Synthesis and Immunological Evaluation of Escherichia coli O1- Derived Oligosaccharide–Protein Conjugates toward Avian Pathogenic Escherichia coli O1 Vaccine Development

Abstract

Avian pathogenic Escherichia coli O1 (APEC O1) is a pathogenic bacterium that causes significant economic losses in the poultry industry and raises concerns about zoonotic infections. The development of effective vaccines against APEC O1 is essential due to antibiotic resistance and the potential for severe symptoms in both chickens and humans. In this context, we have been focusing on the O1A, O1B, and O1C antigen structures derived from E. coli O1 lipopolysaccharide (LPS). In this study, the first synthesis of the pentasaccharide repeating units of the O1B and O1C antigens was successfully achieved. The synthesis and immunological evaluation of their conjugates with bovine serum albumin (BSA) were conducted. Only the O1A-pentasaccharide structure is a glycotope candidate for APEC O1. Keyhole limpet hemocyanin (KLH)–O1A-pentasaccharide conjugate was also synthesized, and its immunogenicity was evaluated by the ELISA assay. The efficient production of antibodies capable of binding to both APEC O1 LPS and the O1A-pentasaccharide structure was observed, indicating that O1A-pentasaccharide is a promising vaccine candidate against APEC O1.

Avian pathogenic Escherichia coli O1 (APEC O1) is one of the pathogenic bacteria that cause significant economic losses in the poultry industry.[1] In addition, it has been reported that there is a genomic similarity to pathogenic E. coli that can cause severe symptoms such as septicemia.[2a] Therefore, there is concern about zoonotic infections which can infect both chickens and humans.[3] The emergence of resistance to antibiotics widely used to treat APEC O1 is problematic, leading to a lack of effective treatments.[2] Therefore, the development of an effective vaccine is urgently required. In 2017, Kong and co-workers reported the development of an APEC O1 vaccine using a genetically modified strain of Salmonella bacteria.[4] They developed a plasmid (pSS27) that expresses the O antigen of E. coli O1 lipopolysaccharide (LPS) and introduced it into an attenuated strain of Salmonella Typhimurium (S740) to prepare a Salmonella bacterium that displays the O antigen of APEC O1 on its surface. When the prepared Salmonella bacterium was administered to chickens, the mortality rate caused by APEC O1 infection was significantly reduced. Therefore, the utility of the O antigen of APEC O1 LPS as an antigen has been demonstrated. However, since this study utilized the expressed glycans in Salmonella bacteria, the detailed structure of the antigenic glycans of APEC O1 was not elucidated. In this context, we focused on the three O1A, O1B, and O1C antigen structures, which have been reported by Jann’s group, as repeating units of O-specific side chains of LPS derived from E. coli O1 (Figure [1]), and conducted research using the O1A antigen derived from virulent E. coli O1.[5] We previously accomplished the first synthesis of the pentasaccharide repeating unit 1 (O1A-penta) of O1A and its substructures, trisaccharide 2 (O1A-tri) and disaccharide 3 (O1A-di), as well as their respective conjugates 4–6 with bovine serum albumin (BSA). We revealed that the pentasaccharide structure of O1A antigen is important for binding with anti-APEC O1 antibody by ELISA assays using 4–6 and APEC O1 immune chicken serum (Figure [2]A).[6]

Herein, we report the first synthesis of the pentasaccharide repeating units 7 and 8 of the O1B and O1C antigens derived from prevalent E. coli O1, which is not reported to be pathogenic, as well as their respective conjugates 9 and 10, utilizing our boron-mediated aglycon delivery (BMAD) method.[7] In addition, ELISA assays using 4, 9, and 10 revealed that the anti-APEC O1 antibody strongly bound only with the O1A-pentasaccharide structure. Moreover, ELISA assays using APEC O1 LPS and a glycoconjugate 11 derived from 1 and keyhole limpet hemocyanin (KLH) and antiserum obtained by immunizing chickens with 11 indicated that 11 effectively stimulated the production of antibodies that bound to APEC O1 LPS and the O1A-pentasaccharide structure, suggesting that 11 has great potential as a lead candidate for vaccine development (Figure [2]B).

The synthesis of 7 is shown in Scheme [1]. First, we prepared the known 1,2-anhydro glucose donor 12 [8] and protected glucosamine 13,[6] and then examined the BMAD reaction between them using bis(4-fluorophenyl)borinic acid (14) in THF at –40 °C to room temperature for 24 hours. The reaction provided the corresponding glycoside 15 in 58% yield [97% yield based on recovered starting material (brsm)] with complete α-stereoselectivity. Glycosylation of 15 with the known rhamnosyl trichloroacetimidate donor 16 [9] using TfOH in toluene at –40 to –20 °C for 19 hours, followed by selective deprotection of the Ac group in 17, provided trisaccharide 18. The anomeric configuration of 17 was determined by ¹ J _CH coupling constants (¹ J _CH = 175 Hz for the newly formed glycosidic linkage).[10] [2+3] Glycosylation of 18 with the known glycosyl N-phenyltrifluoroacetimidate 19 [6] using TfOH in toluene at –40 to –20 °C for 15 hours proceeded smoothly to provide the desired pentasaccharide 20 in good yield with complete α-stereoselectivity (¹ J _CH = 168 Hz for the newly formed glycosidic linkage). Next, the azido group in 20 was converted into an N-Ac group by the Staudinger reaction followed by acetylation. Treatment of the resulting pentasaccharide with Zn–Cu and Ac₂O in 1,4-dioxane and AcOH converted the N-Troc group into an N-Ac group to form 21. Global deprotection provided 7.

The synthetic scheme for 8 is shown in Scheme [2]. First, 1,2-anhydro donor 25 and acceptor 28 were prepared from d-galactal (22) and d-glucosamine hydrochloride (26), respectively (Scheme [2]A). Selective protection of the 3-OH in 22 with a PMB group using ⁿ Bu₂SnO, followed by silyl acetal protection of the 4,6-diol in 23,[11] gave 24. Epoxide formation using dimethyldioxirane afforded 1,2-anhydro donor 25. Next, compound 27 [6] was prepared according to known methods in 6 steps from 26. Selective protection of the primary alcohol in 27 with a TBDPS group gave 28. The BMAD reaction of 25 and 28 was examined using catalytic amounts of tetrahydroxydiboron (29) in MeCN at 70 °C for 24 hours. The reaction provided the desired α(1,3)-glycoside 30 with high regio- and complete α-stereoselectivity in 51% yield along with 13% of the α(1,4)-isomer. The anomeric configuration of the newly formed glycosidic linkage of 30 was determined by the coupling constant (³ J _HH = 3.6 Hz). The position of the glycosidic linkage was confirmed by acetylation and subsequent ¹H NMR analysis of the corresponding acetylated compound 30a. TBS protection of the 2′-OH in 30, followed by deprotection of the PMB group, gave disaccharide acceptor 31. Glycosylation of 31 with rhamnosyl donor 16 using TfOH in CH₂Cl₂ at –40 to –20 °C for 3 hours afforded the α-trisaccharide (¹ J _CH = 168 Hz for the newly formed glycosidic linkage) in 73% yield (91% yield brsm). Selective deprotection of the Ac group using K₂CO₃ gave trisaccharide acceptor 32. The [2+3] glycosylation of 32 with disaccharide donor 19 was performed using catalytic amounts of TfOH in toluene at –40 °C for 3 hours, affording the desired α-pentasaccharide 33 (¹ J _CH = 168 Hz for the newly formed glycosidic linkage) in 56% yield (69% yield brsm) as a single isomer. The azido group of 33 was reduced by the Staudinger reaction, and the resulting amine was subsequently protected with an Ac group. The Troc group in the pentasaccharide was then converted into an N-Ac group using the same reaction with Zn–Cu couple in a mixture of 1,4-dioxane, Ac₂O, and AcOH to give 34. Finally, deprotection of the silyl protecting group, followed by removal of the Bn, PMB, and Cbz groups, afforded compound 8 (Scheme [2]B).

With pentasaccharides 7 and 8 in hand, BSA–glycan conjugates 9 and 10 were synthesized using the p-nitrophenol (PNP) activation method,[12] as summarized in Scheme [3]. Specifically, pentasaccharides 7 and 8 were reacted with an excess of the activated ester 35 to produce monoamide–monoester products 36 and 37, respectively. After purification using a size exclusion column to remove any remaining reactant 35, compounds 36 and 37 were individually mixed with BSA in phosphate-buffered saline at pH 7.5. The conjugation reactions were carried out at 25 °C for 18 hours, resulting in the formulation of conjugates 9 and 10. The resulting conjugates were purified by dialysis to eliminate salts and residual sugars, followed by lyophilization. The carbohydrate loading values[13] of 9 and 10 were determined by MALDI-TOF MS analysis to be an average of 12.8 and 11.4 oligosaccharide residues per BSA molecule, respectively (Supporting Information, Figure S1).

