Introduction

Fusobacterium nucleatum is a Gram-negative anaerobic bacterium found in human mouth [1]. It is one of the most prevalent species in oral environment and regarded as a dental pathogen to cause serious periodontal diseases including periodontitis [2,3,4,5,6], gingivitis [2, 5, 7,8,9], and endodontic infections [10,11,12]. Besides periodontal disease, F. nucleatum is notorious for dissemination to cause a wide spectrum of diseases, some of which are lethal [1, 13]. It was reported that F. nucleatum causes diseases including adverse pregnancy outcomes [14,15,16,17], GI disorders [18,19,20], rheumatoid arthritis [21], respiratory tract infections [22], Lemierre’s syndrome [22, 23], and Alzheimer’s diseases [24]. For instances, it was reported that the bacteria originating from mother’s subgingival plague could translocate to fetus thus causes term stillbirth in human pregnancies [14].

Although F. nucleatum infection results in numerous serious diseases, little is known about the virulence factor. So far, only one protein, FadA, was identified to be crucial in colonization and infection of the bacteria [1, 13, 20]. FadA is not only an adhesin but also an invasin that binds to cadhesin, which is widely distributed in tissues and cells, and this might be the reason of migration of F. Nucleatum [20, 25, 26]. LPS of the bacteria is also thought to be a virulence factor participating in the infection. It was shown that serum antibody levels to F. nucleatum LPS did correlate to poor oral health and the degree of periodontitis [27]. Recently, the structure of O-antigen of F. nucleatum strain 25586 was identified and two novel sugars, 5-acetamidino-3,5,9-trideoxy-L-glycero-L-gluco-non-2-ulosonic acid (fusaminic acid), and 2-acetamido-2,6-dideoxy-L-altrose (6dAlt2NAc), were isolated and found as components of the O-antigen (structures are shown in Fig. 1) [28]. Fusaminic acid attracts our interests due to its structural similarity to pseudaminic acid and legionaminic acid. These nonulosonic acids are unique to bacteria species, as the congener of sialic acids, and considered as an important virulence factor, although their precise roles in evasion of the bacteria have not been elucidated yet [29]. Moreover, being of crucial importance to Gram-negative bacteria, LPS containing these molecules could be candidate targets for developing new antimicrobial agents. However, the major difficulty in investigating the biological significance of the related nonulosonic acid is the inaccessibility of the target molecules in homogeneous form as well as sufficient amount. Herein we report a de novo total synthesis of fusaminic acid based on the strategies applied in our pseudaminic acid total synthesis reported recently [30].

Fig. 1
figure 1

Structures of Fus5Ac, 6dAltNAc, Pse5Ac7Ac, Leg5Ac7Ac, and polysaccharide in Fusobacterium nucleatum 25586 O-antigen

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Results and discussion

Although the isolated fusaminic acid contains an amidine group, we planned to synthesize the general N-acetyl derivative, Fus5Ac 1. The retrosynthesis of Fus5Ac is shown in Scheme 1. The α-keto acid structure can be obtained by ozonolysis of the acrylate 6, which can be prepared from the corresponding aldehyde by indium-mediated Barbier type allylation. The aldehyde can be derived via the Fukuyama reduction from thioester 7, which can be constructed via the aldol-type reaction of thioester derived isonitrile 8 [31] and aldehyde 9. Aldehyde 9 can be prepared from commercial available (S)-(-)-ethyl lactate via Weinreb-Nahm ketone synthesis followed by 1,2-anti selective reduction and ozonolysis. Our planned synthesis involves 3 chain elongation steps with four new chiral centers formed during this process. The absolute configuration of the chiral centers generated has to be carefully determined. Armed with the experience from our former synthesis of pseudaminic acid, we anticipated that the stereoselectivity of the key transformations could be modulated through utilization of different protecting groups with different electronic and steric hindrance effect and optimization of reaction parameters.

Scheme 1
scheme 1

Retrosynthesis of fusaminic acid 1

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To choose suitable protecting groups for the intermediates, we first analyzed the stereoselectivity model of the addition of aldehyde 9. In our recent total synthesis of pseudaminic acid, the selectivity in the aldol-type addition was achieved by lithium chelation of the protected β-hydroxyl group and the aldehyde carbonyl group [30]. Less-hindered protection of β-hydroxy group and bulky protection of α-hydroxyl group of aldehyde 9 were expected to attribute to the diastereoselectivity. Taking these into consideration, we chose p-methoxybenzyl (PMB) and t-butyldiphenylsilyl (TBDPS) ether, respectively (shown in Scheme 2). The synthesis commenced with (S)-(-)-ethyl lactate PMB ether protection, followed by saponification and coupling with N, O-dimethylhydroxylamine to obtain the Weinreb amide 13 in good yield. The latter was transformed to vinyl ketone 15 via vinylmagnesium bromide addition, and the corresponding diol 17 was obtained by stereoselective reduction of the ketone via zinc borohydride [32, 33]. Chelation of zinc ion resulted in 1,2-anti selectivity and the ratio of the diastereomers 17a/17b was around 10:1. The resultant diastereomeric mixture was inseparable and then directly silylated with TBDPSCl followed by ozonolysis to give aldehyde 9. With the key aldehyde in hand, we tried to convert it to the thioester 19a by treating the aldehyde with the isonitrile thioester in the presence of lithium triflate and diisopropylethylamine, followed by acidolysis of the 4,5-trans oxazoline intermediate in aqueous acetic acid [30, 34,35,36,37,38,39,40]. Fortunately, we succeeded in getting target diastereomers in good to moderate yield, but the difficult separation remained a problem. Considering the significance of confirming the chiral centers generated, and the plausible complexity in the generation of the new chiral center in the following Barbier allylation step, it would be necessary to obtain the uniform diastereomer. To our delight, we found that changing PMB ether to benzyl ether simplified the purification without seriously impacting the stereoselectivity, and more importantly, prevented partially decomposition of PMB ether in ozonolysis and acidolysis. We planned to convert the thioester to the corresponding hexosamine derivative, 2-acetamido-2,6-dideoxy-L-altrose, to confirm the absolute configuration. It is noted that the altrose derivative, 6dAlt2NAc, is also the novel rare monosaccharide found in the LPS of Fusobacterium nucleatum strain 25586 [28].

Scheme 2
scheme 2

Synthesis of key intermediate thioester 19a. aReagents and conditions: a NaH, PMBCl for 11, BnBr for 12, DMF/THF, 0 oC to rt, 2 h, 81% for 11, 83% for 12. b LiOH, THF-MeOH-H2O, 2 h, quant. c PivCl, Et3N, 0 oC, 1 h, then N,O-dimethylhydroxyamine, DCM, 0 oC to rt, 3 h, 92% for 13, 93% for 14. d vinylmagnesium bromide, THF, −10 oC, 1 h, 87% for 15 and 16. e Zn(BH4)2, diethyl ether, −20 oC, 1 h.; f TBDPSCl, imidazole, DMF, ovn, 89% for 17, dr 10 : 1, 87% for 18, dr 5 : 1, over 2 steps. g O3, DCM, −78 oC, 0.5 h, then Ph3P. h CNCH2COSEt (8), LiOTf, DIPEA, DCE-DMF, 2 h. i 80% HOAc (aq), 8 h, 45% over three steps

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Our initial attempts to directly convert the thioester to the altrose hemiacetal failed due to the difficulty in removing the benzyl ether. Catalytic hydrogenation or oxidation with sodium bromate combined with sodium dithionite [41] could not free the hydroxyl group without affecting the thioester. While deprotecting the benzyl ether after the Fukuyama reduction [42] was also challenging due to the difficulty in manipulating the corresponding α-N-formyl aldehyde. Thus, we firstly converted the thioester to the aldehyde and trapped the resultant aldehyde with ylide to give the α,β-unsaturated ester (shown in Scheme 3). With this stable olefin as the surrogate to the aldehyde, we had the chance to manipulate the protecting groups. Different solvents such as DCM, acetone, THF were tried to prevent partially triethylsilyl (TES) installation in the Fukuyama reduction, while the silylation seemed to be inevitable [43]. Therefore, we directly removed all silyl ethers by tetrabutylammonium fluoride (TBAF) to give product 20. The formamide 20 was converted to acetamide 21 by acidolysis with methanolic hydrochloride followed by acetylation with acetic anhydride and trimethylamine in 86% yield [44]. The hydroxyl groups were masked by benzoyl chloride in order to prevent side reactions in the following dihydroxylation and vicinal diol cleavage steps. Osmium tetraoxide was used for dihydroxylation, and benzyl ether could be removed by catalytic hydrogenation without affecting any other functional groups at this step. Finally, the hemiacetal 25 was formed by converting the diol 24 to aldehyde via the Malaprade reaction. With the altrose derivative 25 in hand, we carefully examined the configuration of the chiral centers newly generated.

