Imposing the trans/gauche conformation on a sialic acid donor with a 5-N,7-O-oxazinanone group: effect on glycosylation stereoselectivity

Abstract

A 5-N,7-O-oxazinanone derivative of a thiosialic acid ester has been synthesized and investigated for the effect of conformational restriction on glycosylation. The cyclic group is found to be powerfully disarming, but to have no beneficial effect on reaction stereoselectivity.

1. Introduction

Sialic acid residues such as N-acetylneuraminic acid (Neu5Ac), normally incorporated into complex glycolipids or glycoproteins at the terminal positions of the oligosaccharide chains, are involved in a wide range of biological processes.¹ In these complex oligosaccharides, N-acetylneuraminic acid is typically linked α-(2,3) or α-(2,6) to galactoside residues, or is polymerized in the form of α-(2,8) or α-(2,9) linkages. Several challenges are associated with sialylations reactions arising from a number of factors: i) Frequent low yields due to the sterically encumbered C2-position. ii) H3 protons on ring C3 have the tendency to undergo 2,3-elimination during the coupling processes. iii) The contra-thermodynamic equatorial nature of the glycosidic bond that necessitates the overriding of the anomeric effect. Many innovative methodologies and strategies including chemical, enzymatic, and chemoenzymatic synthesis of these sialoconjugates have been developed for α-sialoside installation.² Recently, we directed our efforts to the α-sialylation field and have investigated sialoside donors 1³ based on the knowledge that fused structures in sugar donors have shown beneficial effects on either reactivities or selectivities in glycoslation reactions.⁴ This is exemplified by compounds such as the use of the 4,6-O-benzylidene-protected mannosyl triflate 2 in β-mannosylation, the corresponding α-selective glucosyl triflate 2,^5,6 the 2-N,3-O-carbamate fused glycosamine 4 as a α-selective glycosyl donor,^7–9 the 2,3-O-carbonate fused thioglycoside 5 as a β-selective glucosyl donor not dependent on neighboring participation group,⁹ and the 3,4-O-carbonate fused rhamnopyranose 6 as a β-selective donor.¹⁰

The Takahashi group also reported on the use of 5-N, 4-O-oxazolidinone protected sialic acids, including donor 7 and acceptors 8 and 9 and achieved an elegant synthesis of α-(2→8)-oligosialosides by this method.¹¹ A similar study on the stereoselective synthesis of α-(2 → 3) and α-(2 → 6) linked galactosyl sialosides was reported by De Meo and co-workers.¹² The 1-adamantanylthio sialoside 10 was developed later in this laboratory to address the poor selectivities encountered with donor 1 when coupling with secondary sugar acceptors. This improved donor could be activated by NIS/TfOH in nitrile solvents at −78 °C to afford coupled products with excellent α-selectivity in high yields.¹³ Inspired by these beneficial cyclic structures in sugar chemistry, we became interested in exploring alternative cyclic protected donors and especially in the use of the 5-N,7-O-oxazinanone derivative 11, which was designed to mimic a benzylidene acetal. We describe here the synthesis of donor 11 and its application in sialylation reactions under diphenyl sulfoxide (Ph₂SO) and trifilic anhydride (Tf₂O) promotion conditions.

2. Results and Discussion

The preparation of donor 11 started from the known thioglycoside 12,¹⁴ from which hydrolytic removal of acetyl group followed by selective acylation of the amine group with FmocOSu gave the intermediate carbamate. Further treatment with 2,2-dimethoxypropane in the presence of catalytic p-toluenesulfonic acid afforded thioglycoside 13 in 31% overall yield for the three steps.¹⁵ Treatment of 13 with TBSCl in DMF smoothly furnished TBS ether 14 in 81% yield.¹⁶ The silyl ether protecting group was selected over the more common acyl group to avoid the extra disarming property of the ester function.¹⁷ Removal of the Fmoc group with piperidine followed by 4-N,7-O-oxazinanone ring formation with 1,1’-carbonyldiimidazole in THF at reflux delivered the sialoside donor 11 in 66% overall yield.¹⁸ Establishment of the oxazinanone moiety in donor 11 was confirmed by chemical shift (δ 151.4–151.7) of the carbamate carbon, which was distinct from that of the alternative five-membered oxazolidinone expected to resonate around δ 160.0.^11,4 Of note in the final transformation is that the employment of 4-nitrophenyl chloroformate, a classical carbamate ring closure reagent,^3,7,11 was ineffective, providing 11 in only 30% yield, along with a significant amount of an unidentified byproduct.