An ELISA assay was conducted to confirm whether the pentasaccharide units of 9 and 10 were recognized by antibodies in APEC O1 immune chicken serum. APEC O1 immune chicken serum was prepared from a chicken that was immunized with the APEC O1 strain ATCC 11775[14] at 10 and 24 days of age. Whole blood was collected, and serum isolation was carried out at 38 days of age. As a negative control, non-immune chicken serum was prepared to assess the background absorbance resulting from nonspecific binding or cross-reactivity. Synthetic glycoconjugates 4, 9, and 10 were loaded on the ELISA plates. BSA and APEC O1 LPS extracted from the strain using the hot phenol method[15] were also loaded on the plates as a negative and positive control, respectively. The binding between antibodies in the chicken serum and each compound was evaluated using anti-IgY-HRP (horseradish peroxidase) and OPD (o-phenylenediamine dihydrochloride). Initially, it was confirmed that APEC O1 LPS was well recognized by APEC O1 immune chicken serum compared to non-immune chicken serum (lane 1 in Figure [3]), indicating that APEC O1 LPS-specific antibodies are contained in APEC O1 immune chicken serum. Next, recognitions of BSA, 4, 9, and 10 were evaluated with both sera. BSA, 9, and 10 were hardly recognized by either serum. In sharp contrast, 4 was selectively and significantly recognized by APEC O1 immune chicken serum (lanes 2–5 in Figure [3]). Taken together, these results clearly indicated that O1A-pentasaccharide including a β-rhamnoside structure is important for the binding of anti-APEC O1 LPS antibodies and that only the O1A-pentasaccharide is a potential glycotope for APEC O1.

Next, to investigate the possibility of a vaccine candidate, we synthesized KLH–O1A-pentasaccharide conjugate 11 using the PNP method, as shown in Scheme [4]. In this case, the carbohydrate loading value of 11 was determined by the anthrone–sulfuric acid assay[16] to be 6.5% of carbohydrate content per KLH molecule. To assess the antigenicity, 11 immune chicken serum was prepared from a chicken that was immunized with 11 at 10, 24, and 38 days of age. Whole blood was collected, and serum isolation was carried out at 45 days of age. An ELISA assay was conducted using 11 immune chicken serum and non-immune chicken serum. The ELISA plate was coated with APEC O1 LPS, BSA, and 4. BSA was hardly recognized by either serum (lane 2 in Figure [4]). In sharp contrast, 4 was well recognized by 11 immune chicken serum compared to non-immune chicken serum (lane 3 in Figure [4]). APEC O1 LPS was also selectively recognized by 11 immune chicken serum (lane 1 in Figure [4]). These results indicated that the pentasaccharide structure of O1A holds great promise as a vaccine candidate against APEC O1.

In conclusion, we successfully achieved the first synthesis of the pentasaccharides 7 and 8, derived from non-pathogenic E. coli O1, as well as their respective BSA conjugates 9 and 10. These challenging α-galactosides, which are integral in both compounds, were synthesized with complete stereoselectivity using BMAD methods. Subsequent ELISA tests employing glycoconjugates 4, 9, and 10 revealed the presence of antibodies specifically binding to O1A-pentasaccharide in APEC O1 immune chicken serum. Accordingly, only the O1A-pentasaccharide structure is a glycotope candidate for APEC O1. We also synthesized KLH–O1A-pentasaccharide conjugate 11 and evaluated its antigenicity to investigate the possibility of a vaccine candidate. ELISA tests confirmed the efficient production of antibodies capable of binding to both APEC O1 LPS and O1A-pentasaccharide structures. Further development of a glycoconjugate vaccine against APEC O1 by using O1A-pentasaccharide is now in progress in our laboratories.

NMR spectra were recorded on a JEOL ECA-500 (500 MHz for ¹H, 125 MHz for ¹³C) spectrometer, JEOL ECA-400 (400 MHz for ¹H, 100 MHz for ¹³C) spectrometer, or JEOL ECZ-400S (400 MHz for ¹H, 100 MHz for ¹³C) spectrometer. ¹H NMR data are reported as follows: chemical shift in parts per million (ppm) downfield or upfield from CDCl₃ (δ 7.26), D₂O (δ 4.79), or TMS (δ 0.00), multiplicity (br = broad, s = singlet, d = doublet, m = multiplet, ABq = AB quartet), coupling constant(s) (Hz), and integration. ¹³C NMR chemical shifts are reported in ppm downfield or upfield from CDCl₃ (δ 77.0) or CD₃OD (δ 49.0). Using D₂O as an NMR solvent, ¹³C NMR chemical shifts are reported in ppm downfield or upfield from acetone (δ 29.8) as an external reference. ESI-TOF MS spectra and MALDI-TOF MS spectra were measured on a Waters LCT premier XE instrument and MALDI-7090 instrument (Shimadzu Co.), respectively. Melting points were determined on a micro hot-stage apparatus (Yanako MP-S3). Optical rotations were measured on a JASCO P-2200 polarimeter. Silica gel TLC was performed on Merck TLC 60F-254 (0.25 mm) or Merck PLC 60F-254 (0.5 mm) plates. Column chromatography separation was performed on silica gel 60N (spherical, neutral, 63–210 μm or 40–50 μm) (Kanto Chemical Co., Inc.). Reverse-phase column chromatography separation was performed on a Wakosil 25C18 (Wako Pure Chemical Industries, Ltd.) or Sep-Pak C18 reversed-phase cartridge (Waters). Size exclusion column chromatography separation was performed using Sephadex^TM LH-20 (GE Healthcare). Air- and/or moisture-sensitive reactions were carried out under an argon atmosphere using oven-dried glassware. For reactions under H₂ atmosphere, a TVS-1-50 autoclave (Taiatsu Techno Co.) was used. UV–vis spectra were measured using a SpectraMax i3 (Molecular Devices) microplate reader.

5-(Benzyloxycarbonylamino)pentyl 3,4,6-Tri-O-(tert-butyldimethylsilyl)-α-d-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-d-glucopyranoside (15)

To a solution of 13 [6] (22.5 mg, 34.1 μmol) and bis(4-fluorophenyl)borinic acid (14) (13.2 mg, 68.2 μmol) in dry THF (303 μL) was added a solution of 1,2-anhydo donor 12 [8] (34.4 mg, 68.2 μmol) in dry THF (303 μL) at –40 °C under Ar atmosphere. After the reaction mixture was stirred for 4 h at the same temperature, it was stirred for another 20 h at room temperature. The reaction was quenched by the addition of 0.05 M NaBO₃ aq. (1.34 mL). To the resultant mixture was added sat. NH₄Cl aq. (2 mL). The aqueous layer was extracted with EtOAc (5 × 2 mL), and then the combined extracts were washed with brine (2 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (n-hexane/EtOAc, 4:1 to 3:1) gave 15 (22.9 mg, 19.6 μmol, 58% yield).

White solid; mp 73.0–74.5 °C; [α]_D ²⁶ +19.9 (c 1.0, CHCl₃); R_f = 0.61 (n-hexane/EtOAc, 2:1).

¹H NMR (400 MHz, CDCl₃): δ = 7.48–7.44 (m, 2 H), 7.41–7.29 (m, 8 H), 6.26 (d, J = 7.6 Hz, 1 H), 5.54 (s, 1 H), 5.19 (d, J = 3.6 Hz, 1 H), 5.14–5.06 (m, 2 H), 4.98 (d, J = 12.0 Hz, 1 H), 4.82 (br s, 1 H), 4.60–4.50 (m, 2 H), 4.35 (dd, J = 5.2, 10.8 Hz, 1 H), 4.10 (dd, J = 9.6, 9.6 Hz, 1 H), 4.01 (dd, J = 8.0, 2.4 Hz, 1 H), 3.92–3.75 (m, 5 H), 3.74–3.54 (m, 4 H), 3.50–3.37 (m, 2 H), 3.23–3.13 (m, 2 H), 1.98 (d, J = 10.0 Hz, 1 H), 1.65–1.30 (m, 6 H), 0.98–0.80 (m, 27 H), 0.15–0.01 (m, 18 H).

¹³C NMR (100 MHz, CDCl₃): δ = 156.4, 154.5, 136.7, 136.6, 129.0, 128.5, 128.3, 128.0, 125.9, 102.3, 101.4, 100.0, 95.9, 81.4, 75.5, 74.2, 73.3, 72.9, 72.4, 70.0, 69.6, 68.6, 66.5, 66.1, 64.5, 57.4, 40.8, 29.4, 28.9, 26.2, 25.9, 25.9, 23.0, 18.5, 18.4, 18.4, –3.8, –3.8, –4.4, –5.0, –5.3, –5.5.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₅₃H₈₈N₂O₁₄Si₃Cl₃: 1165.4609; found: 1165.4603.

5-(Benzyloxycarbonylamino)pentyl 2-O-Acetyl-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→2)-3,4,6-tri-O-(tert-butyldimethylsilyl)-α-d-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-d-glucopyranoside (17)

To a solution of 15 (24.4 mg, 20.9 μmol) and 16 [9] (17.6 mg, 31.4 μmol) in dry toluene (418 μL) was added 4 Å MS (17.6 mg) at room temperature under Ar atmosphere. After the reaction mixture was stirred vigorously for 1 h, TfOH (0.37 μL, 4.18 μmol) was added to the reaction mixture at –40 °C. After the reaction mixture was stirred for 19 h at –20 °C, the reaction was quenched by the addition of sat. NaHCO₃ aq. (1 mL). To the resultant mixture were added EtOAc (5 mL) and sat. NaHCO₃ aq. (4 mL). The aqueous layer was extracted with EtOAc (5 × 5 mL), and then the combined extracts were washed with brine (5 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (toluene/acetone, 30:1) gave 17 (17.2 mg, 11.0 μmol, 53% yield).

White solid; mp 76.5–78.3 °C; [α]_D ²⁶ +1.7 (c 1.0, CHCl₃); R_f = 0.61 (toluene/acetone, 8:1).