Scheme 3
scheme 3

Synthesis of hexosamine derivativea. aReagents and condition: aPd/C, Et3SiH, THF, 1 h. b Ph3P=COOtBu, DCM, 0.5 h. c TBAF, HOAc, THF, 2 h, 62% over three steps. d 3% HCl in MeOH, 0 oC to rt, 6 h. e Ac2O, Et3N, 86% over two steps. f BzCl, pyridine, DMAP, DCM, ovn, 92%. g OsO4, NMO, THF-H2O, ovn. h Pd/C, H2, MeOH, 3 h. i NaIO4, DCM-H2O, 6 h, 67% over three steps

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As the 1,2-anti selective reduction of α-hydroxyl ketone via zinc borohydride was an established method, the absolute configuration of C-4 obtained from enantiopure starting material (S)-(-)-ethyl lactate could be settled [33]. Moreover, the isonitrile addition to the aldehyde would form 4,5-trans oxazoline in the presence of base and the following acidolysis did not affect the chiral center, thus the relative configuration of C-2 was also determined [34,35,36,37,38,39,40]. Although starting with a 5:1 mixture of the diastereomers of the vinyl alcohol, we could get the major diastereomer of the thioester in 45% yield in pure form, which excluded the contribution of the minor syn diol from the reduction step to the isolated thioester. Therefore, the thioester could only be 19a or 19b. Compound 19a is the precursor of 2-acetamido-2,6-dideoxy-L-altrose while 19b derives to 2-acetamido-2,6-dideoxy-L-glucose, also known as N-acetyl-L-quinovosamine (shown in Fig. 2). Peaks of this N-acetyl quinovosamine in 1H-NMR spectrum should fit well to N-acetyl glucosamine pattern, of which peaks have large vicinal coupling constant as all protons in have axial orientation. On the contrary, for the altrose derivative in which protons orient both axially and equatorially, small vicinal coupling constants should be. We assigned protons of 24 and found several small coupling constant values such as JH4,H5 (3 Hz, see Experimental section). Although some of the small coupling constants may come from the deviation from the typical chair-like conformation, the observed coupling patterns significantly different from the quinovosamine derivatives excluded the possibility of the structure 19b. We decided to use the major diastereomer 19a to continue the synthesis of fusaminic acid and planned to further confirm the configurations by comparing the splitting pattern of the synthetic final product with the isolated one.

Fig. 2
figure 2

Elucidation of the configurations of isonitrile adducts via derivatization to hexosamine

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The indium-mediated Barbier type allylation is the key step for carbon chain elongation due to its compatibility with various functional groups [45]. Successful conversion of the aldehyde to the desired acrylate was achieved by mixing the aldehyde generated via the Fukuyama reduction with organoindium species generated beforehand in situ in the mixture of ethanol and aqueous ammonium chloride [46]. After simple aqueous work-up, the reaction mixture was subjected to TBAF in THF solution buffered with acetic acid to remove the TBDPS group. Two diastereomers were formed with ratio of 5.5:1 as shown in 1H NMR spectrum, which were readily separated by silica gel column chromatography to give the major diastereomer 26a in 59 yield%. Regarding the previous experience in pseudaminic acid synthesis and similar compounds in the Seeberger’s [46] and Ito’s [47] syntheses, we proposed that the major diastereomer 26a should possess the desired configuration at the newly formed chiral center [30, 40, 41]. The formamide was converted to acetamide as described above with partially intramolecular lactonization (27/27a), and the five-membered ring lactone could be hydrolyzed in 1 M NaOH solution to provide free acid 28 after careful neutralization. However, direct ozonolysis of the acid 28 gave unsuccessful result, while numerous byproducts including glycal were generated. To our delight, we found that protection of the carboxylic acid as benzyl ester 27 could significantly suppress side reactions, and the following ozonolysis gave hemiketal 29 in good yield. After final hydrogenation to remove benzyl groups, we successfully obtained the target molecule, Fus5Ac 1 (Shown in Scheme 4).

Scheme 4
scheme 4

De novo synthesis of fusaminic acida. aReagents and conditions: a Pd/C, Et3SiH, THF, 1h. b Benzyl bromomethacrylate, indium powder, NH4Cl, EtOH, 2 h. c TBAF, HOAc, THF, 2 h, 59% over three steps. d 3% HCl in MeOH, 0 oC to rt, 6 h. e Ac2O, Et3N, 89% over two steps; f 1M NaOH aqueous solution, 1 h, 87%.; g K2CO3, BnBr, DMF, 5 h, 79%.; h O3, −78 oC, DCM-MeOH, 0.5 h, then Me2S, 75%.; i Pd(OH)2/C, H2, MeOH-H2O, 12 h, 89%.; j Ac2O, pyridine, DMAP, 3 h, 83%.; k p-TolSH, BF3.Et2O, dry DCM, ovn, 62%

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In order to further confirm the configuration of Fus5Ac synthesized, we derived 29 to thioglycoside 31 via thioglycosylation of acetate 30. Relatively large JC1, H3a value (~6.4 Hz) indicates the equatorial orientation of the thiol aglycone (β). The small vicinal proton coupling constants of JH3a,H4, JH3e,H4, JH4,H5, and JH5,H6 (3.5, 2.0, < 0.1, 2.0 Hz, respectively) indicate the equatorial orientation of both H-4 and H-5. Combining with relatively large JH6, H7 (7.5 Hz), we identified C4-C7 as gluco configuration [48]. Moreover, the spin system of the synthetic fusaminic acid 1 was highly consistent with the isolated sample as well as 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-gluco-non-2-ulosonic acid (compound 42 in ref. [48]) [28].

In summary, we have developed the de novo total synthesis of a newly discovered nonulosonic acid unique to bacteria, fusaminic acid. The synthesis started with commercial available (S)-(-)-ethyl lactate, involving 18 steps to give Fus5Ac in 8% overall yield. The key steps include the diastereoselective addition of the thioester derived isonitrile to aldehyde and indium-mediated diastereoselective Barbier-type allylation. We derived the key thioester intermediate to 6dAltNAc and synthesized fusaminic acid thioglycoside donor for structure confirmation. The success in generating the thioglycoside broadens the application of the synthesis as it provides the opportunity to assembly complex fusaminylated glycan and glycoconjugates.

