H, H-1_C), 4.03-3.98 (m, 2 H, H-4_B, H-3_A), 3.85 (br s, 1 H, H-4_C), 3.79 (s, 3 H, OCH ₃), 3.76-3.73 (m, 2 H, H-6ab_A), 3.71-3.59 (m, 4 H, H-6ab_B, H-2_A, H-5_C), 3.55-3.39 (m, 5 H, H-6ab_C, H-5_A, H-3_C, H-4_A), 3.32-3.26 (m, 1 H, H-5_B), 2.09, 2.05, 1.99 (3 s, 9 H, 3 COCH ₃).

^¹³C NMR (75 MHz, CDCl₃): δ = 169.8, 169.5, 169.1 (3 COCH₃), 155.3-114.5 (Ar-C), 102.9 (C-1_A), 101.3 (C-1_C), 100.2 (C-1_B), 82.8 (C-2_A), 81.7 (C-5_C), 80.7 (C-5_A), 76.2 (C-3_A), 75.7 (PhCH₂), 74.9 (C-3_C), 74.8 (PhCH₂), 74.4 (PhCH₂), 73.5 (2 C, PhCH₂), 73.4 (C-4_A), 73.1 (2 C, C-3_B, C-5_B), 72.6 (C-4_C), 72.1 (PhCH₂), 71.2 (C-2_C), 70.4 (C-2_B), 68.3 (C-6_C), 67.9 (C-6_A), 66.5 (2 C, C-6_B, C-4_B), 55.5 (OCH₃), 21.0, 20.8, 20.7 (3 C, 3 COCH₃).

ESI-MS: m/z = 1299.4 [M + Na]⁺.

Anal. Calcd for C₇₃H₈₀O₂₀ (1276.52): C, 68.64; H, 6.31. Found: C, 68.45; H, 6.55.

4-Methoxyphenyl (3,4,6-Tri- O -benzyl-β- d -galactopyranosyl)-(1→6)-(β- d -galactopyranosyl)-(1→4)-2,3,6-tri- O -benzyl-β- d -glucopyranoside (11)

A soln of 10 (2.5 g, 2 mmol) in 0.1 M NaOMe in MeOH (mL) was allowed to stir at r.t. for 4 h and was then neutralized with Amberlite-IR 120 (H⁺) resin. The mixture was filtered and then evaporated to dryness; this gave the crude product, which was passed through a short column of silica gel (toluene-EtOAc, 1:1); this gave pure 11.

Yield: 2.3 g (100%); R _f = 0.3 (toluene-EtOAc, 1:2); white solid; mp 168-69 ˚C; [α]_D ^²5 +3.6 (c 1.5, CHCl₃).

IR (KBr): 3449, 3020, 2926, 2364, 1505, 1216, 1064, 763, 669 cm^-¹.

^¹H NMR (300 MHz, CDCl₃): δ = 7.37-7.17 (m, 30 H, Ar-H), 6.98 (d, J = 9.0 Hz, 2 H, Ar-H), 6.78 (d, J = 9.0 Hz, 2 H, Ar-H), 4.97 (d, J = 11.2 Hz, 1 H, PhCH ₂), 4.92 (d, J = 11.0 Hz, 1 H PhCH ₂), 4.85 (d, J = 4.86 Hz, 1 H, H-1_A), 4.82-4.77 (m, 3 H, PhCH ₂), 4.71-4.63 (m, 3 H, PhCH ₂), 4.54 (d, J = 12.1 Hz, 1 H, PhCH ₂), 4.53 (d, J = 11.7 Hz, 1 H, PhCH ₂), 4.42 (d, J = 12.4 Hz, 1 H, PhCH ₂), 4.41 (d, J = 6.3 Hz, 1 H, H-1_B), 4.38 (d, J = 11.8 Hz, 1 H, PhCH ₂), 4.07 (d, J = 7.7 Hz, 1 H, H-1_C), 4.06-4.02 (m, 1 H, H-3_A), 3.99-3.82 (m, 4 H, H-6ab_A, H-2_C, H-6a_B), 3.78-3.76 (m, 1 H, H-4_B), 3.75 (s, 3 H, OCH ₃), 3.68-3.66 (m, 2 H, H-2_A, H-2_B), 3.62-3.58 (m, 1 H, H-6b_B), 3.56-3.49 (m, 3 H, H-4_C, H-3_B, H-6a_C), 3.46-3.44 (m, 1 H, H-6b_C), 3.41-3.32 (m, 4 H, H-3_C, H-5_C, H-4_A, H-5_A), 3.30-3.28 (m, 1 H, H-5_B).

^¹³C NMR (75 MHz, CDCl₃): δ = 155.2-114.5 (Ar-C), 103.5 (C-1_C), 102.8 (C-1_A), 102.7 (C-1_B), 83.0 (C-2_A), 82.2 (C-5_A), 81.7 (C-5_C), 76.2 (C-3_A), 75.5 (PhCH₂), 75.0 (C-4_C), 74.7 (PhCH₂), 74.6 (PhCH₂), 73.5 (2 C, C-4_A, C-3_C), 73.4 (2 C, 2PhCH₂), 73.3 (C-5_B), 73.1 (C-3_B), 72.7 (PhCH₂), 72.1 (C-2_B), 71.2 (C-2_C), 68.4 (C-6_C), 68.3 (C-6_B), 68.1 (C-4_B), 67.8 (C-6_A), 55.4 (OCH₃).

ESI-MS: m/z = 1168.2 [M + NH₄]⁺.

Anal. Calcd for C₆₇H₇₄O₁₇ (1150.49): C, 69.90; H, 6.48. Found: C, 69.70; H, 6.72.