¹H NMR (400 MHz, CDCl₃): δ = 7.40–7.18 (m, 15 H), 7.10–7.02 (m, 2 H), 6.95–6.89 (m, 2 H), 5.82 (d, J = 8.8 Hz, 1 H), 5.57 (br s, 1 H), 5.43 (br s, 1 H), 5.28 (s, 1 H), 5.18–5.05 (m, 2 H), 4.93–4.78 (m, 4 H), 4.69–4.56 (m, 2 H), 4.44 (d, J = 11.2 Hz, 1 H), 4.40–4.33 (m, 2 H), 4.14 (dd, J = 4.4, 10.0 Hz, 1 H), 4.07 (dd, J = 9.6, 9.6 Hz, 1 H), 4.02–3.93 (m, 3 H), 3.92–3.76 (m, 8 H), 3.61 (dd, J = 10.4, 2.8 Hz, 1 H), 3.58–3.49 (m, 2 H), 3.47 (m, 1 H), 3.36 (m, 1 H), 3.26 (m, 1 H), 3.22–3.11 (m, 3 H), 2.08 (s, 3 H), 1.65–1.33 (m, 6 H), 0.99–0.82 (m, 27 H), 0.50 (d, J = 4.0 Hz, 3 H), 0.23–0.03 (m, 18 H).

¹³C NMR (100 MHz, CDCl₃): δ = 169.8, 159.3, 156.4, 154.3, 138.8, 137.1, 136.6, 130.4, 130.0, 128.6, 128.5, 128.2, 128.2, 128.1, 128.0, 127.7, 126.1, 113.8, 102.7 (J _CH = 154 Hz), 100.3 (J _CH = 175 Hz), 98.5 (J _CH = 168 Hz), 96.7, 95.6, 82.2, 79.5, 75.2, 74.6, 73.2, 72.6, 72.3, 71.9, 70.5, 69.8, 68.2, 68.2, 67.8, 66.6, 66.2, 64.5, 56.8, 55.2, 40.9, 29.5, 29.0, 26.1, 26.0, 26.0, 23.0, 21.0, 18.5, 18.4, 18.2, 17.6, –3.7, –4.0, –4.6, –4.7, –5.1, –5.4.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₇₆H₁₁₄N₂O₂₀Si₃Cl₃: 1563.6338; found: 1563.6370.

5-(Benzyloxycarbonylamino)pentyl 4-O-Benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→2)-3,4,6-tri-O-(tert-butyldimethylsilyl)-α-d-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-d-glucopyranoside (18)

To a solution of 17 (30.1 mg, 19.2 μmol) in MeOH (3.84 mL) was added K₂CO₃ (26.6 mg, 192 μmol) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 24 h at the same temperature, the reaction was quenched by the addition of sat. NH₄Cl aq. (1 mL). To the resulting mixture were added EtOAc (5 mL) and H₂O (4 mL). The aqueous layer was extracted with EtOAc (5 × 5 mL), and then the combined extracts were washed with brine (5 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (toluene/acetone, 15:1) gave 18 (26.8 mg, 17.6 μmol, 91% yield).

White solid; mp 71.8–73.3 °C; [α]_D ²⁶ +8.9 (c 0.93, CHCl₃); R_f = 0.45 (toluene/acetone, 12:1).

¹H NMR (400 MHz, CDCl₃): δ = 7.40–7.20 (m, 15 H), 7.17–7.09 (m, 2 H), 6.95–6.88 (m, 2 H), 5.78 (d, J = 10.4 Hz, 1 H), 5.41 (br s, 1 H), 5.36 (s, 1 H), 5.18–5.05 (m, 2 H), 4.92 (br s, 1 H), 4.87–4.75 (m, 3 H), 4.72–4.56 (m, 3 H), 4.47–4.37 (m, 2 H), 4.18 (dd, J = 4.4, 10.0 Hz, 1 H), 4.13–4.05 (m, 2 H), 4.03–3.91 (m, 3 H), 3.91–3.73 (m, 8 H), 3.66–3.57 (m, 3 H), 3.47 (m, 1 H), 3.37 (m, 1 H), 3.30 (m, 1 H), 3.26–3.12 (m, 3 H), 2.25 (br s, 1 H), 1.62–1.34 (m, 6 H), 0.99–0.84 (m, 27 H), 0.54 (br s, 3 H), 0.15–0.02 (m, 18 H).

¹³C NMR (100 MHz, CDCl₃): δ = 159.4, 156.4, 154.3, 138.8, 137.1, 136.6, 130.0, 129.8, 128.7, 128.5, 128.2, 128.0, 127.6, 126.1, 114.0, 102.5, 100.5, 99.7, 96.8, 95.6, 82.2, 79.8, 79.0, 75.2, 74.5, 73.2, 72.5, 72.0, 71.8, 71.0, 69.7, 68.3, 67.6, 66.5, 66.1, 64.5, 56.8, 55.2, 40.9, 29.5, 29.0, 26.3, 26.0, 23.0, 18.5, 18.4, 18.2, 17.6, –3.7, –3.8, –4.5, –4.7, –5.1, –5.4.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₇₄H₁₁₂N₂O₁₉Si₃Cl₃: 1521.6233; found: 1521.6263.

5-(Benzyloxycarbonylamino)pentyl 2-Azido-3,4,6-tri-O-benzyl-2-deoxy-β-d-mannopyranosyl-(1→2)-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→2)-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→2)-3,4,6-tri-O-(tert-butyldimethylsilyl)-α-d-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-d-glucopyranoside (20)

To a solution of 19 [6] (114 mg, 113 μmol) and 18 (115 mg, 75.6 μmol) in dry toluene (1.51 mL) was added 4 Å MS (114 mg) at room temperature under Ar atmosphere. After the reaction mixture was stirred vigorously for 1 h, TfOH (1.34 μL, 15.1 μmol) was added to the reaction mixture at –40 °C. After the reaction mixture was stirred for 15 h at –20 °C, the reaction was quenched by the addition of sat. NaHCO₃ aq. (1 mL). To the resultant mixture were added EtOAc (5 mL) and sat. NaHCO₃ aq. (4 mL). The aqueous layer was extracted with EtOAc (5 × 5 mL), and then the combined extracts were washed with brine (5 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (toluene/acetone, 24:1) gave 20 (126 mg, 54.0 μmol, 71% yield).

White solid; mp 63.2–65.0 °C; [α]_D ²⁶ –16.1 (c 1.0, CHCl₃); R_f = 0.67 (toluene/acetone, 8:1).

¹H NMR (400 MHz, CDCl₃): δ = 7.38–7.06 (m, 39 H), 6.95–6.89 (m, 2 H), 6.82–6.76 (m, 2 H), 5.56 (br s, 1 H), 5.39 (br s, 1 H), 5.28 (s, 1 H), 5.20–5.05 (m, 2 H), 5.03 (d, J = 1.2 Hz, 1 H), 4.89–4.76 (m, 6 H), 4.69 (br s, 1 H), 4.67–4.61 (m, 2 H), 4.61–4.57 (m, 2 H), 4.56–4.44 (m, 6 H), 4.44 (br s, 1 H), 4.36 (d, J = 12.0 Hz, 1 H), 4.32 (d, J = 11.6 Hz, 1 H), 4.20 (br s, 1 H), 4.14–4.07 (m, 2 H), 4.06 (br d, J = 3.6 Hz, 1 H), 3.99 (br s, 1 H), 3.97–3.77 (m, 8 H), 3.77–3.74 (m, 4 H), 3.73–3.67 (m, 2 H), 3.67–3.63 (m, 4 H), 3.64–3.38 (m, 7 H), 3.34 (m, 1 H), 3.29–3.14 (m, 4 H), 3.11 (dd, J = 9.2, 9.2 Hz, 1 H), 1.63–1.33 (m, 6 H), 1.21 (d, J = 6.4 Hz, 3 H), 0.96–0.84 (m, 27 H), 0.54 (br s, 3 H), 0.15–0.03 (m, 18 H).

¹³C NMR (100 MHz, CDCl₃): δ = 159.3, 159.2, 156.4, 138.9, 138.6, 138.1, 138.1, 137.7, 137.2, 136.6, 130.5, 130.4, 129.7, 129.3, 128.6, 128.5, 128.3, 128.3, 128.2, 128.0, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 126.1, 114.0, 113.9, 102.5 (J _CH = 161 Hz), 100.9 (J _CH = 168 Hz, J _CH = 161 Hz), 100.0 (J _CH = 168 Hz), 96.8 (J _CH = 175 Hz), 95.6, 82.0, 80.8, 80.5, 80.4, 79.3, 78.1, 75.7, 75.2, 74.7, 74.1, 73.8, 73.5, 72.7, 72.4, 71.9, 71.3, 70.7, 69.9, 69.0, 68.3, 68.2, 66.6, 66.1, 61.7, 57.1, 55.2, 55.1, 40.9, 29.7, 29.6, 29.1, 26.4, 26.0, 26.0, 23.1, 18.5, 18.4, 18.4, 18.2, 17.8, –3.6, –4.1, –4.3, –4.8, –5.0, –5.4.

HRMS (ESI-TOF): m/z [M + Na]⁺ calcd for C₁₂₂H₁₆₂N₅O₂₈Si₃Cl₃Na: 2356.9672; found: 2356.9785.