Experimental section

General

Commercially available reagents were used without further purification, unless otherwise stated. The anhydrous solvents were either prepared from AR grade solvents via standard methods (DCM, THF, etc.), or purchased in anhydrous form (DMF, pyridine, etc.). The analytical TLC was performed on silica gel 60-F254 precoated on glass plate (E. Merck), with detection by fluorescence and/or or by staining with acidic ceric ammonium molybdate. The normal phase column chromatography was performed on silica gel (230–400 mesh, Merck), while the reverse phase chromatography was performed on C18 silica gel (Davisil 633NC18E, Grace Materials Technologies). The 1H and 13C NMR spectra were recorded on Advance DRX Bruker 400/500 MHz spectrometers at 25 oC. The 2D NMR spectra were recorded on Advance DRX Bruker 500 MHz spectrometers at 25 oC. The high-resolution mass spectrometry was performed on a Waters Micromass Q-TOF Premier Mass Spectrometer.

Ethyl (S)-2-(benzyloxy)propanoate (12)

To a mixture of anhydrous THF (20 mL) and DMF (20 mL) in round bottle flask was added sodium hydride (1.76 g, 44 mmol, 1.1 equiv, 60% suspension in mineral oil) followed by (S)-(−)-ethyl lactate (4.63 mL, 40 mmol, 1.0 equiv) at 0 oC. After strring for 10 min, benzyl bromide (7.13 mL, 60 mmol, 1.5 equiv) was added. The reaction mixture was warmed to room temperature and monitored by TLC. After full conversion (about 2 h) of the starting material, the reaction was quenched by sat. NH4Cl (aq), then diluted with ethyl acetate (500 mL) and water (100 mL). The organic layer was separated and washed sequentially with 1 M HCl (aq, 100 mL), sat. NaHCO3 (aq, 100 mL), and brine (100 mL). The organic layer was dried over anhydrous Na2SO4, and concentrated under vacuum. The residue was purified by silica gel flash chromatography using n-hexane: ethyl acetate 12: 1 v/v as eluent to obtain compound 12 as colorless oil (6.90 g, 83%). 1H NMR (500 MHz, CDCl3) δ 7.28–7.40 (5 H, m), 4.72 (1 H, d, J = 11.5 Hz), 4.47 (1 H, d, J = 11.5 Hz), 4.19–4.28 (2 H, m), 4.07 (1 H q, J = 7.0 Hz), 1.46 (3 H, d, J = 6.5 Hz), 1.31 (3 H, t, J = 7.0 Hz); 13C NMR (125 MHz, CDCl3) δ 173.2, 137.6, 128.4, 14.2, 128.0, 127.8, 74.1, 72.0, 60.8, 18.7; HR-ESI-MS (m/z): calcd for C12H16O3Na+ (M + Na+): 231.0992, found 231. 0998.

(S)-2-(benzyloxy)-N-methoxy-N-methylpropanamide (14)

To a stirred solution of 12 (6.90 g, 33.2 mmol, 1.0 equiv) in MeOH (15 mL) and THF (60 mL), the solution of LiOH (2.0 eq in 15 mL H2O) was added. The mixture was stirred at r.t. for 2 h. After the full conversion was achieved, the mixture was acidified by 1 M HCl (aq). The mixture was diluted with ethyl acetate (500 mL), and the organic layer was separated. The aqueous layer was extract with ethyl acetate (200 mL) again, and the organic layer was combined. The organic layer was washed with brine (2 × 100 mL) and dried over anhydrous Na2SO4. The solvent was removed under vacuum and the residue was further dried under oil pump for 3 h. The residue above was used in the next step without purification.

The saponification product was dissolved in anhydrous DCM (200 mL) and cooled to 0 oC. Triethylamine (11.6 mL, 82.9 mmol, 2.5 equiv) was added to the solution above followed by PivCl (4.49 mL, 36.5 mmol, 1.1 eq) was added dropwise and the solution was kept stirring at 0 oC for 1 h. Then N, O-dimethyl hydroxylamine hydrochloride (3.88 g, 39.8 mmol, 1.2 equiv) was added and the reaction mixture was warmed to r.t. and kept stirring for another 3 h. After full conversion (monitored by TLC), the mixture was diluted with ethyl acetate (500 mL) and washed with 1 M HCl (aq, 100 mL), sat. NaHCO3 (aq, 100 mL) and brine (100 mL) subsequently. The solvent was removed under vacuum and the residue obtained was purified by silica gel flash chromatography using n-hexane: ethyl acetate 3:1 v/v as eluent to obtain compound 14 as slightly yellow oil (7.20 g, 93%). 1H NMR (500 MHz, CDCl3) δ 7.24–7.36 (5 H, m), 4.64 (1 H, d, J = 12.0 Hz), 4.36–4.41 (2 H, m), 4.07 (1 H, q, J = 7.0 Hz,), 3.55 (3 H, s), 3.18 (3 H, s), 1.38 (3 H, d, J = 6.5 Hz,); 13C NMR (125 MHz, CDCl3) δ 137.8, 128.3, 127.9, 127.7, 71.1, 61.2, 17.9; HR-ESI-MS (m/z): calcd for C12H17NO3Na+ (M + Na+): 246.1101, found 246. 1109.

(S)-4-(benzyloxy)pent-1-en-3-one (16)

To a solution of Weinreb amide 14 (7.20 g, 30.9 mmol) in anhydrous THF (100 mL) was added vinylmagnesium bromide (1.0 M solution in THF, 46.3 mL, 1.5 equiv) dropwise at −20 oC. The reaction mixture was gradually warmed to r.t. and kept stirring for 2 h. After full conversion achieved, the reaction mixture was poured into a cold 1 M HCl aqueous solution. The mixture was extracted with ethyl acetate (500 mL) and washed with sat. NaHCO3 (aq, 100 mL) and brine (100 mL). The solvent was removed under vacuum and the residue obtained was purified by silica gel flash chromatography using n-hexane: ethyl acetate 15: 1 v/v as eluent to obtain compound 14 as colorless oil (5.10 g, 87%). 1H NMR (500 MHz, CDCl3) δ 7.34–7.38 (4 H, m), 7.28–7.33 (1 H, m), 6.81 (1 H, dd, J = 17.5, 11.0 Hz), 6.45 (1 H, dd, J = 17.5, 1.5 Hz), 5.80 (1 H, dd, J = 10.5, 1.5 Hz), 4.58 (1 H, d, J = 12.0 Hz), 4.46 (1 H, d, J = 11.5 Hz), 4.13 (1 H, q, J = 7.0 Hz), 1.39 (3 H, d, J = 7.0 Hz); 13C NMR (125 MHz, CDCl3) δ 201.0, 137.5, 130.93, 129.68, 128.39, 127.81, 127.76, 79.7, 71.7, 17.6; HR-ESI-MS (m/z): calcd for C12H14O2Na+ (M + Na+): 213.0886, found 246. 0894.

(((R)-4-(benzyloxy)pent-1-en-3-yl)oxy)(tert-butyl)diphenylsilane (18a/b)

To a solution of the vinyl ketone 16 (5.10 g, 26.8 mmol, 1.0 equiv) in anhydrous diethyl ether (200 mL) was added zinc borohydride (0.2 M solution in diethyl ether, 135 mL, 1.0 equiv) dropwise at −20 oC. The reaction was kept stirring at the temperature for 3 h. When full conversion was achieved, the reaction was quenched by carefully adding 1 M HCl (aq), then diluted with ethyl acetate (200 mL) and water (100 mL). The organic phase was separated and washed with sat. NaHCO3 (aq, 100 mL) and brine (100 mL). The solvent was removed under vacuum and the residue was purified by silica gel flash chromatography using n-hexane: ethyl acetate 5: 1 v/v as eluent to obtain (4 S)-4-(benzyloxy)pent-1-en-3-ol as colorless oil (5 (anti): 1 (syn) mixture of diastereomers, 4.75 g, 92%). 1H NMR (500 MHz, CDCl3) δ 7.36–7.41 (4 H, m), 7.30–7.35 (1 H. m), 5.90 (1 H, ddd, J = 17.5, 11.0, 6.0 Hz), 5.36 (1 H, dt, J = 17.0, 1.5 Hz), 5.25 (1 H, dt, J = 11.0, 1.5 Hz), 4.66 (1 H, d, J = 12.0 Hz), 4.57 (1 H, d, J = 12.0 Hz), 4.25–4.29 (1 H, m), 3.64 (1 H, ddd, J = 12.5, 6.5, 3.5 Hz), 2.61 (1 H, d, J = 4.0 Hz), 1.20 (3 H, d, J = 6.5 Hz); 13C NMR (125 MHz, CDCl3) δ 138.4, 136.7, 128.5, 127.7, 116.4, 77.5, 74.6, 70.8, 14.0; HR-ESI-MS (m/z): calcd for C12H16O2Na+ (M + Na+): 215.1043, found 245. 1050.

