Unusual C-C Bond Cleavage of an α-Trifloxy Sialic Acid Hemiacetal Under Lattrell-Dax Conditions

Abstract

We describe the novel oxidative fragmentation of methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-5-deoxy-3-O-trifluoromethanesulfonyl-β-d-erythro-l-gluco-2-nonulopyranos)onate 2 on stirring with sodium nitrite in DMF to give the novel 3-acetamido-2,5,6,7-tetra-O-acetyl-d-glycero-d-galacto-heptono-1,4-lactone 3 in excellent yield. Stirring of the same triflate with sodium carbonate on the other hand affords the novel methyl (5-acetamido-7,8,9-tri-O-acetyl-3,6-anhydro-5-deoxy-d-manno-3-ene-2-nonulos)onate 19 also in excellent yield. Reduction of the heptono lactone with sodium borohydride followed by acetylation gives a peracetylated aminodeoxyheptitol 6 that adopts the zig zag conformation of its carbon backbone.

Graphical Abstract

Introduction

The Lattrell-Dax reaction is a widely applied process for the inversion of configuration of a secondary alcohol that involves conversion of the said alcohol to the corresponding triflate ester followed by displacement with nitrite ion.^{1, 2} The sensitivity of the reaction to vicinal functionality, with esters being preferred over ethers, has been systematically studied by Ramström and coworkers,³ leading to a proposal for the underlying chemoselectivity of the ambident nitrite nucleophile and the facile hydrolysis of the resulting nitrite ester in the presence of an appropriately-placed ester.⁴ In the absence of a vicinal ester, side reactions including elimination of the trifloxy group and ring contraction,^{5, 6} and even the formation of nitroalkanes have been reported,⁴ but we are not familiar with any instances of oxidative C-C bond cleavage such as we report here.

Results and Discussion.

In the course of an ongoing investigation we had occasion to prepare the neuraminic acid derivative 1, with the d-erythro-l-gluco configuration, according to the literature procedure.^{7, 8} Seeking to invert the configuration at C3 to give the d-erythro-l-manno configuration overall, we prepared and isolated the 3-O-triflate 2 in 82% yield by reaction with triflic anhydride in the presence of pyridine in the usual manner. Subsequent exposure to sodium nitrite in DMF between 0 °C and room temperature over 16 h gave not the anticipated inversion product, but a substance identified as the d-glycero-d-galacto-γ-heptonolactone 3 in 93% yield (Scheme 1). Multiple derivatives of d-glycero-d-galacto heptose have been prepared by Kiliani-Fischer and Sowden-Fischer homologation of N-acetyl-d-mannosamine derivatives en route to the synthesis of N-acetylneuraminic acid,^9–12 and by asymmetric synthesis methods,¹³ as well as by ozonolytic cleavage of the enol form of 5-acetamido-3-deoxy-d-glycero-d-galacto-2-nonulosono-1,4-lactone (N-acetylneuraminic acid 1,4-lactone),¹⁴ but to our knowledge this is the first description of the furanolactone form.

Scheme 1.

Formation and Oxidative Cleavage of Triflate 2.

To verify the configuration of 3, diol 1 was stirred with Dess-Martin periodinane in dichloromethane between 0 °C and room temperature for 6 h after which the aldehydo-oxalate 4 was isolated in 85% yield (Scheme 2). Although not a common transformation, the Dess-Martin periodinane is known to cleave vicinal diols,^{15, 16} including α-hydroxy hemiacetals,^17–19 analogously to the more common periodate ion.²⁰ The acyclic aldehyde 4 displayed a ³J_2,3-coupling constant of 2.0 Hz consistent with the depicted zigzag conformation of its carbon backbone as previously reported²¹ for the N-acetylneuraminic acid derived acid 5 and most readily understood in terms of the arabino configuration spanning C1-C5, which lacks destabilizing 1,5-syn interactions.^{22, 23}

Scheme 2.