5-(Benzyloxycarbonylamino)pentyl 2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-d-mannopyranosyl-(1→2)-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→2)-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→2)-3,4,6-tri-O-(tert-butyldimethylsilyl)-α-d-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-d-glucopyranoside (S1)

To a solution of 20 (172 mg, 73.5 μmol) in THF (1.23 mL) was added PPh₃ (96.4 mg, 368 μmol) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 15 h at 40 °C, H₂O (15.8 μL, 882 μmol) was added to the reaction mixture. After the reaction mixture was refluxed for 3 h, it was concentrated in vacuo. The residue was used for the next reaction without further purification.

To a solution of the above residue in CH₂Cl₂/MeOH (1:1, v/v, 7.32 mL) was added Ac₂O (208 μL, 2.21 mmol) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 2 h at the same temperature, the reaction was quenched by the addition of sat. NaHCO₃ aq. (5 mL). To the resultant mixture were added CHCl₃ (15 mL) and sat. NaHCO₃ aq. (10 mL). The aqueous layer was extracted with CHCl₃ (2 × 15 mL), and then the combined extracts were washed with brine (15 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (toluene/acetone, 10:1) gave S1 (150 mg, 63.7 μmol, 87% yield).

White solid; mp 63.3–67.3 °C; [α]_D ²⁶ –7.3 (c 1.0, CHCl₃); R_f = 0.45 (toluene/acetone, 8:1).

¹H NMR (400 MHz, CDCl₃): δ = 7.38–7.17 (m, 37 H), 7.15–7.07 (m, 2 H), 6.96–6.90 (m, 2 H), 6.80–6.75 (m, 2 H), 6.12 (d, J = 8.0 Hz, 1 H), 5.85 (d, J = 8.4 Hz, 1 H), 5.41 (br s, 1 H), 5.28 (s, 1 H), 5.17–5.05 (m, 2 H), 4.99 (br s, 1 H), 4.92–4.78 (m, 6 H), 4.77–4.70 (m, 3 H), 4.67 (d, J = 11.6 Hz, 1 H), 4.62–4.54 (m, 2 H), 4.53–4.42 (m, 6 H), 4.40 (d, J = 11.2 Hz, 1 H), 4.37–4.31 (m, 2 H), 4.14–4.06 (m, 2 H), 4.04 (br s, 1 H), 4.00 (br s, 1 H), 3.98–3.67 (m, 16 H), 3.64 (s, 3 H), 3.63–3.56 (m, 2 H), 3.53 (dd, J = 8.8, 8.8 Hz, 1 H), 3.48–3.39 (m, 4 H), 3.34 (m, 1 H), 3.25 (m, 1 H), 3.22–3.14 (m, 2 H), 3.08 (dd, J = 8.8, 8.8 Hz, 1 H), 1.92 (s, 3 H), 1.62–1.34 (m, 6 H), 1.17 (d, J = 6.4 Hz, 3 H), 0.98–0.82 (m, 27 H), 0.49 (br s, 3 H), 0.15–0.03 (m, 18 H).

¹³C NMR (100 MHz, CDCl₃): δ = 170.8, 159.3, 159.1, 156.4, 154.2, 138.8, 138.7, 138.2, 138.1, 137.9, 137.2, 136.6, 130.5, 130.4, 129.5, 129.3, 128.6, 128.5, 128.3, 128.3, 128.2, 128.1, 128.0, 127.9, 127.7, 127.7, 127.6, 127.4, 126.1, 114.0, 113.8, 102.4, 100.7, 100.2, 100.0, 96.7, 95.6, 82.0, 80.7, 80.4, 79.4, 79.1, 78.4, 75.3, 75.2, 75.2, 74.7, 74.7, 74.2, 73.8, 73.3, 73.2, 72.5, 71.6, 71.3, 70.7, 69.8, 69.3, 68.5, 68.3, 68.2, 66.6, 66.1, 57.0, 55.1, 55.1, 49.2, 40.9, 29.7, 29.6, 29.0, 26.5, 26.0, 26.0, 23.4, 23.0, 18.5, 18.4, 18.4, 18.2, 17.9, –3.5, –4.1, –4.3, –4.8, –5.0, –5.4.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₁₂₄H₁₆₇N₃O₂₉Si₃Cl₃: 2351.0053; found: 2351.0066.

5-(Benzyloxycarbonylamino)pentyl 2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-d-mannopyranosyl-(1→2)-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→2)-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→2)-3,4,6-tri-O-(tert-butyldimethylsilyl)-α-d-galactopyranosyl-(1→3)-2-acetamido-4,6-O-benzylidene-2-deoxy-β-d-glucopyranoside (21)

To a solution of S1 (46.8 mg, 19.9 μmol) in AcOH/1,4-dioxane (1:1, v/v, 1.99 mL) were added Ac₂O (75.2 μL, 795 μmol) and Zn–Cu (468 mg) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 3 h at the same temperature, it was filtered through a Celite pad, and to the filtrate was added sat. NaHCO₃ aq. (5 mL). The aqueous layer was extracted with EtOAc (2 × 5 mL), and then the combined extracts were washed with brine (5 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (toluene/acetone, 9:1) gave 21 (34.2 mg, 15.4 μmol, 78% yield).

White solid; mp 72.8–74.6 °C; [α]_D ²⁶ –10.3 (c 1.0, CHCl₃); R_f = 0.27 (toluene/acetone, 6:1).

¹H NMR (500 MHz, CDCl₃): δ = 7.40–7.17 (m, 37 H), 7.15–7.10 (m, 2 H), 6.96–6.91 (m, 2 H), 6.80–6.75 (m, 2 H), 6.16 (d, J = 8.5 Hz, 1 H), 6.09 (d, J = 8.5 Hz, 1 H), 5.37 (br s, 1 H), 5.27 (s, 1 H), 5.16–5.06 (m, 2 H), 4.97 (br s, 1 H), 4.94 (br s, 1 H), 4.88–4.78 (m, 4 H), 4.77–4.70 (m, 3 H), 4.66 (d, J = 11.5 Hz, 1 H), 4.59–4.53 (m, 2 H), 4.53–4.41 (m, 5 H), 4.40–4.32 (m, 3 H), 4.14–4.07 (m, 2 H), 4.04–3.95 (m, 3 H), 3.90–3.80 (m, 6 H), 3.80–3.67 (m, 9 H), 3.66–3.56 (m, 5 H), 3.51 (dd, J = 9.0, 9.0 Hz, 1 H), 3.47–3.39 (m, 4 H), 3.37 (m, 1 H), 3.28 (m, 1 H), 3.23–3.13 (m, 2 H), 3.10 (dd, J = 9.0, 9.0 Hz, 1 H), 1.95 (s, 3 H), 1.91 (s, 3 H), 1.65–1.34 (m, 6 H), 1.16 (d, J = 6.0 Hz, 3 H), 0.97–0.84 (m, 27 H), 0.54 (br s, 3 H), 0.16–0.05 (m, 18 H).

¹³C NMR (100 MHz, CDCl₃): δ = 170.7, 169.3, 159.2, 159.1, 156.5, 138.9, 138.7, 138.2, 138.1, 137.9, 137.3, 136.6, 130.5, 130.4, 129.5, 129.3, 128.6, 128.5, 128.3, 128.3, 128.2, 128.1, 128.0, 128.0, 127.9, 127.9, 127.7, 127.6, 127.4, 126.1, 114.0, 113.8, 102.2, 100.7, 100.2, 100.2, 96.7, 82.2, 80.7, 80.4, 79.4, 79.1, 78.4, 75.3, 75.1, 74.7, 74.2, 74.1, 73.3, 73.2, 72.5, 71.6, 71.0, 70.7, 69.4, 69.3, 68.5, 68.3, 68.2, 66.5, 66.0, 55.1, 55.1, 49.2, 41.0, 29.7, 29.5, 28.9, 26.4, 26.0, 26.0, 23.4, 23.0, 18.5, 18.5, 18.3, 18.2, 17.9, –3.5, –4.2, –4.4, –4.9, –5.0, –5.4.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₁₂₃H₁₆₈N₃O₂₈Si₃: 2219.1117; found: 2219.1177.

5-(Benzyloxycarbonylamino)pentyl 2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-d-mannopyranosyl-(1→2)-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→2)-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→2)-α-d-galactopyranosyl-(1→3)-2-acetamido-4,6-O-benzylidene-2-deoxy-β-d-glucopyranoside (S2)

To a solution of 21 (33.6 mg, 15.1 μmol) in dry THF (1.51 mL) was added TBAF (90.8 μL, 90.8 μmol) at room temperature under Ar atmosphere. After the reaction mixture was refluxed for 19 h, the reaction was quenched by the addition of sat. NH₄Cl aq. (5 mL). To the resultant mixture was added EtOAc (5 mL). The aqueous layer was extracted with EtOAc (5 × 5 mL), and then the combined extracts were washed brine (5 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (CHCl₃/MeOH, 16:1) gave S2 (23.6 mg, 12.6 μmol, 83% yield).

White solid; mp 77.8–79.3 °C; [α]_D ²⁶ –16.5 (c 1.0, CHCl₃); R_f = 0.42 (CHCl₃/MeOH, 8:1).