To a solution of the alcohol above (4.75 g, 24.7 mmol) in DMF solution (30 mL) was added imidazole (3.36 g, 43.4 mmol, 2.0 equiv) and TBDPSCl (9.63 mL, 37.0 mmol, 1.5 equiv). The mixture was kept stirring for 8 h and monitored by TLC. After full conversion, the mixture was diluted with ethyl acetate (500 mL) and washed with 1 M HCl (aq, 100 mL), sat. NaHCO3 (aq, 100 mL) and brine (100 mL) subsequently. The solvent was removed under vacuum and the residue obtained was purified by silica gel flash chromatography using n-hexane: ethyl acetate 40: 1 v/v as eluent to obtain compound 18a/b as colorless oil (5 (anti): 1 (syn) mixture of diastereomers, 10.63 g, 97%). 1H NMR (500 MHz, CDCl3) δ 7.85–7.88 (2 H, m), 7.79–7.84 (2 H, m), 7.35–7.55 (11 H, m), 6.03 (1 H, ddd, J = 17.0, 10.0, 7.0 Hz), 5.16 (1 H, dt, J = 10.5, 1.0 Hz), 5.07 (1 H, dt, J = 17.0, 1.0 Hz), 4.73 (1 H, d, J = 12.0 Hz), 4.66 (1 H, d, J = 12.0 Hz), 4.33–4.36 (1 H, m), 3.66 (1 H, ddd, J = 13.0, 6.5, 3.0 Hz), 1.23–1.28 (12 H, m); 13C NMR (125 MHz, CDCl3) δ 139.2, 137.5, 136.25, 136.21, 134.23, 134.13, 129.65, 129.59, 128.3, 127.61, 127.53, 127.42, 127.34, 116.7, 78.8, 78.2, 71.5, 27.2, 19.6, 15.8; HR-ESI-MS (m/z): calcd for C28H34O2SiNa+ (M + Na+): 453.2220, found 453.2231.

S-Ethyl (2 R,3 R,4 R,5 S)-5-(benzyloxy)-4-((tert-butyldiphenylsilyl)oxy)-2-formamido-3-hydroxyhexanethioate (19a)

The solution of 18a/b (10.6 g, 24.7 mmol) in DCM (150 mL) was cooled to −78 oC. The O3 (generated from O2 and carried by the flow of O2) was bubbled through this solution. The color of the solution truned purple, which indicated the saturation of O3 in DCM. The excess amount of O3 was blown off by the flow of O2 and the purple color disappeared. To this solution, Me2S (2 mL) was added to reduce the peroxide intermediate. After 1 h reduction at r.t., the solution was diluted with DCM (500 mL) and washed with water (100 mL) to remove DMSO (generated from Me2S). The solvent was removed under vacuum and the residue was used in the next step without further purification.

To the solution of the above aldehyde in DCE (125 mL), isonitrile 8 (3.83 g, 29.6 mmol, 1.2 equiv) was added, followed by the solution of LiOTf (4.62 g, 29.6 mmol, 1.2 equiv) in anhydrous DMF (25 ml). The final concentration of the aldehyde was controlled at 0.2 M. To this mixture, DIPEA (0.86 ml, 4.94 mmol, 0.2 equiv) was added to initiate the reaction. The mixture was stirred at r.t. for 2 h, then was diluted with DCM (500 ml) and thoroughly washed with water and brine. The organic phase was concentrated under vacuum. The residue was dissolved in 80% acetic acid aqueous solution (30 mL) and kept stirring for 8 h. The acetic acid was removed under vacuum and the residue was diluted with ethyl acetate (500 mL). The mixture was washed with sat. NaHCO3 (aq, 3 × 100 mL) and brine (100 mL), and dried over anhydrous Na2SO4. The solvent was removed under vacuum and the residue was purified by silica gel flash chromatography using n-hexane: ethyl acetate 3: 1 v/v as eluent to obtain 19a as white solid. (6.44 g, 45%). 1H NMR (500 MHz, CDCl3, 3: 1 mixture of two rotamers) δ 7.66–7.71 (4 H, m, major), 7.60–7.65 (1.33 H, m, minor), 7.19–7.47 (14.6 H, m, major and minor), 6.27 (1 H, d, J = 8.5 Hz, major), 6.05 (0.33 H, t, J = 11.0 Hz, minor), 4.98 (1 H, d, J = 9.0 Hz, major), 4.51–4.56 (1.66 H, m, major and minor), 4.44 (1 H, d, J = 11.5 Hz, major), 4.42 (0.33 H, d, J = 12.5 Hz, minor), 4.30 (1 H, d, J = 12.5 Hz, major), 4.19 (0.33 H, d, J = 10.0 Hz, minor), 3.72 (1 H,qd, J = 6.5, 2.0 Hz, major), 3.81 (0.33 H, qd, J = 6.5, 1.5 Hz, minor), 3.67 (0.33 H, dd, J = 8.5, 1.5 Hz, minor), 3.58 (1 H, dd, J = 8.0, 1.5 Hz, major), 3.21 (1 H, d, J = 3.5 Hz, major), 3.05 (0.33 H, d, J = 4.0 Hz, minor), 2.88–2.95 (2.66 H, m, major and minor), 1.26 (3 H, t, J = 7.5 Hz, major), 1.25 (1 H, t, J = 7.5 Hz, minor), 1.06 (9 H, s, major), 1.05 (3 H, s, minor), 1.02 (1 H, d, J = 7.0 Hz, minor), 0.92 (3 H, d, J = 7.0 Hz, major); 13C NMR (125 MHz, CDCl3) δ 199.7 (major), 164.3 (minor), 161.3 (major), 138.2 (major), 138.1(minor), 136.3 (major), 136.14 (major), 136.09 (minor), 133.9 (major), 133.1 (minor), 132.9 (minor), 132.5 (major), 130.5 (minor), 130.3 (minor), 130.2 (major), 129.8 (major), 128.64 (minor), 128.58 (major), 128.2 (minor), 128.0 (major), 127.9 (minor), 127.8 (major), 127.7 (major and minor), 127.5 (major and minor), 79.3 (minor), 78.9 (major), 76.3 (major), 75.5 (minor), 74.0 (major), 73.9 (minor), 71.7 (minor), 71.4 (major), 62.5 (minor), 59.4 (major), 24.0 (minor), 23.7 (major), 19.6 (major and minor), 17.5 (minor), 17.2 (major), 14,37 (major), 14.35 (minor); HR-ESI-MS (m/z): calcd for C32H41NO5SiSNa+ (M + Na+): 602.2367, found 602.2376.

tert-Butyl (4 S,5 R,6 R,7 S,E)-7-(benzyloxy)-4-formamido-5,6-dihydroxyoct-2-enoate (21)