4-Methoxyphenyl (Methyl 5-Acetamido-4,7,8,9-tetra- O -acetyl-3,5-dideoxy-d-glycero-α- d -galacto-2-nonulopyranosylonate)-(2→3)-[(3,4,6-tri- O -benzyl-β- d -galactopyranosyl)-(1→6)]-(β- d -galactopyranosyl)-(1→4)-2,3,6-tri- O -benzyl-β- d -glucopyranoside (12)

To a soln of 11 (1 g, 0.87 mmol) and thioglycoside donor 7 (0.9 g, 1.7 mmol) in anhyd MeCN-CH₂Cl₂ (5:1; 20 mL) was added 3 Å MS (2 g), and the mixture was allowed to stir at r.t. under argon for 30 min. The mixture was cooled to -30 ˚C and NIS (500 mg, 2.3 mmol) and TMSOTf (15 µL) were added. After the mixture had stirred at the same temperature for 20 h, it was filtered through a Celite bed and washed with CH₂Cl₂ (100 mL). The organic layer was washed with 5% Na₂S₂O₃ (100 mL), sat. aq NaHCO₃ (100 mL), and H₂O (100 mL) in succession, dried (Na₂SO₄), and evaporated to dryness. The crude mass was purified by chromatography (silica gel, toluene-EtOAc, 1:2); this gave pure 12.

Yield: 650 mg (50%); R _f = 0.2 (toluene-EtOAc, 1:3); white solid; mp 127-29 ˚C; [α]_D ^²5 +8.4 (c 1.5, CHCl₃).

IR (KBr): 3020, 2925, 2855, 2364, 1742, 1506, 1370, 1217, 1062, 765, 669 cm^-¹.

^¹H NMR (300 MHz, CDCl₃): δ = 7.43-7.22 (m, 30 H, Ar-H), 7.01 (d, J = 9.0 Hz, 2 H, Ar-H), 6.77 (d, J = 9.1 Hz, 2 H, Ar-H), 5.46-5.44 (m, 1 H, H-8_D), 5.35-5.29 (m, 1 H, H-7_D), 5.04 (d, J = 12.1 Hz, 1 H, PhCH ₂), 4.98 (d, J = 12.5 Hz, 1 H, PhCH ₂), 4.88 (d, J = 5.0 Hz, 1 H, H-4_D), 4.85 (d, J = 8.6 Hz, 1 H, H-1_A), 4.82 (d, J = 10.3 Hz, 1 H, PhCH ₂), 4.79 (d, J = 10.6 Hz, 1 H, PhCH ₂), 4.72-4.62 (m, 4 H, PhCH ₂), 4.60 (d, J = 4.4 Hz, 1 H, H-1_B), 4.54 (d, J = 11.4 Hz, 1 H, PhCH ₂), 4.52 (d, J = 11.2 Hz, 1 H, PhCH ₂), 4.49 (d, J = 11.8 Hz, 1 H, PhCH ₂), 4.39 (d, J = 11.8 Hz., 1 H, PhCH ₂), 4.28 (dd, J = 10.1, 3.9 Hz, 1 H, H-9a_D), 4.18-3.97 (m, 7 H, H-1_C, H-4_B, H-9b_D, H-5_D, H-6_D, H-3_A, H-3_B), 3.87-3.78 (m, 4 H, H-2_C, H-2_B, H-6ab_A), 3.75-3.69 (m, 3 H, H-2_A, H-4_C, H-6a_B), 3.73 (s, 3 H, OCH ₃), 3.66-3.64 (m, 1 H, H-6b_B), 3.63 (s, 3 H, COOCH ₃), 3.56-3.53 (m, 2 H, H-4_A, H-6a_C), 3.50-3.45 (m, 2 H, H-3_C, H-6b_C), 3.37-3.29 (m, 3 H, H-5_A, H-5_B, H-5_C), 2.71 (dd, J = 12.3, 4.5 Hz, 1 H, H-3e_D), 2.10-1.89 (5 s, 15 H, 5 COCH ₃), 2.00-1.98 (m, 1 H, H-3a_D).

^¹³C NMR (75 MHz, CDCl₃): δ = 170.8, 170.4, 170.3, 169.9 (2 C), 168.1 (5 COCH₃, COOCH₃), 155.0-114.4 (Ar-C), 103.4 (C-1_C), 102.5 (C-1_A), 102.3 (C-1_B), 97.5 (C-2_D), 82.9 (C-2_A), 82.1 (C-5_A), 81.6 (C-5_C), 76.8 (C-3_A), 76.4 (C-4_B), 76.3 (PhCH₂), 75.6 (C-3_C), 75.1 (C-3_B), 74.8 (PhCH₂), 74.6 (PhCH₂), 73.4 (PhCH₂), 73.2 (C-4_A), 73.1 (C-5_B), 73.0 (PhCH₂), 72.6 (2 C, PhCH₂, C-6_D), 71.2 (C-2_B), 69.9 (C-2_C), 68.7 (C-6_C), 68.5 (C-8_D), 68.3 (C-6_B), 68.2 (C-4_D), 67.5 (C-6_A), 66.9 (2 C, C-7_D, C-4_C), 62.1 (C-9_D), 55.5 (OCH₃), 53.0 (COOCH₃), 49.3 (C-5_D), 37.7 (C-3_D), 23.0 (NHCOOCH₃), 21.1, 20.7, 20.6, 20.5 (4 COCH₃).

ESI-MS: m/z = 1646.5 [M + Na]⁺.

Anal. Calcd for C₈₇H₁₀₁NO₂₉ (1623.64): C, 64.31; H, 6.27; found: C, 64.12; H, 6.50.