With access to the 4-N,7-O-oxazinanone donor 11 established, we next investigated its coupling to a range of acceptors. To our surprise, promotion systems such as NIS/TfOH, which were adopted for activation of the 4-N,3-O-oxalidinone protected donors 1, 7 and 10 was ineffective towards donor 11 with the starting material recovered completely in repeated experiments. The BSP/TTBP/Tf₂O^6,19 promotion system, developed in this laboratory for glycosylation of thioglycoside donors also failed to activate 11. Accordingly we turned our attention to the more potent activation system Ph₂SO/Tf₂O, initially developed by Gin and co-workers²⁰ for dehyrative glycosylation and later extended by van Boom and coworkers to the glycosylation of thioglycosides,²¹ and applied to thiosialoside activation in this laboratory.²² Fortunately, following our previous protocol, treatment of sialoside donor 11 with Ph₂SO (3 equiv)/Tf₂O (1.2 equiv) in the presence of the hindered non-nucleophilic base 2,4,6-tri-tert-butylpyrimidine (TTBP) followed by subsequent addition of 1-butanol smoothly gave the coupled product 15 in excellent yield and moderate selectivity (Table 1, entry 1). Coupling to the tertiary alcohol 1-admantanol and to the protected secondary alcohol threonine favored the β-sialosides (Table 1, entry 2 and 5), and similar results were obtained in the case of secondary carbohydrate accepors (Table 1, entry 3 and 4). The anomeric stereochemistry of all coupling products was assigned based on the ³J_C1,H-3ax coupling constants.²³ While it is not clear whether the β-selectivity observed in the majority of these couplings is the result of the presence of the oxazinanone ring, or of the enforced use of the diphenyl sulfoxide activation system, as was the case with donor 1,³ it is clear that the cyclic protecting group is highly disarming. Thus, the failure to activate 11 by the NIS or BSP methods stands in contrast to the activation of 1 and other ester protected thiosialosides by these systems, and signals the importance of the conformational restriction imposed on 11 by the fused ring. We suggest that the disarming effect of the oxazinanone ring is due to the superposition of the most electron-withdrawing tg conformation of the C6–C7 bond with the presence of electron-withdrawing carbonyl group. This conformational effect on reactivity is closely related to that determined by Bols to be at the heart of the benzylidene effect in donors such as 2 and 3.²⁴

Table 1.

Ph₂SO/Tf₂O Promoted Coupling of Donor 1

	Acceptor	Coupled Product	% Yielda
			α:β ratiob
1	BuOH		85
			1.4:1
2			93
			β only
3			81
			β only
4			63
			β only
5			80
			β only

^aIsolated yields after chromatography.

^bDetermined by ¹H-NMR analysis on the crude reaction mixture, the anomeric stereochemistry is assigned based on the ³J_C1,H3ax coupling constant.

Hydrolysis of the oxazinanones was realized with the use of lithium hydroxide in hot aqueous ethanol. The use of either sodium methoxide in methanol or sodium hydroxide in aqueous THF at room temperature was found to complicate the reaction, affording undesired byproducts or more complex mixtures.

3. Conclusion

The 5-N,7-O-oxazinanone group is powerfully disarming in sialic acid donors owing to the combination its innate electron-withdrawing effect and the imposition of the tg conformation on the C6C7 bond.