To a 50 mL round bottle flask, thioester 19a (579 mg, 1.0 mmol, 1.0 equiv) and Pd/C (10% Pd on activated carbon, 100 mg, 0.1 equiv based on Pd) were added. After agron protection of the flask, anhydrous DCM (5 mL) was added, and the mixture was stirred mildly. Et3SiH (0.60 mL, 3.8 mmol, 3.8 equiv) was added dropwise during 20 min, then the mixture was mildly stirred at r.t. for 2 h. When full conversion achieved, the mixture was filtered through celite. To the filtrate was added (tert-butoxycarbonylmethylene)triphenylphosphorane (451 mg, 1.2 mmol, 1.2 eq), and the mixture was kept stirring for 1 h. After full conversion as indicated by TLC, the solvent was removed under vacuum. The residue was re-dissolved in THF (10 mL) and a mixture of TBAF (1.0 M solution in THF, 3.0 mL, 3.0 equiv) and acetic acid (90 μL, 1.5 mmol, 1.5 equiv) were added. The mixture was then kept stirring for 2 h and monitored by TLC. After full conversion, the reaction mixture was diluted with ethyl acetate (200 mL), washed with brine (2 × 50 mL), and dried over anhydrous Na2SO4. The solvent was removed under vacuum and the residue was purified by by silica gel flash chromatography using DCM: ethyl acetate 3:1 v/v as eluent to obtain 21 as white foam. (235 mg, 62%). 1H NMR (500 MHz, CDCl3) δ 8.26–8.28 (1 H, m), 7.29–7.37 (5 H, m), 6.89 (1 H, dd, J = 15.5, 4.5 Hz), 6.25 (1 H, d, J = 9.0 Hz), 5.94 (1 H, dd, J = 16.0, 2.0 Hz), 5.09 (1 H, q, J = 4.5 Hz), 4.70 (1 H, d, J = 11.0 Hz), 4.43 (1 H, d, J = 11.5 Hz), 3.77 (1 H, dd, J = 8.5, 1.0 Hz), 3.71 (1 H, dd, J = 8.0, 6.0 Hz), 3.17 (1 H, t, J = 8.5 Hz), 1.48 (9 H, s), 1.38 (3 H, d, J = 6.0 Hz); 13C NMR (125 MHz, CDCl3) δ 165.2, 162.4, 143.7, 137.3, 128.85, 128.35, 128.19, 124.8, 81.0, 79.6, 72.3, 71.0, 50.0, 28.3, 16.8; HR-ESI-MS (m/z): calcd for C20H29NO6Na+ (M + Na+): 402.1887, found 402.1896.

tert-Butyl (4 S,5 R,6 R,7 S,E)-4-acetamido-7-(benzyloxy)-5,6-dihydroxyoct-2-enoate (22)

To a 50 mL round bottle flask containing compound 21 (235 mg, 0.62 mmol, 1.0 equiv), a solution of HCl (aq) in MeOH (3%, 12.0 mL prepared from conc. HCl 1.0 mL and MeOH 11.0 mL) cooled to 0 oC was added. The reaction mixture was gradually warmed to r.t. and kept stirring for 6 h. After full conversion as indicated by TLC, the mixture was cooled to 0 oC again and triethylamine was added until the solution turned basic. Acetic anhydride (120 μL, 1.24 mmol, 2.0 equiv) was added and the solution was kept stirring for 1 h in basic environment. After full conversion, the reaction was diluted with ethyl acetate (200 mL), washed with1 M HCl (aq, 50 mL), sat. NaHCO3 (aq, 50 mL) and brine (50 mL), and dried over anhydrous Na2SO4. The solvent was removed under vacuum and the residue was purified by silica gel flash chromatography using DCM: ethyl acetate 3:1 v/v as eluent to obtain 22 as white foam (209.6 mg, 86%). 1H NMR (500 MHz, CDCl3) δ 7.27–7.37 (5 H, m), 6.88 (1 H, dd, J = 16.0, 4.5 Hz), 6.01 (1 H, d, J = 9.0 Hz), 5.91 (1 H, d, J = 16.0 Hz), 4.96–5.01 (1 H, m), 4.71 (1 H, d, J = 11.0 Hz), 4.42 (1 H, d, J = 11.0 Hz), 3.75 (1 H, d, J = 8.5 Hz), 3.66–3.73 (1 H, m), 3.08 (1 H, t, J = 8.5 Hz), 2.10 (3 H, s), 1.48 (9 H, s), 1.39 (3 H, d, J = 6.0 Hz); 13C NMR (125 MHz, CDCl3) δ 172.1, 165.2, 144.4, 137.3, 128.8, 128.3, 128.2, 124.5, 81.0, 80.0, 72.4, 71.1, 51.2, 28.2, 23.2, 17.1; HR-ESI-MS (m/z): calcd for C21H31NO6Na+ (M + Na+): 416.2044, found 416.2058.

(2 S,3 S,4 R,5 S,E)-5-acetamido-2-(benzyloxy)-8-(tert-butoxy)-8-oxooct-6-ene-3,4-diyl dibenzoate (23)

To a solution of compound 22 (235 mg, 0.53 mmol, 1.0 eq) in anhydrous pyridine (10 mL) was added benzoyl chloride (173 μL, 1.32 mmol, 2.5 equiv) dropwise, followed by DMAP (4 mg, 0.03 mmol, 0.05 eq). The reaction was kept stirring for 16 h. When full conversion was achieved, the mixture was diluted with ethyl acetate (200 mL), and washed with 1 M HCl (aq, 3 × 50 mL), sat. NaHCO3 (aq, 50 mL) and brine (50 mL) and dried over anhydrous Na2SO4. The solvent was removed under vacuum and the residue was purified by silica gel flash chromatography using n-hexane: ethyl acetate 3: 1 v/v as eluent to obtain 23 as white foam (318.2 mg, 92%). 1H NMR (500 MHz, CDCl3) δ 8.00 (2 H, d, J = 7.5 Hz), 7.93 (2 H, d, J = 7.5 Hz), 7.59 (2 H, t, J = 7.0 Hz), 7.40–7.47 (4 H, m), 7.25–7.36 (10 H, m), 6.84 (1 H, dd, J = 16.0, 5.0 Hz), 6.20 (1 H, d, J = 9.0 Hz), 5.78 (1 H, d, J = 15.5 Hz), 5.69 (1 H, t, J = 5.0 Hz), 5.60 (1 H, t, J = 5.0 Hz), 5.21–5.28 (1 H, m), 4.63 (1 H, d, J = 11.0 Hz), 4.58 (1 H, d, J = 11.0 Hz), 3.92–3.99 (1 H, m), 1.86 (3 H, s), 1.42 (9 H, s), 1.34 (3 H, d, J = 6.0 Hz); 13C NMR (125 MHz, CDCl3) δ 169.4, 165.53, 165.51, 165.1, 142.8, 137.7, 133.68, 133.53, 129.90, 129.65, 129.32, 128.71, 128.70, 128.63, 128.22, 128.02, 124.6, 80.8, 74.00, 73.92, 73.0, 71.4, 50.5, 28.2, 23.3, 16.1; HR-ESI-MS (m/z): calcd for C35H39NO8Na+ (M + Na+): 624.2568, found 624.2580.

2-Acetamido-3,4-di-O-benzoyl-2,6-dideoxy-L-altrose (25)

To a mixture of compound 23 (318 mg, 0.53 mmol, 1.0equiv) in THF (6 mL) and H2O (2 mL) was added OsO4 (0.04 M solution in H2O, 265 μL, 0.02 equiv) and 4-methylmorpholine 4-oxide (NMO, 4.8 M solution in H2O, 550 μL, 5.0 equiv). The mixture was kept stirring for 8 h. When full conversion was achieved, the mixture was quenched with sat. Na2S2O3 (aq), then diluted with ethyl acetate (200 mL), and washed with brine (50 mL). The organic solvent was dried over Na2SO4 and concentrated under vacuum. The residue was purified by silica gel flash chromatography using n-hexane: ethyl acetate 2: 3 v/v to obtain 24 as white foam. (286.4 mg, 85%)

To a round bottom flask containing compound 24 (143.2 mg, 0.225 mmol), Pd/C (10% Pd on activated carbon, 50 mg) was added. The flask was filled with argon and methanol (10 mL) was added. The mixture was kept stirring under 1 atm H2 atmosphere and monitored by TLC. After full conversion, the mixture was filtered through celite to remove the catalyst. The filtrate was concentrated under vacuum and used in the next step without further purification.