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

A soln of 8 (3 g, 3.4 mmol) in 0.1 M NaOMe in MeOH (100 mL) was allowed to stir at r.t. for 2 h and then neutralized with Amberlite-IR 120 (H⁺) resin. The mixture was filtered and evaporated to dryness to give the crude product, which was passed through a short column of silica gel (hexane-EtOAc, 2:1); this gave pure 13.

Yield: 2.73 g (100%); R _f = 0.3 (hexane-EtOAc, 1:1); yellow oil; [α]_D ^²5 -25.3 (c 1.5, CHCl₃).

IR (neat): 3416, 2919, 2851, 2362, 1734, 1505, 1459, 1218, 1059, 766, 698 cm^-¹.

^¹H NMR (300 MHz, CDCl₃): δ = 7.48-7.23 (m, 20 H, Ar-H), 7.02 (d, J = 9.0 Hz, 2 H, Ar-H), 6.82 (d, J = 9.0 Hz, 2 H, Ar-H), 5.46 (s, 1 H, PhCH), 5.10 (d, J = 11.0 Hz, 1 H, PhCH ₂), 5.03 (d, J = 11.0 Hz, 1 H, PhCH ₂), 4.95 (d, J = 11.1 Hz, 1 H, PhCH ₂), 4.88 (d, J = 7.3Hz, 1 H, H-1_A), 4.83 (d, J = 11.0 Hz, 1 H, PhCH ₂), 4.71 (d, J = 12.1 Hz, 1 H, PhCH ₂), 4.59 (d, J = 11.0 Hz, 1 H, PhCH ₂), 4.56 (d, J = 7.5 Hz, 1 H, H-1_B), 4.11-4.01 (m, 4 H, H-3_A, H-4_B, H-6a_A, H-6a_B), 3.89-3.85 (m, 2 H, H-6b_A, H-6b_B), 3.78 (s, 3 H, OCH ₃), 3.76-3.72 (m, 3 H, H-2_A, H-5_A, H-5_B), 3.69-3.56 (m, 2 H, H-2_B, H-4_A), 3.48-3.39 (m, 1 H, H-3_B).

^¹³C NMR (75 MHz, CDCl₃): δ = 155.3-114.5 (Ar-C), 103.4 (C-1_B), 102.9 (C-1_A), 101.2 (PhCH), 83.4 (C-2_A), 81.8 (C-5_A), 77.5 (C-3_A), 75.5 (PhCH₂), 75.2 (C-4_B), 75.0 (PhCH₂), 74.7 (C-2_B), 73.3 (2 C, PhCH₂, C-4_A), 72.8 (C-3_B), 72.0 (C-5_B), 68.8 (C-6_A), 68.2 (C-6_B), 55.4 (OCH₃).

ESI-MS: m/z = 829.3 [M + Na]⁺.

Anal. Calcd for C₄₇H₅₀O₁₂ (806.33): C, 69.96; H, 6.25. Found: C, 69.75; H, 6.50.

4-Methoxyphenyl (2,3,4,6-Tetra- O -acetyl-β- d -galactopyranosyl)-(1→3)-(2- O -acetyl-4,6- O -benzylidene-β- d -galactopyranosyl)-(1→4)-2,3,6-tri- O -benzyl-β- d -glucopyranoside (14)

To a soln of 13 (2 g, 2.5 mmol) and thioglycoside donor 6 (1.1 g, 2.7 mmol) in CH₂Cl₂ (40 mL) was added 4 Å MS (5 g), and the mixture was allowed to stir at r.t. under argon for 30 min. The mixture was cooled to -50 ˚C and NIS (790 mg, 3.5 mmol) and TMSOTf (10 µL) were added. After the mixture had stirred at the same temperature for 30 min, it was filtered through a Celite bed and washed with CH₂Cl₂ (100 mL). The organic layer was washed with 5% Na₂S₂O₃ (100 mL), sat. aq NaHCO₃ (100 mL), and H₂O (100 mL) in succession, dried (Na₂SO₄), and evaporated to dryness. The crude mass was acetylated with Ac₂O (5 mL) and py (5 mL) at r.t. The acetylated crude mass was purified by chromatography (silica gel, hexane-EtOAc, 3:1); this gave pure 14.

Yield: 2.2 g (74%); R _f = 0.4 (hexane-EtOAc, 1:1); white solid; mp 130-32 ˚C; [α]_D ^²5 -3.7 (c 1.5, CHCl₃).

IR (KBr): 3465, 2871, 1751, 1507, 1370, 1226, 1061, 751, 699 cm^-¹.

^¹H NMR (300 MHz, CDCl₃): δ = 7.50-7.15 (m, 20 H, Ar-H), 6.98 (d, J = 9.0 Hz, 2 H, Ar-H), 6.77 (d, J = 9.0 Hz, 2 H, Ar-H), 5.50 (s, 1 H, PhCH), 5.35-5.34 (m, 1 H, H-4_C), 5.29-5.23 (m, 1 H, H-2_B), 5.15 (dd, J = 10.4, 8.0 Hz, 1 H, H-2_C), 5.06 (d, J = 11.1 Hz, 1 H, PhCH ₂), 4.98 (d, J = 11.1 Hz, 1 H, PhCH ₂), 4.93 (t, J = 5.3 Hz, 1 H, H-3_C), 4.82 (d, J = 11.1 Hz, 1 H, PhCH ₂), 4.80 (d, J = 7.5 Hz, 1 H, H-1_A), 4.78-4.71 (2 d, J = 11.8 Hz, 2 H, PhCH ₂), 4.62 (d, J = 7.9 Hz, 1 H, H-1_B), 4.58 (d, J = 8.0 Hz, 1 H, H-1_C), 4.51 (d, J = 11.9 Hz, 1 H, PhCH ₂), 4.24-4.08 (m, 4 H, H-6ab_C, H-6a_A, H-4_B), 3.97-3.87 (m, 3 H, H-3_A, H-6b_A, H-5_C), 3.82-3.72 (m, 3 H, H-6ab_B, H-5_A), 3.75 (s, 3 H, OCH ₃), 3.69-3.64 (m, 2 H, H-5_B, H-2_A), 3.60 (dd, J = 10.3, 3.5 Hz, 1 H, H-3_B), 3.47-3.44 (m, 1 H, H-4_A), 2.19, 2.06, 2.05, 2.04, 1.99 (5 s, 15 H, 5 COCH ₃).