Oxidative Cleavage of 1 with Dess-Martin Periodinane and Structure of the Neuraminic Acid Derivative 5.

Treatment of 4 with sodium borohydride in ethanol followed by acetylation gave the peracetyl amino-deoxy d-glycero-d-galacto-heptitol 6 in 77% over the two steps. The identical heptitol 6 was obtained on reduction of γ-lactone 3 with NaBH₄ followed by acetylation in 74% yield, thereby confirming the configuration of 3. Finally, hydrolysis of 6 with sodium hydroxide in hot methanol enabled the isolation of the acetamido-deoxy d-glycero-d-galacto heptitol 7 in 82% yield (Scheme 3). Heptitol 7 and its peracetylated derivative 6 both adopt an extended zigzag conformation of the carbon backbone in solution as readily apparent from their NMR spectra, consistent with their relationship to the deoxy nonuronic acid 5, and d-glycero-d-galacto heptitol itself and its zigzag conformation in solution;²⁴ clearly the substitution of a hydroxy group in a linear polyol by an acetamido group with the same configuration does not affect the overall conformation significantly. In the same vein, it is noteworthy that the peracetylated 6 and the free polyol 7 adopt the same backbone conformation, just as free and protected galacto and glucopyranose derivatives show only minor protecting group-induced variations in the population distribution of their side chain conformations.²⁵

Scheme 3.

Synthesis of peracetyl amino polyol 6 from 3 and 4, and reduction to acetamido heptitol 7.

Turning to the mechanism of formation of 3, we hypothesize that triflate 2, which is stable in DMF at room temperature for extended periods of time in the absence of added reagents, reacts with sodium nitrite in the standard Lattrell-Dax manner to give the inverted nitroso ester 8, after which several possibilities can be considered. In a first possibility the nitroso ester may undergo fragmentation with C-C bond cleavage (Scheme 4) in a variation on the Grob fragmentation,²⁶ that is facilitated by the antiperiplanar arrangement of all necessary orbitals, and driven by rupture of the weak²⁷ N-O bond. Alternatively, although less likely in view of the use of Pyrex glassware and the UV/visible spectrum of nitrite esters, a radical fragmentation initiated by homolytic cleavage of the N-O bond is possible (Scheme 5).²⁸ Recombination of the radical 10 formed on fragmentation with the nitrosyl radical would then give the tetrahedral intermediate 11, susceptible to collapse either to the aldehyde-oxalate 9 or the hydroxy-aldehyde 12, no doubt in equilibrium with the hemiacetal 13.

Scheme 4.

Hypothetical Grob-like Fragmentation of the Derived Nitrite Ester 4.

Scheme 5.

Hypothetical Radical Fragmentation of the Derived Nitrite Ester 8.

Both mechanisms require further oxidation of the aldehyde functionality (as in 9 or 12), or in the case of the radical fragmentation, of lactol 13. One possibility for this oxidation invokes nucleophilic attack of the nitrite anion on the aldehyde to give an α-hydroxy nitrite derivative 14, which fragments to the acid 15 (Scheme 6). A parallel mechanism is readily envisaged from lactol 13 and passing through the glycosyl nitrite ester 16. Yet another possibility involves the oxidative fragmentation of nitrite ester 8 to give the ketone 17 followed by formation of the α-hydroxy nitrite ester 18 and Grob type fragmentation, akin to that envisaged for the conversion of 8 to 9. The oxidative fragmentation of glycosyl nitrite ester 16 has the advantage of directly affording the observed lactone 3, while all other mechanisms passing through an acid 15 necessarily invoke subsequent lactonization. The direct Grob fragmentation of Scheme 4 also requires hydrolysis of the labile oxalate ester either in situ or on work up. Regarding the proposed oxidation of aldehydes to carboxylic acids by sodium nitrite, the high yield oxidation of aliphatic aldehydes to carboxylic acids with sodium nitrite in trifluoroacetic acid under air at 0 °C has been described, but is considered to involve the nitrosonium ion as active oxidant.²⁹ The clean and functional group tolerant oxidant of primary, allylic and benzylic alcohols with sodium nitrite in acetic anhydride stops at the level of the aldehyde,³⁰ and clearly does not involve free nitrite ion. The mechanism proposed for the oxidative fragmentation of aldehydes and ketones in Scheme 6 on the other hand employs the nitrite anion itself and finds parallel in the Pinnick oxidation of aldehydes by the chlorate anion.^{31, 32}

Scheme 6.