To the solution of the above residue in DCM (10 mL) was added a solution of NaIO4 (241 mg, 1.13 mmol, 5.0 equiv) in H2O (5 mL). The reaction mixture was kept vigorous stirring for 6 h. After full conversion, the mixture was diluted with ethyl acetate (200 mL), and washed with brine (50 mL). The organic solvent was dried over Na2SO4 and concentrated under vacuum. The residue was purified by silica gel flash chromatography using DCM: methanol 40: 1 v/v as eluent to obtain 25 as white foam (76.2 mg, 82%). 1H NMR (500 MHz, CDCl3) δ 7.60 (2 H, t, J = 7.5 Hz, ArH), 7.52 (2 H, t, J = 7.5 Hz, ArH), 7.47 (3 H, t, J = 7.5 Hz, ArH), 7.37 (3 H, t, J = 7.5 Hz, ArH), 6.60 (1 H, d, J = 8.0 Hz, NH), 5.66 (1 H, qd, J = 6.5, 3.0 Hz, H-5), 5.50 (1 H, dd, J = 9.5, 3.0 Hz, H-4), 4.65 (1 H, d, J = 9.0 Hz, H-3), 4.44 (1 H, dd, J = 8.0, 2.0 Hz, H-2), 4.10 (1 H, s, OH), 2.05 (3 H, s, CH3CO), 1.52 (3 H, d, J = 6.5 Hz, H-6); HR-ESI-MS (m/z): calcd for C22H23NO7Na+ (M + Na+): 436.1367, found 436.1375.

Benzyl (4 R/S,5 S,6 R,7 R,8 S)-8-(benzyloxy)-5-formamido-4,6,7-trihydroxy-2-methylenenonanoate (26a/b)

To a 100 mL round bottom flask, thioester 19a (2.89 g, 5.0 mmol, 1.0 equiv) and Pd/C (10% Pd on activated carbon, 500 mg, 0.1 equiv based on Pd) were added. After agron protection of the flask, anhydrous DCM (25 mL) was added, and the mixture was stirred mildly. Et3SiH (3.0 mL, 19 mmol, 3.8 equiv) was added dropwise during 20 min, then the mixture was mildly stirred at r.t. for 2 h. When full conversion was achieved, the mixture was filtered through celite and the filtrate was concentrated under vacuum to give crude aldehyde 20. The aldehyde was used in the next step without further purification.

To a 50 mL round bottom flask, indium power (1.72 g, 15 mmol, 3.0 equiv) was added, followed by EtOH (20 mL), benzyl bromomethylacrylate (5.15 g, 22.5 mmol, 4.5 equiv) and sat. NH4Cl solution (3 mL). The mixture was sonicated for 20 min at 50 oC to generate the corresponding indium reagent. This so-obtained solution of indium reagent was added into the solution of the above crude aldehyde 20 in EtOH (10 mL) in one portion, and the mixture was stirred at r.t. for 2 h. The reaction was quenched by sat. NaHCO3, then dilute with ethyl acetate (500 mL). The organic layer was separated and the aqueous layer was extract with another 500 mL ethyl acetate. The organic layer was combined and washed with 1 M HCl (aq, 200 mL), sat. NaHCO3 (aq, 200 mL), and brine (200 mL), and was dried over anhydrous Na2SO4. The organic solvent was concentrated under vacuum and the residue was directly used in the next step.

To a solution of the above residue in THF (100 mL) was added a mixture of TBAF (1.0 M solution in THF, 15.0 mL, 3.0 equiv) and acetic acid (0.44 mL, 15.0 mmol, 1.5 equiv). The reaction mixture was kept stirring for 2 h and monitored by TLC. When full conversion was achieved, the reaction mixture was diluted with ethyl acetate (500 mL), then was thoroughly washed with brine (200 mL) and dried over Na2SO4. The organic solvent was concentrated under vacuum and the residue was purified by flash column chromatography using DCM: ethyl acetate 3: 2 v/v as eluent to obtain compound 26a/b as white foam. (6 (4 R): 1 (4 S) mixture of diastereomers, 1.35 g for the major diastereomer, 59% over 3 steps). For major diastereomer 26a: [α] + 27.25 (c 0.6, DCM); 1H NMR (500 MHz, CDCl3) δ 8.32 (1 H, s), 7.28–7.37 (10 H, m), 6.54 (1 H, d, J = 9.0 Hz), 6.31 (1 H, s), 5.66 (1 H, s), 5.20 (1 H, d, J = 12.5 Hz), 5.18 (1 H, d, J = 12.5 Hz), 4.94 (1 H, s), 4.74 (1 H, s), 4.70 (1 H, d, J = 11.5 Hz), 4.41 (1 H, d, J = 11.5 Hz), 4.27 (1 H, t, J = 8.5 Hz), 4.15 (1 H, d, J = 9.0 Hz), 3.88 (1 H, s), 3.81 (1 H, d, J = 9.0 Hz), 3.63–3.70 (1 H, m), 3.10 (1 H, t, J = 8.0 Hz), 2.56 (1 H, dd, J = 14.0, 7.5 Hz), 2.41 (1 H, dd, J = 14.0, 6.0 Hz), 1.38 (3 H, d, J = 6.0 Hz); 13C NMR (125 MHz, CDCl3) δ 166.93, 163.34, 137.21, 136.38, 136.01, 128.88, 128.82, 128.73, 128.40, 128.39, 128.28, 128.19, 80.34, 80.28, 72.7, 71.8, 71.0, 66.8, 49.9, 37.0, 17.2; HR-ESI-MS (m/z): calcd for C25H31NO7Na+ (M + Na+): 480.1993, found 480.2002. For minor diastereomer 26b: [α] + 17.3 (c 0.3, DCM); 1H NMR (500 MHz, CDCl3) δ 8.21 (1 H, s), 7.27–7.42 (10 H, m), 6.66 (1 H, d, J = 8.5 Hz), 6.34 (1 H, s), 5.73 (1 H, s), 5.20 (2 H, s), 4.84 (1 H, s), 4.71 (1 H, d, J = 11.5 Hz), 4.64 (1 H, s), 4.42 (1 H, d, J = 11.0 Hz), 4.15 (1 H, dd, J = 9.0, 4.5 Hz), 3.99 (1 H, d, J = 9.0 Hz), 3.85–3.92 (1 H, m), 3.65–3.73 (1 H, m), 3.46 (1 H, d, J = 9.0 Hz), 3.15 (1 H, t, J = 8.5 Hz), 2.65 (1 H, dd, J1 = 14.0 Hz, J2 = 3.5 Hz), 2.54 (1 H, dd, J1 = 14.0 Hz, J2 = 9.0 Hz), 1.39 (3 H, d, J = 6.0 Hz); 13C NMR (125 MHz, CDCl3) δ 167.12, 162.69, 137.36, 136.74, 135.91, 128.84, 128.75, 128.58, 128.44, 128.35, 128.28, 128.19, 80.1, 75.1, 72.8, 71.6, 70.9, 66.9, 50.9, 37.7, 17.0; HR-ESI-MS (m/z): calcd for C25H31NO7Na+ (M + Na+): 480.1993, found 480.2003.