^¹³C NMR (75 MHz, CDCl₃): δ = 170.1, 170.0, 169.9, 168.9, 168.5 (5 COCH₃), 155.3-114.5 (Ar-C), 102.7 (C-1_A), 101.6 (C-1_B), 100.9 (C-1_C), 100.6 (PhCH), 82.9 (C-2_A), 81.6 (C-5_B), 77.3 (C-3_A), 77.2 (C-3_B), 75.7 (C-4_B), 75.5 (PhCH₂), 75.1 (C-4_A), 74.9 (PhCH₂), 73.6 (PhCH₂), 70.9 (C-2_B), 70.8 (C-3_C), 70.7 (C-5_C), 68.4 (2 C, C-2_C, C-5_A), 67.9 (C-6_B), 66.8 (C-4_C), 61.1 (2 C, C-6_A, C-6_C), 55.4 (OCH₃), 21.0, 20.6 (2 C), 20.5 (2 C) (5 COCH₃).

ESI-MS: m/z = 1201.3 [M + Na]⁺.

Anal. Calcd for C₆₃H₇₀O₂₂ (1178.43): C, 64.17; H, 5.98. Found: C, 64.0; H, 6.20.

4-Methoxyphenyl (2,3,4,6-Tetra- O -acetyl-β- d -galactopyranosyl)-(1→3)-(2- O -acetyl-β- d -galactopyranosyl)-(1→4)-2,3,6-tri- O -benzyl-β- d -glucopyranoside (15)

HClO₄˙SiO₂ (500 mg) was added to a soln of 14 (2 g, 1.7 mmol) in MeCN-H₂O (9:1; 50 mL), and the mixture was allowed to stir at r.t. for 30 min. It was then filtered through a Celite bed and evaporated to dryness. The crude product was purified through a short pad of silica gel (toluene-EtOAc, 1:1); this gave pure 15.

Yield: 1.7 g (92%); R _f = 0.4 (toluene-EtOAc, 1:2); colorless oil; [α]_D ^²5 -12.3 (c 1.5, CHCl₃).

IR (neat): 3482, 2920, 2361, 1749, 1506, 1370, 1225, 1062, 755 cm^-¹.

^¹H NMR (300 MHz, CDCl₃): δ = 7.39-7.25 (m, 15 H, Ar-H), 6.98 (d, J = 9.0 Hz, 2 H, Ar-H), 6.78 (d, J = 9.0 Hz, 2 H, Ar-H), 5.35-5.34 (m, 1 H, H-4_C), 5.19-5.12 (m, 2 H, H-2_B, H-2_C), 4.99-4.94 (m, 3 H, H-3_C, 2 PhCH ₂), 4.81 (d, J = 8.1 Hz, 1 H, H-1_A), 4.78 (d, J = 10.6 Hz, 1 H, PhCH ₂), 4.76 (d, J = 12.3 Hz, 1 H, PhCH ₂), 4.73 (d, J = 12.0 Hz, 1 H, PhCH ₂), 4.53 (d, J = 8.0 Hz, 1 H, H-1_B), 4.49 (d, J = 11.2 Hz, 1 H, PhCH ₂), 4.46 (d, J = 7.8 Hz, 1 H, H-1_C), 4.19-4.05 (m, 2 H, H-6ab_C), 3.94-3.89 (m, 3 H, H-4_B, H-5_C, H-3_A), 3.77 (s, 3 H, OCH ₃), 3.75-3.68 (m, 2 H, H-6ab_A), 3.67-3.58 (m, 3 H, H-5_B, H-6a_B, H-2_A), 3.55-3.44 (m, 3 H, H-3_B, H-6b_B, H-4_A), 3.32-3.28 (m, 1 H, H-5_A), 2.16, 2.06, 1.98 (3 s, 15 H, 5 COCH ₃).

^¹³C NMR (75 MHz, CDCl₃): δ = 170.2, 170.0, 169.9, 169.0, 168.6 (5 COCH₃), 155.3-114.5 (Ar-C), 102.8 (C-1_A), 101.5 (C-1_B), 100.3 (C-1_C), 82.4 (C-2_A), 81.3 (C-5_B), 80.1 (C-3_B), 76.6 (C-3_A), 75.5 (PhCH₂), 75.1 (C-4_A), 74.9 (PhCH₂), 74.6 (C-5_A), 73.6 (PhCH₂), 71.1 (C-2_C), 70.9 (C-3_C), 70.6 (C-4_B), 68.7 (C-5_C), 68.5 (C-2_B), 67.9 (C-6_A), 66.9 (C-4_C), 61.6 (C-6_B), 61.3 (C-6_C), 55.5 (OCH₃), 20.8 (2 C), 20.6, 20.5, 20.4 (5 COCH₃).

ESI-MS: m/z = 1113.2 [M + Na]⁺.

Anal. Calcd for C₅₆H₆₆O₂₂ (1090.40): C, 61.64; H, 6.10. Found: C, 61.46; H, 6.32.