4. Experimental Section

4.1. General Methods

¹H and ¹³C NMR spectra were recorded at 500 and 125 MHz, respectively, with chemical shifts reported downfield from tetramethylsilane. All solvents were dried by standard procedures. Commercial reagents were used without purification. Air and/or moisture-sensitive reactions were carried out under an atmosphere of argon or nitrogen using oven-dried glassware. High-resolution mass spectra were recorded with electrospray ionization.

4.2. Methyl [Phenyl 3,5-dideoxy-8,9-isopropylidene-5-(9-fluorenylmethoxycarbonylamino)-2-thio-d-glycero-β-d-galacto-non-2-ulopyranoside]onate (13)

A mixture of methyl (phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-d-glycerol-β-d-galacto-non-2-ulopyranoside)onate 12 (2.0 g, 2.9 mmol) and methanesulfonic acid (1.4 mL, 23.2 mmol) in MeOH (30 mL) was heated at 60 °C for 24 h. The reaction mixture was then cooled down to room temperature and concentrated under vacuum, affording a residue, which was redissolved in acetonitrile (20 mL) and water (3 mL) followed by neutralization with triethylamine until the pH was 7–8. 9-Fluorenylmethyl succinimidyl carbonate (1.2 g, 3.5 mmol) was added to the above solution at room temperature. After being stirred for 3 h, the solvent was removed under vacuum to give a residue, which was diluted with ethyl acetate (100 mL). The organic layer was washed with 0.5 M HCl solution, aq NaHCO₃, brine, dried over anhydrous Na₂SO₄, and concentrated. The crude residue was used for the next step without purification. To a stirred solution of the residue in DMF (8 mL) was added p-toluenesulfonic acid monohydrate (0.1 g, 0.5 mmol) followed by 2,2-dimethoxypropane (3 mL, 8.4 mmol). The resulting reaction mixture was stirred at room temperature for 30 min, then diluted with ethyl acetate (100 mL). The organic layer was washed twice with water, aq NaHCO₃, brine, dried over anhydrous Na₂SO₄, and concentrated. The residue was purified by chromatography on silica gel (hexane/ethyl acetate, 1:1) to give the title compound 13 (0.52 g, 0.9 mmol, 31%). [α]¹⁴ _D -190.0 (c, 1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.75 (d, J = 7.5 Hz, 2H), 7.52–7.58 (m, 4H), 7.27–7.40 (m, 7H), 5.05 (d, J = 7.5 Hz, 1H), 4.41–4.51 (m, 2H), 4.30 (d, J = 10.5 Hz, 1H), 4.20 (t, J = 6.5 Hz, 1H), 4.13 (t, J = 7.5 Hz, 1H), 4.03–4.08 (m, 1H), 3.94–4.01 (m, 2H), 3.74 (q, J = 9.0 Hz, 1H), 3.65 (br. s, 2H), 3.43 (s, 3H), 3.00 (br. s, 1H), 2.78 (dd, J = 4.5, 13.5 Hz, 1H), 2.08 (dd, J = 11.5, 13.5 Hz, 1H), 1.95 (br. s, 1H), 1.41 (s, 3H), 1.40 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 168.6, 157.6, 143.7, 143.5, 141.3, 136.2, 127.7, 129.6, 128.7, 127.8, 127.2, 125.0, 120.1, 109.0, 90.2, 74.6, 72.8, 70.4, 67.7, 67.2, 67.1, 54.6, 52.4, 47.1, 41.1, 26.9, 25.5; ESIHRMS Calcd for C₃₄H₃₇NO₉SNa [M+Na]⁺: 658.2082. Found 658.2083.