Hypothetical Mechanism for Aldehyde to Acid Oxidation, and Structure of a Potential Glycosyl Nitrite Ester 16, Ketone 17, and α-Hydroxy Nitrite Ester 18.

In an attempt to shed light on the mechanism of the fragmentation reaction and to probe the requirement for nitrite, we stirred triflate 2 with sodium carbonate in DMF under otherwise identical conditions. After 1.5 h we isolated the alternative ring contraction product 19 in 90% yield, whereas prolonged stirring at 50 °C gave the furan 20 in 74% yield, presumably by adventitious aerial oxidation of the vinylogous enolate of 19 (Scheme 7). Finally, we conducted the nitrite-mediated reaction of Scheme 1 in the presence of radical inhibitors. In the presence two equivalents of butylated hydroxytoluene, the dihydrofuran 19 was formed in >90% yield in the crude reaction mixture after 1.5 h, whereas in the presence of tetramethylpiperidinoxyl (TEMPO) a complex mixture that was not pursued further was observed after 5 h. Together these experiments point to the need for sodium nitrite for the oxidative cleavage of 2 to 3 and suggest the possibility of the intervention of radicals at one or more stages of the mechanism.

Scheme 7.

Formation of Dihydrofuran 19 and Furan 20 from Triflate 2 Under Basic Conditions

Conclusion

We describe the unexpected oxidative fragmentation of the N-acetylneuraminic acid-derived triflate 2 to the novel d-glycero-d-galacto-γ-heptonolactone 3 in excellent yield on simple stirring in DMF with sodium nitrite. Treatment of triflate 2 with sodium carbonate in DMF on the other hand provokes ring contraction and elimination to give the dihydrofuran 19, also in excellent yield. Prolonged stirring of 2 in DMF with gentle heating in the presence of sodium carbonate results in the more highly oxidized furan 20 in 74% yield. Reduction of lactone 3 with sodium borohydride followed by acetylation gives a 77% yield of the novel peracetylated polyol 3-amino-3-deoxy d-glycero-d-galacto-heptitol 6 which, like its hydrolyzed variant 7, displays a zig zag conformation of its carbon backbone.

Experimental

General Experimental

All experiments were carried out under a dry argon atmosphere unless otherwise specified. Chromatographic purifications were carried over silica gel (230–400 mesh). Thin layer chromatography was performed with precoated glass backed plates (w/UV 254). TLC plates were visualized by UV irradiation (254 nm) and by charring with sulfuric acid in ethanol (20:80, v/v) or with ceric ammonium molybdate solution [Ce(SO4)₂: 4 g, (NH₄)₆Mo₇O₂₄: 10 g, H₂SO₄: 40 mL, H₂O: 360 mL]. Optical rotations were measured at 589 nm and 21 °C on a digital polarimeter with a path length of 10 cm. NMR spectra were recorded in CDCl₃, C₆D₆, D₂O, or CD₃OD as indicated using a 500, 600, or 900 MHz instrument and assignments made with the help of COSY, HMBC, and HSQC spectra. Chemical shifts (δ) are given in ppm, with multiplicities abbreviated as follows: s (singlet), m (multiplet), br (broad), d (doublet), t (triplet), q (quartet) and sept (septet). High-resolution (HRMS) mass spectra were recorded in the electrospray mode with an Orbitrap analyzer. Heating of reaction mixtures was carried out on an aluminum heating block of appropriate size.

Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-5-deoxy-3-O-(trifluoromethyl)sulfonyl-β-d-erythro-l-gluco-2-nonulopyranos)onate (2):

A stirred solution of 2,3-dihydroxy N-acetylneuraminic acid derivative 1 (350 mg, 0.69 mmol, 1.0 equiv) in anhydrous DCM (4.6 mL) was cooled to 0 °C (crushed ice bath) and treated with anhydrous pyridine (0.17 mL, 2.07 mmol, 3.0 equiv). Trifluoromethanesulfonic anhydride (0.14 mL, 0.83 mmol, 1.2 equiv) was then added dropwise over a period of 5 min. After stirring at 0 °C for 0.5 h, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with aqueous 1M HCl solution (5 mL), saturated aqueous NaHCO₃ solution (5 mL) and saturated aqueous NaCl solution (5 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered and concentrated to dryness under reduced pressure. Triflate 2 was obtained by silica gel column chromatography using gradient elution from 90 % EtOAc in to 100 % EtOAc; white powder (361 mg, 82 % yield), analytical TLC on silica gel, EtOAc, R_f =0.6. ${\left[\alpha \right]}_D^{22}-10.39\left(c1.3,{\text{CHCl}}_{\text{3}}\right)$. IR (cm⁻¹): 1754, 1662, 1372, 1216; ¹H NMR (600 MHz, CDCl₃) δ 6.05 (d, J = 10.2 Hz, 1H), 5.38 (d, J = 10.0 Hz, 1H), 5.35 – 5.33 (m, 1H), 5.27 (d, J = 9.8 Hz, 1H), 5.18 (ddd, J = 7.8, 5.8, 2.3 Hz, 1H), 4.46 – 4.41 (m, 1H), 4.39 (t, J = 10.3 Hz, 1H), 4.30 (dd, J = 10.7, 2.4 Hz, 1H), 3.97 (dd, J = 12.5, 7.3 Hz, 1H), 3.94 (s, 3H), 2.15 (s, 3H), 2.11 (s, 3H), 2.08 (s, 3H), 2.00 (s, 3H), 1.89 (s, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 170.9, 170.7, 170.2, 170.0, 166.6, 93.4, 80.2, 70.6, 70.2, 67.2, 62.3, 54.5, 49.2, 23.0, 20.9, 20.7, 20.6, 20.4; ¹⁹F NMR (282 MHz, CDCl₃) δ −74.98; HRMS (ESI/Q-TOF): m/z [M+Na]⁺ Calcd for C₂₁H₂₈O₁₆NF₃NaS 662.0973; Found 662.0953.

3-Acetamido-2,5,6,7-tetra-O-acetyl-d-glycero-d-galacto-heptono-1,4-lactone (3):

A stirred solution of compound 2 (75 mg, 0.12 mmol, 1.0 equiv) in anhydrous DMF (0.6 mL) was cooled to 0 °C (crushed ice bath) and treated with NaNO₂ (24 mg, 0.32 mmol, 3.0 equiv). After stirring at RT for 16 h, the reaction mixture was diluted with ethyl acetate (10 mL) and washed with 5% aqueous NaCl solution (3 mL) followed by saturated aqueous NaCl solution (3 mL×3). The organic layer was dried over anhydrous Na₂SO₄, filtered and concentrated to dryness under reduced pressure. Pure product 3 was obtained by silica gel column chromatography using gradient elution from 80 % EtOAc to 100 % EtOAc; white powder (45 mg, 93% yield), analytical TLC on silica gel, EtOAc, R_f=0.65. ${\left[\alpha \right]}_D^{22}+1.2\left(c0.67,{\text{CHCl}}_{\text{3}}\right)$. IR (cm⁻¹): 1806, 1748, 1372, 1218; ¹H NMR (600 MHz, CDCl₃) δ 6.16 (d, J = 7.4 Hz, 1H), 5.87 (d, J = 9.2 Hz, 1H), 5.34 (dd, J = 6.6, 2.2 Hz, 1H), 5.27 (td, J = 6.1, 2.7 Hz, 1H), 4.76 (dd, J = 8.7, 2.2 Hz, 1H), 4.40 (dd, J = 12.6, 2.8 Hz, 1H), 4.26 – 4.13 (m, 2H), 2.17 (s, 3H), 2.16 (s, 3H), 2.09 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 171.2, 170.6, 170.2, 169.9, 169.8, 168.7, 76.1, 70.4, 69.8, 67.9, 61.6, 53.3, 23.2, 20.8, 20.7, 20.6, 20.5; HRMS (ESI/Q-TOF): m/z [M+Na]⁺ Calcd for C₁₇H₂₃O₁₁NNa 440.1163; Found 440.1153.