¹H NMR (400 MHz, CDCl₃): δ = 7.38–7.13 (m, 37 H), 7.09–7.04 (m, 2 H), 6.92–6.87 (m, 2 H), 6.80–6.75 (m, 2 H), 6.40–6.22 (m, 2 H), 5.59 (d, J = 3.6 Hz, 1 H), 5.31 (s, 1 H), 5.15–5.06 (m, 3 H), 5.04–4.97 (m, 2 H), 4.92–4.73 (m, 5 H), 4.67 (d, J = 10.8 Hz, 1 H), 4.65–4.55 (m, 4 H), 4.54 (d, J = 12.4 Hz, 1 H), 4.52–4.35 (m, 5 H), 4.33 (d, J = 11.2 Hz, 1 H), 4.23 (dd, J = 9.6, 9.6 Hz, 1 H), 4.16 (dd, J = 4.4, 10.4 Hz, 1 H), 4.04–3.95 (m, 3 H), 3.94–3.82 (m, 6 H), 3.82–3.71 (m, 7 H), 3.71–3.63 (m, 5 H), 3.62–3.50 (m, 4 H), 3.49–3.42 (m, 2 H), 3.39–3.25 (m, 3 H), 3.22–3.05 (m, 3 H), 2.08–1.94 (m, 9 H), 1.64–1.30 (m, 6 H), 1.21 (d, J = 6.0 Hz, 3 H), 0.54 (d, J = 6.0 Hz, 3 H).

¹³C NMR (100 MHz, CDCl₃): δ = 171.1, 159.2, 156.6, 138.7, 138.5, 138.2, 138.0, 137.5, 136.9, 136.5, 130.5, 129.5, 129.3, 129.0, 128.7, 128.5, 128.3, 128.3, 128.1, 128.0, 127.9, 127.7, 127.6, 113.9, 113.8, 101.4, 100.9, 100.5, 100.2, 99.0, 96.6, 82.5, 80.3, 80.1, 79.8, 79.1, 75.5, 75.0, 74.7, 74.6, 73.4, 73.3, 72.5, 72.3, 71.4, 70.6, 70.4, 70.2, 70.0, 68.8, 68.6, 68.3, 68.1, 66.6, 65.8, 62.6, 55.1, 49.3, 41.0, 29.7, 29.5, 23.3, 23.2, 23.1, 17.8, 17.6.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₁₀₅H₁₂₆N₃O₂₈: 1876.8528; found: 1876.8615.

5-Aminopentyl 2-Acetamido-2-deoxy-β-d-mannopyranosyl-(1→2)-α-l-rhamnopyranosyl-(1→2)-α-l-rhamnopyranosyl-(1→2)-α-d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β-d-glucopyranoside (7)

A solution of S2 (12.7 mg, 6.77 μmol) in dry MeOH (3.38 mL) was stirred under H₂ atmosphere (7 atm) in the presence of 20% Pd(OH)₂/C (38.1 mg) in an autoclave at room temperature for 3 h. After restoring the atmospheric pressure to normal pressure, the resultant mixture was filtered through a disposable membrane filter (DISMIC-13cp), and then the filtrate was concentrated in vacuo. Purification of the residue by reverse-phase silica gel column chromatography (H₂O) gave 7 (5.0 mg, 5.19 μmol, 77% yield).

Colorless syrup; [α]_D ²⁶ –9.6 (c 0.44, H₂O); R_f = 0.33 (n-butanol/MeOH/25% NH₃ aq., 4:4:5).

¹H NMR (500 MHz, D₂O): δ = 5.43 (br s, 1 H), 5.11–5.04 (m, 2 H), 4.74 (br s, 1 H), 4.45–4.38 (m, 2 H), 3.99 (br s, 1 H), 3.90 (br s, 1 H), 3.85 (m, 1 H), 3.82–3.71 (m, 7 H), 3.71–3.64 (m, 6 H), 3.62–3.57 (m, 3 H), 3.56–3.49 (m, 2 H), 3.47 (m, 1 H), 3.41 (dd, J = 8.0, 8.0 Hz, 1 H), 3.38–3.28 (m, 2 H), 3.26–3.13 (m, 2 H), 2.89–2.81 (m, 2 H), 1.97–1.87 (m, 6 H), 1.57–1.42 (m, 4 H), 1.33–1.21 (m, 2 H), 1.15 (d, J = 4.4 Hz, 3 H), 1.09 (d, J = 4.4 Hz, 3 H).

¹³C NMR (100 MHz, D₂O): δ = 176.3, 174.8, 101.8, 101.5, 101.3, 100.6, 98.0, 79.4, 78.2, 77.4, 76.5, 76.3, 73.7, 72.7, 72.4, 72.2, 71.9, 71.5, 70.7, 70.4, 70.1, 70.0, 69.8, 69.7, 69.6, 67.0, 61.1, 60.9, 60.8, 54.8, 53.8, 49.4, 39.8, 28.6, 26.9, 22.8, 22.7, 22.5, 17.3, 17.1.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₃₉H₇₀N₃O₂₄: 964.4344; found: 964.4345.

4,6-O-Di-tert-butylsilylene-3-O-(4-methoxybenzyl)-d-galactal (24)

To a solution of 23 [11] (1.16 g, 4.34 mmol) in dry DMF (17.4 mL) was added ^t Bu₂Si(OTf)₂ (1.55 mL, 4.78 mmol) at –40 °C under Ar atmosphere. After the reaction mixture was stirred for 1 h at the same temperature, it was treated with pyridine (910 μL, 11.3 mmol) and stirred for 1 h at room temperature. The reaction was quenched by the addition of sat. NaHCO₃ aq. (30 mL). To the resultant mixture was added EtOAc (30 mL). The aqueous layer was extracted with EtOAc (2 × 30 mL), and then the combined extracts were washed with brine (30 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (n-hexane/EtOAc, 7:1) gave 24 (1.60 g, 3.93 μmol, 90% yield).

White solid; mp 71.0–72.5 °C; [α]_D ²⁶ +67.5 (c 1.0, CHCl₃); R_f = 0.85 (CHCl₃/acetone, 9:1).

¹H NMR (400 MHz, CDCl₃): δ = 7.34–7.29 (m, 2 H), 6.89–6.84 (m, 2 H), 6.32 (dd, J = 6.4, 2.4 Hz, 1 H), 4.75–4.68 (m, 2 H), 4.59 (m, 1 H), 4.48 (d, J = 11.2 Hz, 1 H), 4.30–4.19 (m, 3 H), 3.81 (br s, 1 H), 3.78 (s, 3 H), 1.15–1.01 (m, 18 H).

¹³C NMR (100 MHz, CDCl₃): δ = 159.1, 144.1, 130.4, 129.2, 113.7, 100.9, 73.1, 70.6, 69.0, 67.5, 66.1, 55.1, 27.7, 27.0, 23.4, 20.9.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₂H₃₅O₅Si: 407.2254; found: 407.2245.

1,2-Anhydro-4,6-O-di-tert-butylsilylene-3-O-(4-methoxybenzyl)-α-d-galactopyranoside (25)

To a solution of 24 (1.12 g, 2.75 mmol) in dry CH₂Cl₂ (27.5 mL) was added dimethyldioxirane (87.8 mL, 4.13 mmol) at –20 °C under Ar atmosphere. After the reaction mixture was stirred for 30 min at the same temperature, it was directly concentrated in vacuo. Purification of the residue by recrystallization at –20 °C gave 25 (1.15 g, 2.72 mmol, 99% yield).

White solid; mp 79.5–81.0 °C; [α]_D ²⁶ +85.5 (c 1.0, CHCl₃).

¹H NMR (400 MHz, CDCl₃): δ = 7.36–7.31 (m, 2 H), 6.93–6.88 (m, 2 H), 4.99 (d, J = 3.6 Hz, 1 H), 4.76 and 4.56 (ABq, J = 11.2 Hz, 2 H), 4.36 (d, J = 2.8 Hz, 1 H), 4.22–4.18 (m, 2 H), 3.82 (s, 3 H), 3.66 (d, J = 3.6 Hz, 1 H), 3.48 (br s, 1 H), 3.04 (dd, J = 3.6, 3.6 Hz, 1 H), 1.13–1.02 (m, 18 H).

¹³C NMR (100 MHz, CDCl₃): δ = 159.3, 129.5, 129.4, 113.9, 77.5, 73.6, 69.6, 67.7, 67.3, 64.5, 55.2, 50.8, 27.7, 27.2, 23.4, 20.4.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₂₂H₃₅O₆ Si: 423.2203; found: 423.2206.

5-(Benzyloxycarbonylamino)pentyl 6-O-tert-Butyldiphenylsilyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-d-glucopyranoside (28)

To a solution of 27 [6] (408 mg, 710 μmol) in dry DMF (7.10 mL) were added imidazole (155 mg, 2.27 mmol) and TBDPSCl (295 μL, 1.14 mmol) at 0 °C under Ar atmosphere. After the reaction mixture was stirred for 16 h at room temperature, the reaction was quenched by the addition of H₂O (10 mL). To the resulting mixture was added EtOAc (20 mL). The aqueous layer was extracted with EtOAc (2 × 20 mL), and then the combined extracts were washed with brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (CHCl₃/MeOH, 15:1) gave 28 (525 mg, 646 μmol, 91% yield).

White solid; mp 42.6–44.5 °C; [α]_D ²⁶ –21.2 (c 1.0, CHCl₃); R_f = 0.67 (CHCl₃/MeOH, 7:1).