Benzyl (4 R,5 S,6 R,7 R,8 S)-5-acetamido-8-(benzyloxy)-4,6,7-trihydroxy-2-methylenenonanoate (27)

To a 50 mL round bottom flask containing compound 26a (1.35 g, 2.95 mmol, 1.0 equiv), a cold solution of HCl (aq) in MeOH (3%, 12.0 mL prepared from conc. HCl 1.0 mL and MeOH 11.0 mL) was added. The reaction mixture was gradually warmed from 0 oC to r.t. and kept stirring for 6 h. When full conversion was achieved as indicated by TLC, the mixture was cooled to 0 oC again and solid sodium bicarbonate was carefully added until the solution turned neutral. Acetic anhydride (120 μL, 1.24 mmol, 2.0 equiv) was added and the solution was kept stirring for 1 h. After full conversion as indicated by TLC, the reaction was diluted with ethyl acetate (500 mL), washed with1 M HCl (aq, 50 mL), sat. NaHCO3 (aq, 50 mL), and brine (50 mL), and dried over anhydrous Na2SO4. The organic solvent was removed under vacuum and the residue was purified by flash column chromatography using DCM: ethyl acetate 3: 2 v/v as eluent to obtain compound 27 as white foam (347 mg, 24.5%) and DCM: ethyl acetate 1: 1 v/v as eluent to obtain lactone 27a as white foam (686 mg, 64%). For lactone 27a: [α] + 12.3 (c 0.5, DCM); 1H NMR (500 MHz, CDCl3) δ 7.27–7.40 (5 H, m), 6.25 (1 H, t, J = 2.0 Hz), 6.09 (1 H, d, J = 9.0 Hz), 5.67 (1 H, s), 4.69–4.75 (1 H, m), 4.70 (1 H, d, J = 11.0 Hz), 4.43 (1 H, d, J = 11.0 Hz), 4.29 (1 H, t, J = 7.0 Hz), 3.67–3.75 (1 H. m), 3.68 (1 H, d, J = 9.0 Hz), 3.21 (1 H, t, J = 8.0 Hz), 3.06 (1 H, dt, 1 = 17.0, 2.0 Hz), 2.76 (1 H, dt, J = 17.0, 3.0 Hz), 2.08 (3 H, s), 1.36 (3 H, d, J = 6.0 Hz); 13C NMR (125 MHz, CDCl3) δ 172.7, 169.8, 137.5, 133.6, 128.8, 128.29, 128.15, 123.0, 79.2, 74.8, 72.1, 71.0, 54.1, 31.4, 23.3, 16.4; HR-ESI-MS (m/z): calcd for C19H25NO6Na+ (M + Na+): 386.1574, found 386.1581.

To a 50 mL round bottom flask containing lactone 27a (686 mg, 1.88 mmol, 1.0 equiv) was added 1 M NaOH (aq, 5 mL). The reaction was kept vigorous stirring for 2 h and monitored by TLC. When full conversion was achieved, ion exchange resin Dowex 50 W X8 (H+) was carefully added to neutralize the reaction. The mixture was filtered through celite to remove the resin and a small portion of the acid 28 (10 mg) was purified by HPLC for characterization. To the aqueous solution of acid 28 was added K2CO3 (259 mg, 1.88 mmol, 1.0 equiv). Water was removed by oil pump and the residue obtained was re-dissolved in DMF (5 mL). Benzyl bromide (446 μL, 3.76 mmol, 2.0 equiv) was added and the reaction mixture was kept stirring for 6 h. When full conversion was achieved as indicated by TLC, the mixture was diluted with ethyl acetate (300 mL), washed with brine (50 mL), and dried over anhydrous Na2SO4. The organic solvent was removed under vacuum and the residue was purified by flash column chromatography using DCM: ethyl acetate 3: 2 v/v as eluent to obtain compound 27 as white foam (761.5 mg, 86%, together with previous potion, 1.10 g, 79% from 26a). For acid 28: 1H NMR (500 MHz, CDCl3) δ 7.28–7.38 (5 H, m), 6.76 (1 H, d, J = 10.5 Hz), 6.38 (1 H, s), 5.73 (1 H, s), 4.70 (1 H, d, J = 11.0 Hz), 4.41 (1 H, d, J = 11.0 Hz), 4.30 (1 H, t, J = 7.0 Hz), 4.12 (1 H, d, J = 9.0 Hz), 3.88 (1 H, dd, J = 9.0, 1.0 Hz), 3.68–3.73 (1 H, m), 3.10 (1 H, t, J = 9.0 Hz), 2.55 (1 H, dd, J = 14.0, 7.5 Hz), 2.43 (1 H, dd, J = 14.0, 6.0 Hz), 2.17 (3 H, s), 1.40 (3 H, d, J = 5.5 Hz). No carbon spectrum recorded due to the unstability of the acid.

For Benzyl ester 27: [α] + 19.96 (c 0.3, DCM); 1H NMR (400 MHz, CDCl3) δ 7.27–7.40 (10 H, m), 6.38 (1 H, d, J = 9.2 Hz), 6.30 (1 H, d, J = 0.8 Hz), 5.65 (1 H, s), 5.27 (1 H, br), 5.21 (1 H, d, J = 12.4 Hz), 5.17 (1 H, d, J = 12.4 Hz), 4.78 (1 H, s), 4.70 (1 H, d, J = 11.2 Hz), 4.40 (1 H, d, J = 11.2 Hz), 4.25 (1 H, t, J = 6.8 Hz), 4.06 (1 H, d, J = 9.2 Hz), 3.09 (1 H, s), 3.79 (1 H, dd, J = 8.8, 0.8 Hz), 3.62–3,71 (1 H, m), 3.06 (1 H, t, J = 8.8 Hz), 2.55 (1 H, dd, J = 14.0, 7.6 Hz), 2.41 (1 H, dd, J = 14.0, 6.0 Hz), 2.11 (3 H, s), 1.39 (3 H, d, J = 6.0 Hz); 13C NMR (100 MHz, CDCl3) δ 172.9, 166.9, 137.23, 136.5, 136.1, 128.87, 128.72, 128.61, 128.39, 128.35, 128.24, 128.20, 80.75, 80.64, 71.98, 71.89, 71.0, 66.8, 51.1, 37.0, 23.2, 17.3; HR-ESI-MS (m/z): calcd for C26H33 NO7Na+ (M + Na+): 494.2149, found 494.2160.

5-Acetamido-3,5,9-trideoxy-L-glycero-L-gluco-non-2-ulosonic acid (1)

The solution of compound 27 (235 mg, 0.5 mmol) in DCM (10 mL) and methanol (5 mL) was cooled to −78 oC. The O3 (generated from O2 and carried by the flow of O2) was bubbled through this solution. The color of the solution turned purple, which indicated the saturation of O3. The excess amount of O3 was blown off by the flow of O2 and the purple color disappeared. To this solution, Me2S (2 mL) was added to reduce the peroxide intermediate. After 1 h reduction at r.t., the solvent was removed under vacuum and the residue was purified by flash column chromatography using DCM: methanol 30: 1 v/v as eluent to obtain compound 29 as white foam, which was the mixture of linear and cyclic tautomers (177.4 mg, 75%).To a round bottom flask containing compound 29 (109.4 mg, 0.375 mmol), Pd(OH)2/C (20% Pd on activated carbon, 50 mg) was added. methanol (10 mL) and water (3 mL) were added to the flask. The mixture was kept stirring under 1 atm H2 atmosphere for 12 h. After filtration, the solvent was removed under vacuum and the residue was further purified by BioGel column using H2O as eluent. The product 1 was obtained after lyophilization as white foam (92.2 mg, 87%). 1H NMR (500 MHz, D2O) δ 4.28 (1 H, dd, J = 9.5, 2.0 Hz, H-6), 4.00–4.08 (3 H, m, H-4, H-5, H-8), 3.69 (1 H, dd, J = 9.5, 7.0 Hz, H-7), 2.05–2.11 (1 H, m, H-3e), 2.05 (3 H, s, CH3CO), 1.83–1.88 (1 H, m, H-3a), 1.15 (3 H, d, J = 6.5 Hz, H-9); 13C NMR (125 MHz, D2O) δ 176.7 (CH3CO), 174.3 (COOH), 96.0 (C-1), 71.5 (C-7), 67.6 (C-8), 66.3 (C-6), 65.9 (C-4), 47.9 (C-5), 32.3 (C-3), 21.7 (CH3CO), 14.6 (C-9); HR-ESI-MS (m/z): calcd for C11H19NO8Na+ (M + Na+): 316.1003, found 316.1009.