3-Acetamido-2,5,6,7-tetra-O-acetyl-3-deoxy-4-O-(methyl oxalyl)-d-glycero-d-galacto-heptose (4):

A stirred solution of compound 1 (200 mg, 0.39 mmol, 1.0 equiv) in CH₂Cl₂ (4.0 mL) was cooled to 0 °C (crushed ice bath) and treated with Dess–Martin periodinane (424 mg, 0.79 mmol, 2.0 equiv). After stirring at RT for 6 h the reaction mixture was concentrated to dryness under reduced pressure. Pure product 4 was obtained by silica gel column chromatography using elution EtOAc; white foam (170 mg, 85 % yield), analytical TLC on silica gel, EtOAc, R_f=0.5. ${\left[\alpha \right]}_D^{22}+5.56\left(c1.33,{\text{CHCl}}_{\text{3}}\right)$. IR (cm⁻¹): 1748, 1667, 1372, 1218; ¹H NMR (600 MHz, CDCl₃) δ 9.43 (s, 1H), 5.80 (d, J = 10.3 Hz, 1H), 5.51 (dd, J = 10.0, 2.2 Hz, 1H), 5.45 (dd, J = 7.2, 2.2 Hz, 1H), 5.17 (td, J = 10.2, 2.1 Hz, 1H), 5.14 (d, J = 2.0 Hz, 1H), 5.09 (ddd, J = 7.2, 5.5, 3.3 Hz, 1H), 4.35 (dd, J = 12.5, 3.4 Hz, 1H), 3.98 (dd, J = 12.5, 5.6 Hz, 1H), 3.93 (s, 3H), 2.18 (s, 3H), 2.14 (s, 3H), 2.04 (d, J = 1.2 Hz, 6H), 1.92 (s, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 193.8, 170.7, 169.9, 169.87, 169.80, 169.2, 156.7, 156.5, 76.3, 70.9, 69.2, 67.2, 61.7, 54.0, 45.6, 22.9, 20.7, 20.7, 20.67, 20.2; HRMS (ESI/Q-TOF): m/z [M+H]⁺ Calcd for C₂₀H₂₈O₁₄N 506.1504; Found 506.1506.

3-Acetamido-1,2,4,5,6,7-hexa-O-acetyl-3-deoxy-d-glycero-d-galacto-heptitol (6):

A stirred solution of compound 4 (100 mg, 0.2 mmol, 1.0 equiv) in H₂O/EtOH (2.0 mL, 1:1 v/v) was cooled to 0 °C (crushed ice bath) and treated with NaBH₄ (22 mg, 0.59 mmol, 3.0 equiv). After stirring at 0 °C for 6 h the reaction mixture was concentrated to dryness under reduced pressure, co-evaporated with MeOH (10 mL×5) and dried under high vacuum to give the crude compound as a white powder.