4-Methoxyphenyl (Methyl 5-Acetamido-4,7,8,9-tetra- O -acetyl-3,5-dideoxy- d -glycero-α- d -galacto-2-nonulopyranosylonate)-(2→6)-[(2,3,4,6-tetra- O -acetyl-β- d -galactopyranosyl)-(1→3)]-(2- O -acetyl-β- d -galactopyranosyl)-(1→4)-2,3,6-tri- O -benzyl-β- d -glucopyranoside (16)

To a soln of 15 (1 g, 0.92 mmol) and thioglycoside donor 7 (960 mg, 1.8 mmol) in anhyd MeCN-CH₂Cl₂ (5:1; 15 mL) was added 3 Å MS (2 g), and the mixture was allowed to stir at r.t. under argon for 30 min. The mixture was cooled to -30 ˚C and NIS (525 mg, 2.3 mmol) and TMSOTf (10 µL) were added. After the mixture had stirred at the same temperature for 3 h, it was filtered through a Celite bed and washed with CH₂Cl₂ (100 mL). The organic layer was washed with 5% Na₂S₂O₃ (100 mL), sat. aq NaHCO₃ (100 mL), and H₂O (100 mL) in succession, dried (Na₂SO₄), and evaporated to dryness. The crude mass was purified by chromatography (silica gel, toluene-EtOAc, 1:2); this gave pure 16.

Yield: 940 mg (67%); R _f = 0.3 (toluene-EtOAc, 1:3); white solid; mp 142-44 ˚C; [α]_D ^²5 -11.4 (c 1.5, CHCl₃).

IR (KBr): 3486, 2925, 1748, 1507, 1371, 1226, 1064, 741, 699 cm^-¹.

^¹H NMR (300 MHz, CDCl₃): δ = 7.39-7.24 (m, 15 H, Ar-H), 7.00 (d, J = 9.0 Hz, 2 H, Ar-H), 6.78 (d, J = 9.1 Hz, 2 H, Ar-H), 5.39-5.32 (m, 3 H, H-7_D, H-8_D, H-4_C), 5.26-5.15 (m, 3 H, H-2_C, H-2_B, H-6_D), 5.02-4.93 (m, 3 H, H-3_C, H-4_D, PhCH ₂), 4.86 (d, J = 7.7 Hz, 1 H, H-1_A), 4.85 (d, J = 11.3 Hz, 1 H, PhCH ₂), 4.77 (d, J = 11.1 Hz, 1 H, PhCH ₂), 4.75 (d, J = 11.0 Hz, 1 H, PhCH ₂), 4.69 (d, J = 8.2 Hz, 1 H, H-1_B), 4.65 (d, J = 8.0 Hz, 1 H, H-5_D), 4.57 (d, J = 8.0 Hz, 1 H, H-1_C), 4.53 (d, J = 12.7 Hz, 1 H, PhCH ₂), 4.35-4.25 (m, 1 H, H-6a_C), 4.24-4.18 (m, 1 H, H-6a_B), 4.13-4.04 (m, 4 H, H-5_C, H-9a_D, H-6b_C, H-6b_B), 3.95-3.90 (m, 3 H, H-4_B, H-5_A, H-9b_D), 3.82-3.72 (m, 2 H, H-6ab_A), 3.76 (s, 6 H, OCH ₃ and COOCH ₃), 3.71-3.62 (m, 2 H, H-5_B, H-2_A), 3.58-3.51 (m, 2 H, H-3_B, H-4_A), 3.38 (t, J = 7.0 Hz, 1 H, H-3_A), 2.55 (dd, J = 12.1, 4.4 Hz, 1 H, H-3e_D), 2.16-1.88 (10 s, 30 H, 10 COCH ₃), 1.99-1.89 (m, 1 H, H-3a_D).

^¹³C NMR (75 MHz, CDCl₃): δ = 170.9, 170.7, 170.5, 170.3 (2 C), 170.2, 170.1, 170.0, 169.3, 168.9, 167.9 (9 COCH₃, COOCH₃, NHCOCH₃), 155.3-114.5 (Ar-C), 102.6 (C-1_A), 101.5 (C-1_C), 100.0 (C-1_B), 99.0 (C-2_D), 82.4 (C-2_A), 81.7 (C-5_B), 79.5 (C-3_B), 76.4 (C-5_A), 74.9 (C-4_A), 74.8 (2 C, 2 PhCH₂), 73.7 (C-4_B), 73.6 (PhCH₂), 72.9 (C-5_C), 72.3 (C-3_A), 71.4 (C-2_B), 71.3 (C-2_C), 70.8 (2 C, C-3_C, C-4_D), 69.3 (C-4_C), 69.1 (C-6_D), 68.5 (C-9_D), 67.6 (C-7_D), 66.8 (C-8_D), 62.4 (C-6_C), 62.1 (C-6_B), 60.9 (C-6_A), 55.6 (OCH₃), 52.9 (COOCH₃), 49.4 (C-5_D), 37.0 (C-3_D), 22.7 (NHCOCH₃), 21.1, 21.0, 20.9, 20.8, 20.7, 20.6 (2 C), 20.5, 20.3 (9 COCH₃).

ESI-MS: m/z = 1581.1 [M + NH₄]⁺.

Anal. Calcd for C₇₆H₉₃NO₃₄ (1563.56): C, 58.34; H, 5.99. Found: C, 58.13; H, 6.25.