¹H NMR (500 MHz, CDCl₃): δ = 7.72–7.66 (m, 4 H), 7.47–7.29 (m, 11 H), 5.44 (br s, 1 H), 5.17–5.07 (m, 2 H), 4.80 (br s, 1 H), 4.77–4.71 (m, 2 H), 4.46 (d, J = 8.0 Hz, 1 H), 3.95–3.90 (m, 2 H), 3.87–3.75 (m, 2 H), 3.63 (ddd, J = 9.5, 9.5, 1.0 Hz, 1 H), 3.47–3.30 (m, 3 H), 3.21–3.13 (m, 2 H), 1.65–1.32 (m, 6 H), 1.06 (s, 9 H).

¹³C NMR (125 MHz, CDCl₃): δ = 156.4, 155.0, 136.4, 135.5, 135.5, 133.0, 132.9, 129.7, 128.4, 128.0, 127.7, 127.6, 100.4, 95.5, 75.0, 74.4, 74.3, 72.4, 69.0, 66.5, 64.4, 57.8, 40.8, 29.3, 28.8, 26.7, 23.0, 19.1.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₃₈H₅₀N₂O₉SiCl₃: 811.2351; found: 811.2325.

5-(Benzyloxycarbonylamino)pentyl 4,6-O-Di-tert-butylsilylene-3-O-(4-methoxybenzyl)-α-d-galactopyranosyl-(1→3)-6-O-tert-butyldiphenylsilyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-d-glucopyranoside (30)

To a solution of 28 (16.8 mg, 20.7 μmol) and tetrahydroxydiboron (29) (0.37 mg, 4.14 μmol) in dry MeCN (207 μL) was added a solution of 1,2-anhydo donor 25 (17.5 mg, 41.4 μmol) in dry MeCN (207 μL) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 24 h, the reaction was quenched by the addition of 0.05 M NaBO₃ aq. (100 μL). To the resultant mixture was added sat. NH₄Cl aq. (2 mL). The aqueous layer was extracted with EtOAc (5 × 2 mL), and then the combined extracts were washed with brine (2 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (toluene/acetone, 9:1) gave 30 (13.0 mg, 10.5 μmol, 51%) and the α(1,4)-isomer (3.3 mg, 2.7 μmol, 13%).

White solid; mp 49.5–51.3 °C; [α]_D ²⁶ + 49.6 (c 1.0, CHCl₃); R_f = 0.33 (toluene/acetone, 9:1).

¹H NMR (400 MHz, CDCl₃): δ = 7.75–7.67 (m, 4 H), 7.44–7.26 (m, 13 H), 6.91–6.86 (m, 2 H), 5.41 (br s, 1 H), 5.14 (d, J = 3.6 Hz, 1 H), 5.12–5.06 (m, 2 H), 4.88 (d, J = 12.0 Hz, 1 H), 4.83 (br s, 1 H), 4.67 (d, J = 11.2 Hz, 1 H), 4.62–4.51 (m, 2 H), 4.46 (d, J = 11.2 Hz, 1 H), 4.39 (d, J = 7.6 Hz, 1 H), 4.24–4.12 (m, 3 H), 4.01 (dd, J = 11.6, 2.8 Hz, 1 H), 3.93–3.84 (m, 2 H), 3.83–3.74 (m, 4 H), 3.68 (dd, J = 9.6, 2.4 Hz, 1 H), 3.64–3.54 (m, 2 H), 3.52–3.37 (m, 3 H), 3.23–3.10 (m, 2 H), 2.80 (br s, 1 H), 1.60–1.33 (m, 6 H), 1.15–0.97 (m, 27 H).

¹³C NMR (100 MHz, CDCl₃): δ = 159.3, 156.4, 154.4, 136.6, 135.6, 135.6, 133.5, 133.4, 129.9, 129.6, 128.5, 128.1, 127.6, 113.9, 102.1, 101.2, 95.4, 85.9, 75.6, 74.4, 71.6, 69.9, 69.5, 69.0, 68.2, 68.1, 67.1, 66.5, 63.7, 56.3, 55.2, 40.8, 29.7, 29.4, 28.7, 27.6, 27.2, 26.7, 26.0, 25.8, 25.7, 25.6, 23.4, 23.0, 20.6, 19.3.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₆₀H₈₄N₂O₁₅Si₂Cl₃: 1233.4476; found: 1233.4427.

5-(Benzyloxycarbonylamino)pentyl 2-O-Acetyl-4,6-O-di-tert-butylsilylene-3-O-(4-methoxybenzyl)-α-d-galactopyranosyl-(1→3)-4-O-acetyl-6-O-tert-butyldiphenylsilyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-d-glucopyranoside (30a)

To a solution of 30 (22.3 mg, 18.1 μmol) in dry pyridine (1.20 mL) was added Ac₂O (602 μL) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 3 h at the same temperature, the reaction was quenched by the addition of sat. NaHCO₃ aq. (5 mL). To the resulting mixture was added EtOAc (5 mL). The aqueous layer was extracted with EtOAc (5 × 5 mL), and then the combined extracts were washed with brine (5 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Purification of the residue by preparative TLC (toluene/acetone, 8:1) gave 30a (19.0 mg, 14.4 μmol, 80% yield).

White solid; mp 47.2–48.9 °C; [α]_D ²⁶ +52.6 (c 1.0, CHCl₃); R_f = 0.61 (toluene/acetone, 8:1).

¹H NMR (500 MHz, CDCl₃): δ = 7.68–7.62 (m, 4 H), 7.44–7.23 (m, 13 H), 6.88–6.83 (m, 2 H), 5.53 (d, J = 9.0 Hz, 1 H), 5.26–5.19 (m, 2 H), 5.17–5.04 (m, 2 H), 4.97 (dd, J = 9.0, 9.0 Hz, 1 H), 4.94–4.81 (m, 2 H), 4.59–4.34 (m, 5 H), 4.23–4.09 (m, 2 H), 4.01 (dd, J = 9.0, 9.0 Hz, 1 H), 3.89 (m, 1 H), 3.83 (br s, 1 H), 3.80 (s, 3 H), 3.72–3.64 (m, 2 H), 3.60–3.48 (m, 2 H), 3.44–3.32 (m, 2 H), 3.25–3.10 (m, 2 H), 2.06 (s, 3 H), 1.86 (s, 3 H), 1.67–1.30 (m, 6 H), 1.09–1.01 (m, 27 H).

¹³C NMR (125 MHz, CDCl₃): δ = 171.2, 169.6, 159.2, 156.5, 153.9, 136.6, 135.6, 133.3, 133.2, 131.0, 130.4, 129.7, 129.1, 128.5, 128.1, 128.0, 127.6, 113.8, 100.9, 97.0, 95.4, 75.6, 75.0, 74.5, 74.4, 71.9, 71.0, 69.2, 68.9, 67.8, 67.0, 66.6, 63.1, 57.0, 55.2, 41.0, 29.7, 29.3, 28.6, 27.6, 27.2, 26.7, 23.4, 23.0, 21.0, 20.8, 20.6, 19.2.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₆₄H₈₈N₂O₁₇Si₂Cl₃: 1317.4687; found: 1317.4735.

5-(Benzyloxycarbonylamino)pentyl 2-O-(tert-Butyldimethylsilyl)-4,6-O-di-tert-butylsilylene-3-O-(4-methoxybenzyl)-α-d-galactopyranosyl-(1→3)-6-O-tert-butyldiphenylsilyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-d-glucopyranoside (S3)

To a solution of 30 (251 mg, 204 μmol) in dry DMF (2.03 mL) were added imidazole (194 mg, 2.85 mmol), TBSCl (3.91 g, 25.9 mmol), and DMAP (49.7 mg, 407 μmol) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 1 h at 60 °C, the reaction was quenched by the addition of H₂O (10 mL). To the resulting mixture was added EtOAc (20 mL). The aqueous layer was extracted with EtOAc (2 × 20 mL), and then the combined extracts were washed with brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (toluene/acetone, 15:1) gave S3 (259 g, 192 μmol, 94% yield).

White solid; mp 53.6–55.2 °C; [α]_D ²⁶ +45.3 (c 1.0, CHCl₃); R_f = 0.67 (toluene/acetone, 9:1).

¹H NMR (500 MHz, CDCl₃): δ = 7.74–7.69 (m, 4 H), 7.44–7.27 (m, 13 H), 6.87–6.82 (m, 2 H), 5.41 (br s, 1 H), 5.18–5.05 (m, 2 H), 4.98 (d, J = 3.5 Hz, 1 H), 4.87–4.78 (m, 2 H), 4.63–4.57 (m, 2 H), 4.50 (br s, 1 H), 4.45–4.39 (m, 2 H), 4.29 (dd, J = 3.5, 9.5 Hz, 1 H), 4.19–4.11 (m, 2 H), 3.99 (dd, J = 10.5, 3.5 Hz, 1 H), 3.92–3.83 (m, 2 H), 3.80 (s, 3 H), 3.77 (br s, 1 H), 3.68–3.59 (m, 3 H), 3.47–3.35 (m, 3 H), 3.21–3.14 (m, 2 H), 1.60–1.33 (m, 6 H), 1.09–0.97 (m, 27 H), 0.88–0.83 (m, 9 H), 0.09–0.02 (m, 6 H).

¹³C NMR (125 MHz, CDCl₃): δ = 158.9, 156.4, 154.3, 136.6, 135.6, 135.6, 135.5, 133.5, 133.5, 130.7, 129.5, 129.5, 129.2, 128.5, 128.0, 128.0, 127.5, 113.5, 103.2, 101.0, 95.5, 84.7, 75.8, 74.4, 71.4, 70.2, 70.1, 69.7, 69.0, 68.3, 67.0, 66.5, 63.6, 56.8, 55.1, 40.8, 29.4, 28.7, 27.6, 27.2, 26.7, 26.1, 23.3, 23.1, 20.6, 19.3, 18.5, –4.4, –4.4.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₆₆H₉₈N₂O₁₅Si₃Cl₃: 1347.5341; found: 1347.5323.