The above obtained crude compound was dissolved in anhydrous pyridine (3 mL) and treated with Ac₂O (0.37 mL, 3.96 mmol, 20.0 equiv). After stirring at 100 °C for 16 h, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with aqueous 1N HCl solution (5 mL×3) followed by saturated aqueous NaCl solution (3 mL×3). Organic layer was dried over anhydrous Na₂SO₄, filtered and concentrated to dryness under reduced pressure. Pure product 6 was obtained by silica gel column chromatography eluting with 80 % EtOAc in to 100 % EtOAc; white foam (77 mg, 77 % yield), analytical TLC on silica gel, 8:2 EtOAc/hexane, R_f=0.6. ${\left[\alpha \right]}_D^{22}-2.67\left(c1.2,{\text{CHCl}}_{\text{3}}\right)$. IR (cm⁻¹): 1746, 1663, 1371, 1215; ¹H NMR (600 MHz, CDCl₃) δ 5.60 (d, J = 10.5 Hz, 1H), 5.34 (dd, J = 8.3, 2.2 Hz, 1H), 5.25 (dd, J = 10.4, 2.2 Hz, 1H), 5.09 (ddd, J = 7.2, 5.2, 1.8 Hz, 1H), 5.01 (ddd, J = 8.5, 5.6, 3.1 Hz, 1H), 4.56 (td, J = 10.4, 1.8 Hz, 1H), 4.23 (dd, J = 12.5, 3.1 Hz, 1H), 4.18 (dd, J = 11.7, 5.2 Hz, 1H), 3.96 (dd, J = 12.5, 5.7 Hz, 1H), 3.89 (dd, J = 11.7, 7.4 Hz, 1H), 2.11 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 2.02 (s, 6H), 1.99 (s, 3H), 1.97 (s, 3H); ¹³C NMR (151 MHz, CDCl3) δ 170.6, 170.5, 170.0, 169.94, 169.91, 169.89, 169.7, 68.6, 68.4, 67.8, 67.6, 62.5, 62.0, 47.3, 23.1, 20.9, 20.8, 20.69, 20.68, 20.6; HRMS (ESI/Q-TOF): m/z [M+Na]⁺ Calcd for C₂₁H₃₁O₁₃NNa 528.1688; Found 528.1690.

3-Acetamido-3-deoxy-d-glycero-d-galacto-heptitol (7):

0.1 M NaOMe solution in MeOH (1 mL) was added to the compound 6 (10 mg) and continue the reaction at RT for 24 h. reaction mixture was diluted with MeOH (3 mL) and neutralized with Amberlyst® 15 hydrogen form. Reaction mixture was filtered and concentrated to dryness under reduced pressure. Pure product 7 was obtained by C-18 column chromatography using elution water; colorless sticky solid (4 mg, 82 % yield). ${[\alpha ]}_{\text{D}}^{22}-17.39\left(c0.23,\text{MeOH}\right)$.

¹H NMR (900 MHz, MeOD) δ 4.19 (t, J = 6.8 Hz, 1H), 4.02 (d, J = 10.0 Hz, 1H), 3.95 (d, J = 10.0 Hz, 1H), 3.82 (dd, J = 11.3, 3.6 Hz, 1H), 3.74 (ddd, J = 9.1, 5.8, 3.6 Hz, 1H), 3.64 (dd, J = 11.2, 5.8 Hz, 1H), 3.51 (dd, J = 11.1, 7.0 Hz, 1H), 3.46 (dd, J = 11.3, 7.1 Hz, 2H), 2.03 (s, 3H); ¹³C NMR (226 MHz, MeOD) δ 173.2, 71.1, 69.9, 69.0, 68.0, 63.8, 63.3, 51.4, 21.1.; HRMS (ESI/Q-TOF): m/z [M+Na]⁺ Calcd for C₉H₁₉O₇NNa 276.1054; Found 276.1056.