4-Methoxyphenyl (Sodium 5-Acetamido-3,5-dideoxy- d -glycero-α- d -galacto-2-nonulopyranosylonate)-(2→3)-[(β- d -galactopyranosyl)-(1→6)]-(β- d -galactopyranosyl)-(1→4)-β- d -gluco­-pyranoside (1)

To a soln of the tetrasaccharide derivative 12 (500 mg, 0.33 mmol) in MeOH (20 mL) was added 20% Pd(OH)₂/C (400 mg), and the mixture was allowed to stir at r.t. for 24 h under a positive pressure of H₂. The mixture was filtered through a Celite bed and concentrated under reduced pressure. The crude mass was dissolved in 0.1 M NaOMe in MeOH (30 mL), and the mixture was allowed to stir at r.t. for 12 h; then a few drops of distilled H₂O was added, and the mixture was allowed to stir for 6 h. The mixture was neutralized with Dowex 50W X8 (H⁺) resin, filtered, and evaporated to dryness and again passed through a short pad of Dowex 50W X8 (Na⁺) resin. The crude product was purified by being passed through a column of Sephadex-LH-20 (MeOH-H₂O, 4:1); this gave tetrasaccharide 1 as its sodium salt.

Yield: 215 mg (70%); R _f = 0.2 (MeCN-MeOH-H₂O, 4:1:0.5); white powder; [α]_D ^²5 -9.3 (c 1.1, H₂O).

IR (KBr): 3443, 3021, 1637, 1216, 767, 669 cm^-¹.

^¹H NMR (300 MHz, D₂O): δ = 7.01 (d, J = 9.0 Hz, 2 H, Ar-H), 6.80 (d, J = 9.0 Hz, 2 H, Ar-H), 4.79 (d, J = 7.3 Hz, 1 H, H-1_A), 4.42 (d, J = 7.9 Hz, 1 H, H-1_B), 4.28 (d, J = 7.0 Hz, 1 H, H-1_C), 4.06-3.96 (m, 2 H, H-8_D, H-7_D), 3.92-3.75 (m, 9 H, H-_4D, H-9a_D, H-6_D, H-5_D, H-3_A, H-6ab_B, H-6ab_A), 3.72-3.65 (m, 3 H, H-4_B, H-6ab_C), 3.71 (s, 3 H, OCH ₃), 3.65-3.55 (m, 5 H, H-2_B, H-9b_D, H-3_B, H-3_C, H-4_C), 3.52-3.43 (m, 6 H, H-2_A, H-2_C, H-5_B, H-5_A, H-5_C, H-4_A), 2.74 (dd, J = 12.1, 3.7 Hz, 1 H, H-3e_D), 1.97 (s, 3 H, NHCOCH ₃), 1.87 (t, J = 11.8 Hz, 1 H, H-3a_D).

^¹³C NMR (75 MHz, D₂O): δ = 174.0 (COONa), 155.6-114.5 (Ar-C), 104.3 (C-2_D), 104.2 (2 C, C-1_C, C-1_B), 101.9 (C-1_A), 81.2 (C-5_A), 76.5 (C-7_D), 75.5 (2 C, C-2_A, C-2_C), 75.2 (C-4_A), 74.3 (2 C, C-4_C, C-5_B), 73.6 (C-5_C), 73.4 (C-4_D), 71.6 (2 C, C-3_A, C-3_C), 69.5 (C-8_D), 69.3 (C-6_D), 69.0 (C-2_B), 68.8 (C-6_A), 68.4 (C-3_B), 67.7 (C-4_B), 63.7 (C-9_D), 61.5 (C-6_B), 61.0 (C-6_C), 55.1 (OCH₃), 52.7 (C-5_D), 39.9 (C-3_D), 22.6 (NHCOCH₃).

ESI-MS: m/z = 924.1 [M + 1]⁺.

Anal. Calcd for C₃₆H₅₄NNaO₂₅ (923.28): C, 46.81; H, 5.89. Found: C, 46.60; H, 6.15.

4-Methoxyphenyl (Sodium 5-acetamido-3,5-dideoxy- d -glycero-α-d-galacto-2-nonulopyranosylonate)-(2→6)-[(β- d -galactopyranosyl)-(1→3)]-(β- d -galactopyranosyl)-(1→4)-β- d -glucopyranoside (2)

To a soln of the tetrasaccharide derivative 16 (600 mg, 0.38 mmol) in MeOH (20 mL) was added 20% Pd(OH)₂/C (400 mg), and the mixture was allowed to stir at r.t. for 24 h under a positive pressure of H₂. The mixture was filtered through a Celite bed and concentrated under reduced pressure. The crude mass was dissolved in 0.1 M NaOMe in MeOH (30 mL), and the mixture was allowed to stir at r.t. for 12 h; then a few drops of distilled H₂O was added, and the mixture was allowed to stir for 6 h. The mixture was neutralized with Dowex 50W X8 (H⁺) resin, filtered, and evaporated to dryness, before it was again passed through a short pad of Dowex 50W X8 (Na⁺) resin. The crude product was purified by being passed through a column of Sephadex-LH-20 (MeOH-H₂O, 4:1); this gave tetrasaccharide 2 as its sodium salt.

Yield: 250 mg (72%); R _f = 0.3 (MeCN-MeOH-H₂O, 4:1:0.5); white powder; [α]_D ^²5 -8.7 (c 1.1, H₂O).

IR (KBr): 3021, 2926, 2401, 1216, 767, 670 cm^-¹.