5-(Benzyloxycarbonylamino)pentyl 2-O-(tert-Butyldimethylsilyl)-4,6-O-di-tert-butylsilylene-α-d-galactopyranosyl-(1→3)-6-O-tert-butyldiphenylsilyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-d-glucopyranoside (31)

To a solution of S3 (243 mg, 180 μmol) in dry CH₂Cl₂/phosphate buffer (9:1, v/v, pH 7.4, 6.0 mL) was added DDQ (81.7 mg, 360 μmol) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 3 h at the same temperature, the reaction was quenched by the addition of sat. NaHCO₃ aq. (20 mL). To the resulting mixture was added CHCl₃ (20 mL). The aqueous layer was extracted with CHCl₃ (2 × 20 mL), and then the combined extracts were washed with brine (20 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (toluene/acetone, 14:1) gave 31 (208 mg, 169 μmol, 94% yield).

White solid; mp 55.6–57.3 °C; [α]_D ²⁶ +33.7 (c 1.0, CHCl₃); R_f = 0.42 (toluene/acetone, 9:1).

¹H NMR (400 MHz, CDCl₃): δ = 7.74–7.68 (m, 4 H), 7.44–7.28 (m, 11 H), 5.45 (br s, 1 H), 5.18–5.04 (m, 2 H), 4.98 (d, J = 3.2 Hz, 1 H), 4.86–4.77 (m, 2 H), 4.63 (d, J = 12.0 Hz, 1 H), 4.45 (d, J = 6.8 Hz, 1 H), 4.39 (br s, 1 H), 4.25–4.11 (m, 2 H), 4.03–3.75 (m, 6 H), 3.72–3.58 (m, 2 H), 3.44 (m, 1 H), 3.41–3.30 (m, 2 H), 3.23–3.13 (m, 2 H), 2.42–2.34 (m, 2 H), 1.62–1.33 (m, 6 H), 1.09–0.90 (m, 36 H), 0.19–0.12 (m, 6 H).

¹³C NMR (125 MHz, CDCl₃): δ = 156.5, 154.3, 136.6, 135.7, 135.6, 133.5, 133.5, 129.6, 128.5, 128.1, 128.0, 127.6, 103.1, 100.8, 95.4, 85.1, 75.7, 74.4, 73.7, 72.2, 71.3, 70.0, 69.0, 67.9, 66.6, 66.5, 63.5, 56.9, 40.9, 29.5, 28.7, 27.5, 27.1, 26.7, 26.2, 23.3, 23.1, 20.6, 19.3, 18.6, –4.0, –4.5.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₅₈H₉₀N₂O₁₄Si₃Cl₃: 1227.4765; found: 1227.4786.

5-(Benzyloxycarbonylamino)pentyl 2-O-Acetyl-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→3)-2-O-(tert-butyldimethylsilyl)-4,6-O-di-tert-butylsilylene-α-d-galactopyranosyl-(1→3)-6-O-tert-butyldiphenylsilyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-d-glucopyranoside (S4)

To a solution of 16 (9.9 mg, 17.6 μmol) and 31 (10.8 mg, 8.79 μmol) in dry CH₂Cl₂ (176 μL) was added 4 Å MS (9.9 mg) at room temperature under Ar atmosphere. After the reaction mixture was stirred vigorously for 1 h, TfOH (0.16 μL, 1.76 μmol) was added to the reaction mixture at –40 °C. After the reaction mixture was stirred for 3 h at –20 °C, the reaction was quenched by the addition of sat. NaHCO₃ aq. (1 mL). To the resultant mixture were added EtOAc (5 mL) and sat. NaHCO₃ aq. (4 mL). The aqueous layer was extracted with EtOAc (5 × 5 mL), and then the combined extracts were washed with brine (5 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (n-hexane/Et₂O, 1:1) gave S4 (10.5 mg, 6.45 μmol, 73% yield).

White solid; mp 51.9–53.9 °C; [α]_D ²⁶ +39.4 (c 0.76, CHCl₃); R_f = 0.61 (n-hexane/Et₂O, 1:2).

¹H NMR (400 MHz, CDCl₃): δ = 7.73–7.66 (m, 4 H), 7.44–7.16 (m, 18 H), 6.79–6.73 (m, 2 H), 5.55 (dd, J = 1.6, 2.4 Hz, 1 H), 5.52 (br s, 1 H), 5.17–5.02 (m, 3 H), 4.93 (d, J = 11.2 Hz, 1 H), 4.89–4.82 (m, 2 H), 4.81–4.65 (m, 2 H), 4.64–4.57 (m, 2 H), 4.47 (br s, 1 H), 4.43–4.36 (m, 2 H), 4.28 (dd, J = 2.8, 10.4 Hz, 1 H), 4.21–4.09 (m, 2 H), 4.04 (m, 1 H), 4.01–3.79 (m, 6 H), 3.79–3.70 (m, 4 H), 3.61 (dd, J = 8.4, 8.4 Hz, 1 H), 3.48–3.30 (m, 4 H), 3.22–3.13 (m, 2 H), 2.13 (s, 3 H), 1.65–1.33 (m, 6 H), 1.29 (d, J = 5.6 Hz, 3 H), 1.10–0.87 (m, 36 H), 0.21–0.14 (m, 6 H).

¹³C NMR (100 MHz, CDCl₃): δ = 170.0, 159.1, 156.5, 154.2, 139.0, 136.6, 135.6, 135.6, 133.4, 133.3, 130.0, 129.8, 129.6, 128.5, 128.0, 127.6, 127.1, 127.0, 113.6, 102.7 (J _CH = 168 Hz), 100.7 (J _CH = 175 Hz, J _CH = 161 Hz), 95.4, 83.9, 79.8, 78.6, 75.5, 74.6, 74.4, 74.3, 71.9, 71.1, 69.1, 68.5, 68.3, 68.2, 66.7, 66.5, 64.0, 57.0, 55.1, 40.9, 29.7, 29.4, 28.7, 27.5, 27.3, 26.7, 26.1, 23.3, 23.1, 21.0, 20.7, 19.3, 18.4, 18.2, –4.5, –4.6.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₈₁H₁₁₆N₂O₂₀Si₃Cl₃: 1625.6495; found: 1625.6570.

5-(Benzyloxycarbonylamino)pentyl 4-O-Benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→3)-2-O-(tert-butyldimethylsilyl)-4,6-O-di-tert-butylsilylene-α-d-galactopyranosyl-(1→3)-6-O-tert-butyldiphenylsilyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-d-glucopyranoside (32)

To a solution of S4 (20.2 mg, 12.4 μmol) in MeOH (2.48 mL) was added K₂CO₃ (17.2 mg, 124 μmol) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 17 h at the same temperature, the reaction was quenched by the addition of sat. NH₄Cl aq. (1 mL). To the resulting mixture were added EtOAc (5 mL) and H₂O (4 mL). The aqueous layer was extracted with EtOAc (5 × 5 mL), and then the combined extracts were washed with brine (5 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (toluene/acetone, 9:1) gave 32 (14.1 mg, 8.89 μmol, 72% yield).

White solid; mp 60.0–61.8 °C; [α]_D ²⁶ +22.0 (c 1.0, CHCl₃); R_f = 0.33 (toluene/acetone, 9:1).

¹H NMR (400 MHz, CDCl₃): δ = 7.74–7.67 (m, 4 H), 7.44–7.18 (m, 18 H), 6.83–6.78 (m, 2 H), 5.50 (br s, 1 H), 5.18–5.05 (m, 2 H), 5.02 (d, J = 2.8 Hz, 1 H), 4.94 (br s, 1 H), 4.89 (d, J = 11.6 Hz, 1 H), 4.85 (br s, 1 H), 4.79–4.66 (m, 2 H), 4.64 (d, J = 11.6 Hz, 1 H), 4.57 (br s, 2 H), 4.49 (d, J = 4.4 Hz, 1 H), 4.42 (br s, 1 H), 4.26 (dd, J = 2.8, 10.8 Hz, 1 H), 4.20–4.09 (m, 3 H), 4.07–3.93 (m, 2 H), 3.92–3.71 (m, 9 H), 3.62 (dd, J = 8.4, 8.4 Hz, 1 H), 3.49–3.28 (m, 4 H), 3.22–3.12 (m, 2 H), 2.41 (br s, 1 H), 1.62–1.31 (m, 6 H), 1.29 (d, J = 6.4 Hz, 3 H), 1.08–0.89 (m, 36 H), 0.14–0.06 (m, 6 H).

¹³C NMR (100 MHz, CDCl₃): δ = 159.3, 156.5, 154.2, 138.9, 136.6, 135.6, 135.6, 133.4, 133.4, 130.0, 129.6, 129.5, 128.5, 128.1, 128.1, 128.0, 127.6, 127.2, 127.1, 113.8, 102.7, 102.0, 100.7, 95.4, 83.8, 80.0, 79.0, 78.6, 75.5, 74.7, 74.4, 74.3, 71.9, 71.5, 69.3, 69.1, 68.5, 68.3, 67.8, 66.7, 66.5, 63.9, 57.0, 55.1, 40.9, 29.7, 29.4, 28.7, 27.4, 27.4, 26.7, 26.1, 23.3, 23.1, 20.8, 19.2, 18.4, 18.2, –4.4.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₇₉H₁₁₄N₂O₁₉Si₃Cl₃: 1583.6389; found: 1583.6425.