Methyl (5-acetamido-7,8,9-tri-O-acetyl-3,6-anhydro-5-deoxy-d-manno-3-ene-2-nonulos)onate (19):

A stirred solution of compound 2 (25 mg, 0.04 mmol, 1.0 equiv) in anhydrous DMF (0.2 mL) was cooled to 0 °C (crushed ice bath) and treated with Na₂CO₃ (12 mg, 0.12 mmol, 3.0 equiv). After stirring at RT for 1.5 h, the reaction mixture was diluted with ethyl acetate (10 mL) and washed with 5% aqueous NaCl solution (3 mL) followed by saturated aqueous NaCl solution (3 mL×3). Organic layer was dried over anhydrous Na₂SO₄, filtered and concentrated to dryness under reduced pressure to give the pure product 19 as a sticky solid (15 mg, 90 % yield), analytical TLC on silica gel, 9:1 EtOAc/hexane, R_f=0.55. ${\left[\alpha \right]}_D^{22}-14.47\left(c0.47,{\text{CHCl}}_{\text{3}}\right)$. IR (cm⁻¹): 1741, 1223; ¹H NMR (900 MHz, CDCl₃) δ 6.45 (d, J = 3.3 Hz, 1H), 5.77 (d, J = 8.6 Hz, 1H), 5.45 – 5.40 (m, 2H), 5.31 (dt, J = 7.7, 3.9 Hz, 1H), 4.58 (t, J = 4.4 Hz, 1H), 4.43 (dd, J = 12.4, 2.2 Hz, 1H), 4.28 – 4.21 (m, 1H), 3.94 (s, 3H), 2.11 (s, 3H), 2.11 (s, 3H), 2.09 (s, 3H), 2.03 (s, 3H); ¹³C NMR (226 MHz, CDCl3) δ 174.9, 170.7, 170.1, 169.8, 169.6, 160.7, 153.5, 116.9, 85.6, 76.9, 70.5, 70.1, 61.6, 55.6, 53.3, 23.0, 20.9, 20.8, 20.6; HRMS (ESI/Q-TOF): m/z [M+H]⁺ Calcd for C₁₈H₂₄O₁₁N 430.1344; Found 430.1335.

Methyl (5-acetamido-7,8,9-tri-O-acetyl-3,6-anhydro-5-deoxy-d-erythro-3,5-diene-2-nonulos)onate (20):

A stirred solution of compound 2 (25 mg, 0.04 mmol, 1.0 equiv) in anhydrous DMF (0.2 mL) was cooled to 0 °C (crushed ice bath) and treated with Na₂CO₃ (12 mg, 0.12 mmol, 3.0 equiv). After stirring at RT for 1.5 h, continue the reaction at 50 °C for 24 h. Next, the reaction mixture was diluted with ethyl acetate (10 mL) and washed with 5% aqueous NaCl solution (3 mL) followed by saturated aqueous NaCl solution (3 mL×3). Organic layer was dried over anhydrous Na₂SO₄, filtered and concentrated to dryness under reduced pressure. Pure product 20 was obtained by preparative thin layer chromatography on silica gel coated glass plate, using elution 80 % EtOAc in (two times run); colorless sticky solid (12 mg, 74 % yield), analytical TLC on silica gel, 9:1 EtOAc/hexane, R_f=0.6. ${\left[\alpha \right]}_D^{22}+51.03\left(c0.29,{\text{CHCl}}_{\text{3}}\right)$. IR (cm⁻¹): 1745, 1516, 1217; ¹H NMR (600 MHz, CDCl₃) δ 8.31 (s, 1H), 8.19 (s, 1H), 5.79 (d, J = 8.1 Hz, 1H), 5.70 (ddd, J = 8.2, 4.3, 2.7 Hz, 1H), 4.52 (dd, J = 12.5, 2.7 Hz, 1H), 4.39 (dd, J = 12.5, 4.4 Hz, 1H), 3.94 (d, J = 1.0 Hz, 3H), 2.22 (s, 3H), 2.14 (d, J = 1.0 Hz, 3H), 2.10 (s, 3H), 1.97 (d, J = 1.0 Hz, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 171.6, 171.2, 170.5, 169.3, 168.3, 161.2, 147.7, 141.9, 127.4, 119.9, 69.1, 64.3, 61.5, 53.3, 23.9, 20.7, 20.6; HRMS (ESI/Q-TOF): m/z [M+Na]⁺ Calcd for C₁₈H₂₁O₁₁NNa 450.1007; Found 450.1005.