^¹H NMR (300 MHz, D₂O): δ = 7.01 (d, J = 8.9 Hz, 2 H, Ar-H), 6.80 (d, J = 8.9 Hz, 2 H, Ar-H), 4.79 (d, J = 7.3 Hz, 1 H, H-1_A), 4.47 (d, J = 7.3 Hz, 1 H, H-1_B), 4.41 (d, J = 7.5 Hz, 1 H, H-1_C), 4.14-4.05 (m, 3 H, H-8_D, H-7_D, H-9a_D), 3.91-3.76 (m, 7 H, H-4_D, H-5_D, H-4_B, H-6_D, H-6ab_A, H-9b_D), 3.75-3.67 (m, 3 H, H-2_C, H-6ab_C), 3.71 (s, 3 H, OCH ₃), 3.64-3.57 (m, 6 H, H-3_A, H-2_B, H-5_B, H-3_C, H-6ab_B), 3.55-3.44 (m, 6 H, H-2_A, H-4_A, H-5_A, H-3_B, H-4_C, H-5_C), 2.73 (dd, J = 12.3, 4.2 Hz, 1 H, H-3e_D), 1.96 (s, 3 H, NHCOCH ₃), 1.75 (t, J = 11.9 Hz, 1 H, H-3a_D).

^¹³C NMR (75 MHz, D₂O): δ = 174.4 (COONa), 173.9 (NHCOCH₃), 155.6-114.5 (Ar-C), 105.3 (C-1_B), 103.7 (C-1_C), 101.9 (C-1_A), 99.5 (C-2_D), 83.4 (C-2_C), 80.5 (C-5_B), 75.7 (C-2_B), 75.2 (2 C, C-3_A, C-3_C), 74.1 (C-4_A), 73.7 (C-5_A), 73.5 (2 C, C-3_B, C-4_C), 71.9 (2 C, C-4_D, C-6_D), 70.4 (C-4_B), 69.3 (C-8_D), 69.2 (C-7_D), 68.8 (C-5_C), 68.3 (C-2_A), 63.6 (C-9_D), 63.1 (C-6_B), 61.5 (C-6_C), 60.9 (C-6_A), 55.1 (OCH₃), 52.6 (C-5_D), 40.8 (C-3_D), 21.9 (NHCOCH₃).

ESI-MS: m/z = 924.2 [M + 1]⁺.

Anal. Calcd for C₃₆H₅₄NNaO₂₅ (923.28): C, 46.81; H, 5.89. Found: C, 46.58; H, 6.18.

Acknowledgment

Instrumentation facilities from SAIF, CDRI, Lucknow is gratefully acknowledged. P.K.M. thanks CSIR, New Delhi for providing a Senior Research fellowship. A.K.M. thanks the Department of Science and Technology (DST), New Delhi for providing a Ramanna Fellowship (SR/S1/RFPC-06/2006).

References

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• 13b Konradsson P. Udodong UE. Fraser-Reid B. Tetrahedron Lett.  1990,  31:  4313 
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• 16a Hasegawa A. Kiso M. In Carbohydrates - Synthetic Methods and Applications in Medicinal Chemistry   Ogura H. Hasegawa A. Suami T. Wiley; New York: 1992.  p.243-266  
• 16b Hasegawa A. Kiso M. In Preparative Carbohydrate Chemistry   Hanessian S. Marcel Dekker; New York: 1997.  p.357-379  
• 16c Boons G.-J. Demchenko AV. Chem. Rev.  2000,  100:  4539 
  Search in Google Scholar
• 16d Kanie O. Kiso M. Hasegawa A. J. Carbohydr. Chem.  1988,  7:  501 
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• 16e Paulsen H. Tietz H. Angew. Chem., Int. Ed. Engl.  1982,  21:  927 
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• 16f Hasegawa A. Nagahama T. Ohki T. Hotta K. Ishida H. Kiso M. J. Carbohydr. Chem.  1991,  10:  493 
  Search in Google Scholar
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  Search in Google Scholar
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References