4.3. Methyl [Phenyl 3,5-dideoxy-8,9-isopropylidene-5-(9-fluorenylmethoxycarbonylamino)-4-O-tert-butyldimethylsilyl-2-thio-D-glycero-β-d-galacto-non-2-ulopyranoside]onate (14)

A mixture of compound 13 (0.34 g, 0.54 mmol), tert-butyldimethylsilyl chloride (0.12 g, 0.80 mmol) and imidazole (0.72 g, 1.08 mmol) in dry DMF (1 mL) was stirred at room temperature for 20 h, and then diluted with ethyl acetate (50 mL). The organic layer was washed twice with water, aq NaHCO₃, brine, dried over anhydrous Na₂SO₄, and concentrated. The residue was purified by chromatography on silica gel (hexane/ethyl acetate, 2:1) to give title compound 14 (0.33 g, 0.44 mmol, 81%). [α]²⁰ _D - 99.5 (c, 1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.77 (d, J = 7.5 Hz, 2H), 7.56–7.61 (m, 4H), 7.26–7.42 (m, 7H), 4.81 (d, J = 7.5 Hz, 1H), 4.51–4.55 (m, 1H), 4.42–4.46 (m, 2H), 4.39 (d, J = 10.5 Hz, 1H), 4.13–4.21 (m, 3H), 4.07 (dd, J = 5.5, 8.5 Hz, 1H), 3.96 (t, J = 9.0 Hz, 1H), 3.77 (t, J = 7.0 Hz, 1H), 3.60–3.67 (m, 2H), 3.43 (s, 3H), 2.68 (dd, J = 4.5, 13.5 Hz, 1H), 2.06 (dd, J = 11.5, 13.5 Hz, 1H), 1.45 (s, 3H), 1.43 (s, 3H), 0.86 (s, 9H), 0.10 (s, 3H), 0.06 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 168.6, 157.3, 143.8, 143.7, 141.4, 136.2, 129.9, 129.6, 128.7, 127.8, 127.2, 127.1, 125.0, 124.9, 120.1, 108.8, 90.3, 74.9, 72.9, 70.3, 67.8, 67.2, 66.9, 54.9, 52.4, 47.2, 42.1, 26.9, 25.7, 25.6, 17.9, −4.3, −4.7; ESIHRMS Calcd for C₄₀H₅₁NO₉SiSNa [M+Na]⁺: 772.2946. Found 772.2934.

4.4. Methyl [Phenyl 5-amino-5-N,7-O-carbonyl-3,5-dideoxy-8,9-isopropylidene-4-O-tert-butyldimethylsilyl-2-thio-d-glycero-β-D-galacto-non-2-ulopyranoside]onate (11)

To a stirred solution of compound 14 (0.4 g, 0.53 mmol) in dry DMF (4 mL) was added piperidine (0.8 mL, 7.7 mmol) at room temperature, the resulting reaction mixture was stirred at room temperature for 1 h, and then diuted with ethyl acetate (50 mL). The organic layer was washed twice with water, dried over anhydrous Na₂SO₄, and concentrated to afford the crude amino alcohol intermediate, which was purified by a short silica gel column (hexane/ethyl acetate, 1:1) to give 0.28 pure sample. To a stirred solution of this amino alcohol (0.28 g, 0.53 mmol) in dry THF (15 mL) was added 1,1’-carbonyldiimidazole (0.19 g, 1.2 mmol) at room temperature. The resultant reaction mixture was then heated to reflux for 48 h before being cooled down to room temperature, and the reaction solvent was removed under vacuum to give a residue, which was purified by chromatography on silica gel (hexane/ethyl acetate, 3:1) to afford donor 11 (0.19 g, 0.35 mmol, 66%) as a white solid. Mp 163–164 °C; [α]¹⁶_D -63.8 (c, 1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.31–7.38 (m, 5H), 5.26 (s, 1H), 4.76 (dd, J = 6.0, 10.5 Hz, 1H), 4.53 (t, J = 5.0 Hz, 1H), 4.45 (q, J = 6.0 Hz, 1H), 4.01–4.05 (m, 2H), 3.56 (s, 3H), 3.31 (t, J = 10.5 Hz, 1H), 2.57 (dd, J = 4.5, 14.0 Hz, 1H), 1.99 (dd, J = 11.0, 14.0 Hz, 1H), 1.37 (s, 3H), 1.34 (s, 3H), 0.91 (s, 9H), 0.17 (s, 3H), 0.13 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 168.0, 151.5, 135.3, 129.7, 129.3, 129.1, 109.7, 90.0, 75.0, 69.8, 66.1, 65.4, 55.1, 52.6, 40.2, 26.4, 25.7, 25.6, 17.9, −4.1, −4.6; ESIHRMS Calcd for C₂₆H₃₉NO₈SiSNa [M+Na]⁺: 576.2058. Found 576.2050.