Benzyl 5-acetamido-8-O-benzyl-2,4,7-tri-O-acetyl-3,5,9-trideoxy-L-glycero-L-gluco-2-nonulopyranosonate (30)

To the solution of compound 29 (112 mg, 0.378 mmol) in pyridine (5 mL), Ac2O (2 mL) and DMAP (3 mg, 0.02 mmol, 0.06 equiv) were added sequentially. The mixture was kept stirring for 3 h. The mixture was concentrated under vacuum and the residue was dissolved in ethyl acetate (100 mL). The solution was washed with 1 M HCl (aq, 20 mL), sat. NaHCO3 (aq, 20 mL), and brine (20 mL) and was dried over anhydrous Na2SO4. The organic solvent was removed under vacuum and the residue was purified by silica gel flash chromatography using n-hexane: ethyl acetate 2.5: 1 v/v as eluent to obtain acetate 30 as white foam (183 mg, 83%). 1H NMR (0.9: 1 mixture of two anomers, peaks selected for the major anomer, 500 MHz, CDCl3,) δ 7.27–7.39 (10 H, m), 6.20 (1 H, d, J = 9.0 Hz), 5.26 (1 H, dd, J = 9.0, 2.5 Hz), 5.20 (1 H, d, J = 12.0 Hz), 5.13 (1 H, d, J = 12.5 Hz), 5.02 (1 H, q, J = 3.5 Hz), 4.80 (1 H, dd, J = 9.0, 2.0 Hz), 4.56 (1 H, d, J = 11.5 Hz), 4.45 (1 H, d, J = 11.5 Hz), 4.16 (1 H, dt, J = 9.0, 2.0 Hz), 3.87 (1 H, qd, J = 6.5, 3.5 Hz), 2.59 (1 H, dd, J = 15.5, 2.5 Hz), 2.32 (2 H, dd, J = 15.0, 3.5 Hz), 2.09 (3 H, s), 2.03 (3 H, s), 1.96 (3 H, s), 1.83 (3 H, s), 1.17 (3 H, d, J = 6.5 Hz); 1H NMR (0.9: 1 mixture of two anomers, peaks selected for the minor anomer, 500 MHz, CDCl3,) δ 7.27–7.39 (9 H, m), 6.20 (0.9 H, d, J = 9.0 Hz), 5.22 (0.9 H, d, J = 12.5 Hz,), 5.18 (0.9 H, dd, J = 9.0, 2.5 Hz), 5.13 (0.9 H, d, J = 12.5 Hz), 4.92–4.94 (0.9 H, m), 4.63 (0.9 H, dd, J = 9.0, 2.0 Hz), 4.57 (0.9 H, d, J = 11.5 Hz), 4.49 (0.9 H, d, J = 11.5 Hz), 4.20 (1 H, dt, J = 9.0, 2.0 Hz), 3.78 (0.9 H, qd, J = 6.5, 2.5 Hz), 2.40 (0.9 H, dd, J = 16.0, 1.5 Hz), 2.11 (2.7 H, s), 2.04 (2.7 H, s), 1.99 (0.9 H, dd, J = 16.0, 4.0 Hz), 1.93 (2.7 H, s), 1.92 (2.7 H, s), 1.24 (d, J = 6.5 Hz, 2.7 H); 13C NMR (125 MHz, CDCl3) δ 170.46, 170.19, 170.06, 169.91, 169.57, 169.16, 168.55, 167.65, 168.42, 167.36, 138.53, 138.46, 134.87, 134.81, 128.89, 128.84, 128.80, 128.70, 128.54, 128.45, 128.44, 128.08, 128.03, 127.79, 127.67, 96.3, 95.8, 75.0, 74.1, 71.5, 71.23, 71.05, 70.7, 70.2, 68.20, 68.16, 67.8, 67.3, 45.05, 44.60, 30.88, 29.89, 23.32, 23.11, 21.49, 21.25, 21.00, 20.97, 20.73, 20.58, 15.5, 14.3; HR-ESI-MS (m/z): calcd for C31H37NO11Na+ (M + Na+): 622.2259, found 622.2271.

4-Methylphenyl 7-acetamido-8-benzyl-4,7-di-O-acetyl-3,5,9-trideoxy-β-thiofusaminoside (31)

To a 25 mL round bottom flask, acetate 30 (183 mg, 0.31 mmol, 1.0 equiv) and 4-toluenethiol (234 mg, 1.88 mmol, 6.0 equiv) were added. Anhydrous DCM (6.0 mL) was added under argon, and the concentration of 30 was kept at 50 mM. The mixture was cooled to 0 oC, then BF3·Et2O (77 μL, 0.62 mol, 2.0 equiv) was added dropwise. The mixture was then kept stirring at 0 oC to r.t. for 16 h. The reaction was quenched by sat. NaHCO3 (aq), and the organic phase was dried over anhydrous Na2SO4. The solvent was removed under vacuum and the residue was purified by silica gel flash chromatography using n-hexane: ethyl acetate 4:1 v/v as eluent to obtain thioglycoside 31 as yellow foam (127.4 mg, 62%) and characterized as β anomer (JC1, H3a = 6.4 Hz). [α] + 10.85 (c 0.7, DCM); 1H NMR (500 MHz, CDCl3,) δ 7.26–7.35 (10 H, m, ArH), 7.15–7.20 (ArH, m, 2 H), 7.02 (2.0 H, d, J = 8.0 Hz, ArH), 6.31 (1 H, d, J = 8.0 Hz, NH), 5.23 (1 H, dd, J = 7.5, 4.5 Hz, H-7), 5.11 (1 H, dd, J = 7.0, 2.0 Hz, H-6), 4.98–5.02 (1 H, m, H-4), 4.86 (1 H, d, J = 12.0 Hz, PhCH2O), 4.83 (1 H, d, J = 12.0 Hz, PhCH2O), 4.57 (1 H, d, J = 11.0 Hz, PhCH2O), 4.48 (1 H, d, J = 11.0 Hz, PhCH2O), 4.14 (1 H, dt, J = 7.5, 1.0 Hz, H-5), 3.89 (1 H, qd, J = 6.5, 4.5 Hz, H-8), 2.68 (1 H, dd, J = 16.0, 2.0 Hz, H-3e), 2.32 (3 H, s, SC6H4CH3), 2.20 (1 H, dd, J = 16.0, 3.5 Hz, H-3a), 2.18 (3 H, s, CH3CO), 2.08 (3 H, s, CH3CO), 1.71 (3 H, s, CH3CO), 1.30 (3 H, d, J = 6.5 Hz, H-9); 13C NMR (125 MHz, CDCl3) δ 170.4, 170.0, 169.8, 167.9, 139.6, 138.1, 135.3, 134.9, 129.7, 128.7, 128.6, 128.4, 127.9, 127.5, 89.3, 74.1, 71.9, 71.3, 68.5, 67.6, 46.1, 32.9, 27.2, 22.9, 21.7, 21.4, 21.0, 15.8; HR-ESI-MS (m/z): calcd for C36H41NO9SNa+ (M + Na+): 686.2394, found 686.2407.