• 1a Newburg DS. Neubaur SH. Handbook of Milk Composition   Academic Press; San Diego: 1995.  p.273-349  
• 1b Stahl B. Thurl S. Zeng J. Karas M. Hillenkamp F. Steup M. Sawatzki G. Anal. Biochem.  1994,  223:  218 
  Search in Google Scholar
• 2a Holmgren J. Svennerholm AM. Lindblad M. Infect. Immun.  1983,  39:  147 
  Search in Google Scholar
• 2b Chaturvedi P. Warren CD. Altaye M. Morrow AL. Ruiz-Palacios G. Pickering LK. Newburg DS. Glycobiology  2001,  11:  365 
  Search in Google Scholar
• 2c Gothefors L. Olling S. Winberg J. Acta Pediatr. Scand.  1975,  64:  807 
  Search in Google Scholar
• 2d Andersson B. Porras O. Hanson LA. Lagergard T. Svanborg-Eden C. J. Infect. Dis.  1986,  153:  232 
  Search in Google Scholar
• 2e Coppa GV. Gabrielli O. Pierani P. Catassi C. Carlucci A. Giorgi P. Pediatrics  1993,  91:  637 
  Search in Google Scholar
• 2f Crane JK. Azar SS. Stam A. Newburg DS. J. Nutr.  1994,  124:  2358 
  Search in Google Scholar
• 2g Gyorgy P. Jeanloz RW. von Nicolai H. Zilliken F. Eur. J. Biochem.  1974,  43:  29 
  Search in Google Scholar
• 2h Ofek I. Beachey EH. Adv. Intern. Med.  1980,  25:  503 
  Search in Google Scholar
• 2i Laegreid A. Otnaess ABK. Fuglesang J. Pediatr. Res.  1986,  20:  416 
  Search in Google Scholar
• 2j Zopf D. Roth S. Lancet  1996,  347:  1017 
  Search in Google Scholar
• 2k Cravioto A. Tello A. Villafan H. Ruiz J. Del Vedovo S. Neeser J.-R. J. Infect. Dis.  1991,  163:  1247 
  Search in Google Scholar
• 2l Newburg DS. Pickering LK. McCluer RH. Cleary TC. J. Infect. Dis.  1990,  162:  1075 
  Search in Google Scholar
• 3 Coppa GV. Gabrielli O. Giorgi P. Catassi C. Montanari MP. Varaldo PE. Nichols BL. Lancet  1990,  335:  569 
  CrossrefSearch in Google Scholar
• 4a Feizi T. Nature  1985,  314:  53 
  Search in Google Scholar
• 4b Wieruszeski JM. Chekkor A. Bouquelet S. Montreuil J. Strecker G. Peter-Katalinic J. Egge H. Carbohydr. Res.  1985,  137:  127 
  Search in Google Scholar
• 4c Strecker G. Fievre S. Wieruszeski JM. Michalski JC. Montreuil J. Carbohydr. Res.  1992,  226:  1 
  Search in Google Scholar
• 4d Dua VK. Goso K. Dube VE. Bush CA. J. Chromatogr.  1985,  328:  259 
  Search in Google Scholar
• 4e Yamashita K. Mizuochi T. Kobata A. Methods Enzymol.  1982,  83:  105 
  Search in Google Scholar
• 5a Gopal PK. Gill HS. Br. J. Nutr., Suppl. 1  2000,  84:  S69 
  Search in Google Scholar
• 5b Sharon N. Ofek I. Glycoconj. J.  2000,  17:  659 
  Search in Google Scholar
• 5c Boedeker EC. Gastroenterology  1982,  83:  489 
  Search in Google Scholar
• 6a Urashima T. Saito T. Nakamura T. Messer M. Glycoconj. J.  2001,  18:  357 
  Search in Google Scholar
• 6b Messer M. Urashima T. Trends Glycosci. Glycotechnol.  2002,  14:  153 
  Search in Google Scholar
• 6c Guerardel Y. Morelle W. Plancke Y. Lemoine J. Strecker G. Carbohydr. Res.  1999,  320:  230 
  Search in Google Scholar
• 6d Saksena R. Deepak D. Khare A. Sahai R. Tripathi LM. Srivastava VML. Biochem. Biophys. Acta  1999,  1428:  433 
  Search in Google Scholar
• 6e Chaturvedi P. Sharma CB. Biochim. Biophys. Acta  1988,  967:  115 
  Search in Google Scholar
• 6f Urashima T. Bubb WA. Messer M. Tsuji Y. Taneda Y. Carbohydr. Res.  1994,  262:  173 
  Search in Google Scholar
• 7 Nakamura T. Urashima T. Trends Glycosci. Glycotechnol.  2004,  16:  135 
  CrossrefSearch in Google Scholar
• 8 Meijer A. Ellervik U. J. Org. Chem.  2004,  69:  6249 
  CrossrefSearch in Google Scholar
• 9 Yashunsky DU. Higson AP. Ross AJ. Nikolaev AV. Carbohydr. Res.  2001,  336:  243 
  CrossrefSearch in Google Scholar
• 10 Berces A. Whitfield DM. Nukada T. Santos IZ. Obuchowska A. Krepinsky JJ. Can. J. Chem.  2004,  82:  1157 
  CrossrefSearch in Google Scholar
• 11 Fried J. Walz DE. J. Am. Chem. Soc.  1949,  71:  140 
  CrossrefSearch in Google Scholar
• 12 Hasegawa A. Ohki H. Nagahama T. Ishida H. Kiso M. Carbohydr. Res.  1991,  212:  277 
  CrossrefSearch in Google Scholar
• 13a Veeneman GH. van Leeuwen SH. van Boom JH. Tetrahedron Lett.  1990,  31:  1331 
  Search in Google Scholar
• 13b Konradsson P. Udodong UE. Fraser-Reid B. Tetrahedron Lett.  1990,  31:  4313 
  Search in Google Scholar
• 14 Agnihotri G. Misra AK. Tetrahedron Lett.  2006,  47:  3653 
  CrossrefSearch in Google Scholar
• 15 Schmidt RR. Jung K.-H. Preparative Carbohydrate Chemistry   Hanessian S. Marcel Dekker; New York: 1997.  p.283-312  
• 16a Hasegawa A. Kiso M. In Carbohydrates - Synthetic Methods and Applications in Medicinal Chemistry   Ogura H. Hasegawa A. Suami T. Wiley; New York: 1992.  p.243-266  
• 16b Hasegawa A. Kiso M. In Preparative Carbohydrate Chemistry   Hanessian S. Marcel Dekker; New York: 1997.  p.357-379  
• 16c Boons G.-J. Demchenko AV. Chem. Rev.  2000,  100:  4539 
  Search in Google Scholar
• 16d Kanie O. Kiso M. Hasegawa A. J. Carbohydr. Chem.  1988,  7:  501 
  Search in Google Scholar
• 16e Paulsen H. Tietz H. Angew. Chem., Int. Ed. Engl.  1982,  21:  927 
  Search in Google Scholar
• 16f Hasegawa A. Nagahama T. Ohki T. Hotta K. Ishida H. Kiso M. J. Carbohydr. Chem.  1991,  10:  493 
  Search in Google Scholar
• 16g Kiso M. Ando K. Inagaki H. Ishida H. Hasegawa A. Carbohydr. Res.  1995,  272:  159 
  Search in Google Scholar
• 16h Castro-Palomino JC. Ritter G. Fortunato SR. Reinhardt S. Old LJ. Schmidt RR. Angew. Chem., Int. Ed. Engl.  1997,  36:  1998 
  Search in Google Scholar
• 16i Iida M. Endo A. Fujita S. Numata M. Sugimoto M. Nunomura S. Ogawa T. J. Carbohydr. Chem.  1998,  17:  647 
  Search in Google Scholar
• 17 Pearlman WM. Tetrahedron Lett.  1967,  8:  1663 
  CrossrefSearch in Google Scholar