4.5. General Procedure for Sialylation with Donor 11

A solution of donor 11 (50 mg, 0.09 mmol, 1 equiv), diphenyl sulfoxide (55 mg, 0.27 mmol, 3 equiv), TTBP (45 mg, 0.18 mmol, 2 equiv), and activated 4 Å molecular sieves in anhydrous CH₂Cl₂ (2 mL) was stirred at room temperature for 1 h under an argon atmosphere, and then cooled down to −78 °C, followed by addition of Tf₂O (18 µL, 0.11 mmol, 1.2 equiv). After 15 min, a solution of the acceptor (0.18 mmol, 2 equiv) in CH₂Cl₂ (1 mL) was added. The reaction mixture was stirred at −78 °C for 30 min, and then warmed up to 0 °C slowly over 1 h, and quenched with aq NaHCO₃, diluted with CH₂Cl₂ (20 mL), and filtered through Celite. The organic layer was washed with brine, dried over anhydrous Na₂SO₄ and concentrated. The crude product was purified by chromatography on silica gel to afford the sialosides, eluting with toluene/ ethyl acetate or toluene/acetone systems.

4.6. Methyl [2-butyl 5-amino-5-N,7-O-carbonyl-3,5-dideoxy-8,9-isopropylidene-4-O-tert-butyldimethylsilyl-d-glycerol-β-d-galacto-non-2-ulopyranoside]onate (15β) and Methyl [2-butyl 5-amino-5-N,7-O-carbonyl-3,5-dideoxy-8,9-isopropylidene-4-O-tert-butyldimethylsilyl-d-glycerol-α-d-galacto-non-2-ulopyranoside]onate (15α)

15β: [α]⁹_D 36.3 (c, 0.25, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 5.19 (s, 1H), 4.60 (t, J = 5.5 Hz, 1H), 4.46 (m, 1H), 4.05–4.14 (m, 2H), 3.93 (dd, J = 5.5, 10.5 Hz, 1H), 3.84 (s, 3H), 3.73–3.77 (m, 1H), 3.49–3.52 (m, 1H), 3.32–3.40 (m, 2H), 2.62 (dd, J = 4.5, 13.0 Hz, 1H), 1.76 (dd, J = 11.5, 13.0 Hz, 1H), 1.47–1.54 (m, 2H), 1.44 (s, 3H), 1.25–1.39 (m, 2H), 1.37 (s, 3H), 0.83–0.92 (m, 3H), 0.88 (s, 9H), 0.11 (s, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 168.7 (³J_C1,H-3ax = 0 Hz), 151.4, 110.0, 100.1, 75.7, 74.7, 70.3, 67.9, 66.1, 64.4, 54.4, 52.8, 40.2, 31.6, 26.4, 25.7, 25.6, 19.1, 17.9, 13.8, −4.2, −4.7; ESIHRMS Calcd for C₂₄H₄₄NO₉Si [M+H]⁺: 518.2780. Found 517.2779. 15α: [α]⁹_D 27.6 (c, 0.5, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 5.22 (s, 1H), 4.58 (dd, J = 4.5, 5.5 Hz, 1H), 4.50–4.53 (m, 1H), 4.07–4.14 (m, 2H), 3.98 (dd, J = 6.0, 10.5 Hz, 1H), 3.88–3.93 (m, 1H), 3.79 (s, 3H), 3.43–3.47 (m, 1H), 3.30–3.34 (m, 2H), 2.30 (dd, J = 4.5, 13.0 Hz, 1H), 1.66 (dd, J = 11.0, 13.0 Hz, 1H), 1.54–1.59 (m, 2H), 1.46 (s, 3H), 1.38 (s, 3H), 1.33–1.41 (m, 2H), 0.93 (t, J = 7.5 Hz, 3H), 0.89 (s, 9H), 0.13 (s, 3H), 0.11 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 167.9 (³J_C1,H-3ax = 6.25 Hz), 151.7, 109.7, 99.8, 75.4, 75.1, 69.4, 65.41, 65.37, 63.8, 54.9, 52.7, 40.3, 31.5, 26.3, 26.1, 25.7, 19.3, 17.9, 13.9, −4.1, −4.6; ESIHRMS Calcd for C₂₄H₄₄NO₉Si [M+H]⁺: 518.2780. Found 517.2773.

4.7. Methyl [2-(1-adamantanyl) 5-amino-5-N,7-O-carbonyl-3,5-dideoxy-8,9-isopropylidene-4-O-tert-butyldimethylsilyl-d-glycerol-β-d-galacto-non-2-ulopyranoside]onate (16)

[α]¹⁶_D 15.1 (c, 1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 5.22 (s, 1H), 4.57–4.59 (m, 1H), 4.50–4.54 (m, 1H), 4.22 (dd, J = 5.5, 10.5 Hz, 1H), 4.12–4.15 (m, 1H), 4.06–4.09 (m, 1H), 3.90 (dt, 4,5, 10.0 Hz, 1H), 3.77 (s, 3H), 3.18 (t, J = 10.0 Hz, 1H), 2.31 (dd, J = 4.5, 13.0 Hz, 1H), 2.10 (br. s, 3H), 1.78–1.85 (m, 6H), 1.54–1.61 (m, 6H), 1.43–1.48 (m, 1H), 1.45 (s, 3H), 1.38 (s, 3H), 0.88 (s, 9H), 0.12 (s, 3H), 0.09 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 170.0 (³J_C1,H-3ax = 2.5 Hz), 151.7, 109.5, 98.2, 78.3, 75.3, 75.1, 69.7, 65.3, 65.1, 55.0, 52.6, 43.2, 42.9, 36.0, 30.9, 26.4, 25.7, 17.9, −4.1, −4.6; ESIHRMS Calcd for C₃₀H₅₀NO₉Si [M+H]⁺: 596.3249. Found 596.3233.

4.8. Methyl O-[Methyl 5-amino-5-N,7-O-carbonyl-3,5-dideoxy-8,9-isopropylidene-4-O-tert-butyldimethylsilyl-d-glycerol-β-d-galacto-non-2-ulopyranosylonate]-(2→4)-2,3-isopropylidene-α-l-rhamnopyranoside (17)

[α]²⁰_D 1.2 (c, 1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 5.23 (s, 1H), 4.80 (s, 1H), 4.52 (br. s, 2H), 4.21–4.23 (m, 2H), 3.98–4.04 (m, 3H), 3.76–3.80 (m, 2H), 3.73 (s, 3H), 3.51 (t, J = 10.0 Hz, 1H), 3.38–3.44 (m, 1H), 3.35 (s, 3H), 2.32 (dd, J = 4.5, 13.5 Hz, 1H), 1.60 (dd, J = 11.0, 13.5 Hz, 1H), 1.44 (s, 3H), 1.43 (s, 3H), 1.38 (s, 3H), 1.29 (d, J = 6.0 Hz, 3H), 1.26 (s, 3H), 0.88 (s, 9H), 0.11 (s, 3H), 0.09 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 168.4 (³J_C1,H-3ax = 1.25 Hz), 151.6, 109.9, 109.2, 99.2, 97.9, 76.0, 75.5, 75.0, 74.9, 69.3, 66.3, 65.5, 63.9, 54.9, 54.5, 52.4, 41.6, 28.1, 26.4, 26.3, 25.8, 25.7, 18.4, 17.9, −4.1, −4.7; ESIHRMS Calcd for C₃₀H₅₂NO₁₃Si [M+H]⁺: 662.3202. Found 662.3199.

4.9. 3-O-[Methyl 5-amino-5-N,7-O-carbonyl-3,5-dideoxy-8,9-isopropylidene-4-O-tert-butyldimethylsilyl-D-glycerol-β-d-galacto-non-2-ulopyranosylonate]-(2→3)- 1,2:5,6-di-isopropylidene-α-D-glucofuranose (18)

[α]¹⁸_D 8.9 (c, 1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 5.82 (d, J = 3.5 Hz, 1H), 5.21 (s. 1H), 4.73 (dd, J = 5.5 Hz, 1H), 4.47–4.52 (m, 2H), 4.43 (d, J = 3.5 Hz, 1H), 4.25 (d, J = 4.0 Hz, 1H), 4.02–4.23 (m, 6H), 3.82–3.89 (m, 1H), 3.82 (s, 3H), 3.29 (t, J = 10.0 Hz, 1H), 2.44 (dd, J = 4.5, 14.0 Hz, 1H), 1.71 (dd, J = 11.0, 13.5 Hz, 1H), 1.47 (s, 3H), 1.43 (s, 3H), 1.41 (s, 3H), 1.36 (s, 3H), 1.31 (s, 3H), 1.25 (s, 3H), 1.21 (s, 3H), 0.89 (s, 9H), 0.12(s, 3H), 0.11 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 168.1 (³J_C1,H-3ax = 3.75 Hz), 151.5, 111.8, 110.1, 109.7, 104.7, 98.5, 82.7, 81.1, 75.5, 75.10, 75.05, 71.4, 69.6, 68.0, 65.5, 65.0, 54.5, 52.8, 40.4, 26.7, 26.4, 26.1, 25.7, 25.6, 24.8, 17.9, −4.0, −4.6; ESIHRMS Calcd for C₃₂H₅₄NO₁₄Si [M+H]⁺: 704.3308. Found 704.3312.

4.10. N-Benzoyl-O-[Methyl 5-amino-5-N,7-O-carbonyl-3,5-dideoxy-8,9-isopropylidene-4-O-tert-butyldimethylsilyl-d-glycerol-β-d-galacto-non-2-ulopyranosylonate]-l-threonine methyl ester (19)

[α]⁹_D 17.0 (c, 1.0, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.38–7.39 (m, 5H), 5.48 (d, J = 9.0 Hz, 1H), 5.22 (s, 1H), 5.13 (d, J = 1.5 Hz, 2H), 4.64 (t, J = 5.0 Hz, 1H), 4.49–4.52 (m, 1H), 4.41 (dd, J = 2.0, 9.5 Hz, 1H), 4.34–4.38 (m, 1H), 4.14–4.17 (m, 1H), 4.07 (dd, J = 6.5, 8.0 Hz, 1H), 3.88 (dd, J = 6.0, 10.5 Hz, 1H), 3.76 (s, 3H), 3.74 (s, 3H), 3.69–3.73 (m, 1H), 3.34 (t, J = 10.0 Hz, 1H), 2.29 (dd, J = 4.5, 13.0 Hz, 1H), 1.56 (dd, J = 11.0, 13.5 Hz, 1H), 1.45 (s, 3H), 1.37 (s, 3H), 1.12 (d, J = 6.0 Hz, 3H), 0.89 (s, 9H), 0.13 (s, 3H), 0.11 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 170.5 (³J_C1,H-3ax = 2.5 Hz), 168.5, 156.4, 151.5, 136.0, 128.6, 128.4, 128.3, 109.8, 98.1, 78.1, 76.4, 75.2, 74.9, 70.1, 69.3, 67.5, 66.4, 65.4, 58.8, 54.4, 52.9, 52.6, 40.9, 26.3, 25.71, 25.66, 17.9, 16.0, −4.2, −4.7; ESIHRMS Calcd for C₃₃H₅₁N₂O₁₃Si [M+H]⁺: 711.3155. Found 711.3147.

4.11. 2-(1-Adamantanyl) 5-amino-3,5-dideoxy-d-glycerol-β-d-galacto-non-2-ulopyranosidic Acid (20)

To a stirred solution of compound 16 (28 mg, 0.05 mmol) in ethanol(2 mL) and water (2 mL) added LiOH (34 mg, 1.5 mmol) at room temperature. The reaction mixture was then heated at 80 °C for 3 h, and cooled down to room temperature followed by removal of the solvent under reduced pressure. The residue was purified by chromatography on silica gel (ethyl acetate/isopropanol/water, 9:4:2) to give title compound 20 (17 mg, 82%).[α]¹⁹_D -19.0 (c, 1.0, MeOH); ¹H NMR (500 MHz, MeOD) δ 4.35 (br. s, 1H), 4.00–4.17 (m, 4H), 3.45 (d, J = 8.0 Hz, 1H), 3.03 (t, J = 8.0 Hz, 1H), 2.45 (d, J = 11.0 Hz, 1H), 1.99–2.14 (m, 9H), 1.58–1.67 (m, 6H), 1.39 (s, 3H), 1.31 (s, 3H), 1.31–1.39 (m, 1H); ¹³C NMR (125 MHz, MeOD) δ 176.3, 109.3, 99.0, 76.4, 73.5, 70.9, 69.9, 67.6, 65.5, 53.5, 43.7, 42.4, 35.9, 31.2, 25.9, 24.1; ESIHRMS Calcd for C₂₂H₃₆NO₈ [M+H]⁺: 442.2435. Found 442.2421.

4.12. Methyl O-(5-amino-dideoxy-d-glycerol-β-d-galacto-non-2-ulopyranosylonate)-(2→4)-2,3-isopropylidene-α-l-rhamnopyranoside (21)

To a stirred solution of compound 18 (30 mg, 0.05 mmol) in ethanol(2 mL) and water (2 mL) added LiOH (33 mg, 1.5 mmol) at room temperature. The reaction mixture was then heated at 80 °C for 3 h , and cooled down to room temperature followed by removal of the solvent under reduced pressure. The residue was purified by chromatography on silica gel (ethyl acetate/isopropanol/water, 9:4:2) to give title compound 21 (19 mg, 83%).[α]¹⁹_D -61.3 (c, 1.0, MeOH); ¹H NMR (500 MHz, MeOD) δ 4.65 (s, 1H), 4,53 (br. s, 1H), 4.36 (br. s, 1H), 4.16 (br.s, 1H), 3.85–4.02 (m, 6H), 3.46 (d, J = 8.5 Hz, 1H), 3.37 (s, 3H), 3.05 (t, J = 9.0 Hz, 1H), 2.48 (d, J = 12.5 Hz, 1H), 1.95 (s, 1H), 1.55 (t, J = 12.0 Hz, 1H), 1.47 (s, 3H), 1.40 (s, 3H), 1.37 (s, 3H), 1.34 (s, 3H), 1.29 (s, 3H); ¹³C NMR (125 MHz, MeOD) δ 174.1, 109.6, 108.5, 100.1, 98.5, 76.7, 75.3, 73.7, 72.9, 70.7, 70.6, 67.5, 67.0, 65.6, 54.2, 53.2, 42.4, 26.7, 26.0, 24.9, 24.3, 17.2; ESIHRMS Calcd for C₂₂H₃₇NO₁₂Na [M+Na]⁺: 530.2208. Found 530.2197.

Scheme 1.

Synthesis of Sialoside Donor 11

Scheme 2.

Hydrolysis of Oxazinanone Groups in Compounds 16 and 17.