5-(Benzyloxycarbonylamino)pentyl 2-Azido-3,4,6-tri-O-benzyl-2-deoxy-β-d-mannopyranosyl-(1→2)-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→2)-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→3)-2-O-(tert-butyldimethylsilyl)-4,6-O-di-tert-butylsilylene-α-d-galactopyranosyl-(1→3)-6-O-tert-butyldiphenylsilyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-d-glucopyranoside (33)

To a solution of 19 (63.0 mg, 62.8 μmol) and 32 (66.4 mg, 41.9 μmol) in dry toluene (838 μL) was added 4 Å MS (63.0 mg) at room temperature under Ar atmosphere. After the reaction mixture was stirred vigorously for 1 h, TfOH (0.74 μL, 8.38 μmol) was added to the reaction mixture at –40 °C. After the reaction mixture was stirred for 3 h at the same temperature, the reaction was quenched by the addition of sat. NaHCO₃ aq. (1 mL). To the resultant mixture were added EtOAc (5 mL) and sat. NaHCO₃ aq. (4 mL). After separation, the aqueous layer was extracted with EtOAc (5 × 5 mL), and then the combined extracts were washed with brine (5 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (n-hexane/EtOAc, 3:1) gave 33 (56.4 mg, 23.5 μmol, 56% yield).

White solid; mp 50.5–52.5 °C; [α]_D ²⁶ +8.2 (c 1.0, CHCl₃); R_f = 0.55 (toluene/acetone, 9:1).

¹H NMR (400 MHz, CDCl₃): δ = 7.73–7.67 (m, 4 H), 7.43–7.11 (m, 40 H), 6.85–6.81 (m, 2 H), 6.75–6.70 (m, 2 H), 5.43 (br s, 1 H), 5.14–5.07 (m, 3 H), 4.97 (d, J = 3.2 Hz, 1 H), 4.91–4.75 (m, 6 H), 4.73–4.43 (m, 13 H), 4.37 (d, J = 12.0 Hz, 1 H), 4.34 (br s, 1 H), 4.24 (dd, J = 2.0, 2.0 Hz, 1 H), 4.20 (dd, J = 2.8, 10.0 Hz, 1 H), 4.15–4.09 (m, 2 H), 4.08 (br d, J = 3.6 Hz, 1 H), 4.02–3.90 (m, 3 H), 3.89–3.72 (m, 8 H), 3.71–3.65 (m, 8 H), 3.63–3.53 (m, 2 H), 3.49 (dd, J = 9.2, 9.2 Hz, 1 H), 3.45–3.40 (m, 2 H), 3.40–3.29 (m, 3 H), 3.26 (m, 1 H), 3.21–3.13 (m, 2 H), 1.61–1.32 (m, 6 H), 1.26–1.20 (m, 6 H), 1.05–0.95 (m, 27 H), 0.90–0.85 (m, 9 H), 0.08–0.04 (m, 6 H).

¹³C NMR (100 MHz, CDCl₃): δ = 159.3, 159.0, 156.4, 154.2, 139.1, 138.4, 138.1, 137.7, 136.6, 135.6, 135.6, 133.5, 133.5, 130.5, 130.4, 129.6, 129.3, 129.2, 128.5, 128.4, 128.3, 128.2, 128.0, 128.0, 127.9, 127.8, 127.8, 127.7, 127.6, 127.6, 127.6, 127.5, 127.1, 127.0, 113.9, 113.7, 103.0 (J _CH = 168 Hz), 101.8 (J _CH = 168 Hz), 101.0 (J _CH = 175 Hz), 100.9 (J _CH = 161 Hz), 100.6 (J _CH = 161 Hz), 95.4, 84.4, 80.8, 80.5, 80.3, 79.5, 78.3, 78.0, 76.4, 75.8, 75.7, 75.6, 75.3, 75.2, 74.4, 74.3, 74.1, 73.4, 72.5, 71.9, 71.0, 69.3, 69.1, 68.5, 68.3, 68.1, 66.7, 66.5, 63.8, 61.7, 57.1, 55.1, 55.0, 40.9, 29.7, 29.5, 28.7, 27.5, 27.3, 26.7, 26.2, 23.3, 23.1, 20.7, 19.2, 18.4, 18.2, 17.8, –4.2, –4.6.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₁₂₇H₁₆₅N₅O₂₈Si₃Cl₃: 2397.0009; found: 2397.0103.

5-(Benzyloxycarbonylamino)pentyl 2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-d-mannopyranosyl-(1→2)-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→2)-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→3)-2-O-(tert-butyldimethylsilyl)-4,6-O-di-tert-butylsilylene-α-d-galactopyranosyl-(1→3)-6-O-tert-butyldiphenylsilyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-β-d-glucopyranoside (S5)

To a solution of 33 (55.4 mg, 23.1 μmol) in THF (384 μL) was added PPh₃ (60.6 mg, 231 μmol) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 3 h at 40 °C, H₂O (5.0 μL, 277 μmol) was added to the reaction mixture. After the reaction mixture was refluxed for 6 h, it was concentrated in vacuo. The residue was used for the next reaction without further purification.

To a solution of the above residue in CH₂Cl₂/MeOH (1:1, v/v, 2.31 mL) was added Ac₂O (65.5 μL, 693 μmol) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 13 h at the same temperature, the reaction was quenched by the addition of sat. NaHCO₃ aq. (5 mL). To the resultant mixture were added CHCl₃ (15 mL) and NaHCO₃ aq. (10 mL). The aqueous layer was extracted with CHCl₃ (2 × 15 mL), and then the combined extracts were washed with brine (15 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (n-hexane/EtOAc, 3:2) gave S5 (50.5 mg, 20.9 μmol, 91% yield).

White solid; mp 58.7–60.3 °C; [α]_D ²⁶ +13.2 (c 1.0, CHCl₃); R_f = 0.39 (toluene/acetone, 9:1).

¹H NMR (500 MHz, CDCl₃): δ = 7.72–7.68 (m, 4 H), 7.42–7.17 (m, 40 H), 6.83–6.79 (m, 2 H), 6.75–6.71 (m, 2 H), 6.02 (br s, 1 H), 5.44 (m, 1 H), 5.14–5.05 (m, 3 H), 4.98 (d, J = 3.0 Hz, 1 H), 4.91–4.85 (m, 3 H), 4.84–4.78 (m, 3 H), 4.77–4.65 (m, 6 H), 4.60–4.53 (m, 3 H), 4.51–4.41 (m, 4 H), 4.40–4.35 (m, 2 H), 4.35 (br s, 1 H), 4.21 (dd, J = 3.5, 10.5 Hz, 1 H), 4.17–4.08 (m, 2 H), 4.05 (br s, 1 H), 4.02 (br s, 1 H), 4.00–3.90 (m, 2 H), 3.89–3.79 (m, 5 H), 3.79–3.71 (m, 6 H), 3.70 (s, 3 H), 3.67 (s, 3 H), 3.62–3.54 (m, 2 H), 3.52–3.35 (m, 4 H), 3.34–3.28 (m, 2 H), 3.20–3.14 (m, 2 H), 1.93 (s, 3 H), 1.59–1.32 (m, 6 H), 1.24–1.21 (d, J = 6.0 Hz, 3 H), 1.21–1.17 (d, J = 6.0 Hz, 3 H), 1.06–0.95 (m, 27 H), 0.90–0.85 (m, 9 H), 0.11–0.04 (m, 6 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.6, 159.2, 159.0, 156.4, 154.2, 139.0, 138.6, 138.2, 138.1, 138.0, 136.6, 135.6, 135.6, 133.4, 130.5, 130.3, 129.6, 129.3, 129.1, 128.5, 128.3, 128.3, 128.1, 128.0, 128.0, 127.9, 127.9, 127.7, 127.6, 127.6, 127.5, 127.1, 127.0, 113.9, 113.6, 102.9, 101.7, 100.6, 100.3, 95.4, 84.1, 80.7, 80.3, 79.2, 78.4, 78.3, 77.5, 75.7, 75.4, 74.9, 74.8, 74.4, 74.3, 74.2, 73.3, 73.2, 72.6, 71.7, 71.1, 69.4, 69.3, 69.1, 68.6, 68.2, 68.2, 66.7, 66.5, 63.9, 57.0, 55.1, 55.0, 49.2, 40.9, 29.7, 29.4, 28.7, 27.5, 27.3, 26.7, 26.2, 23.4, 23.3, 23.1, 20.7, 19.2, 18.4, 18.2, 18.0, –4.2, –4.6.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₁₂₉H₁₆₉N₃O₂₉Si₃Cl₃: 2413.0210; found: 2413.0247.

5-(Benzyloxycarbonylamino)pentyl 2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-d-mannopyranosyl-(1→2)-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→2)-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→3)-2-O-(tert-butyldimethylsilyl)-4,6-O-di-tert-butylsilylene-α-d-galactopyranosyl-(1→3)-2-acetamido-6-O-tert-butyldiphenylsilyl-2-deoxy-β-d-glucopyranoside (34)

To a solution of S5 (52.6 mg, 21.8 μmol) in AcOH/1,4-dioxane (1:1, v/v, 2.18 mL) were added Ac₂O (82.3 μL, 871 μmol) and Zn–Cu (526 mg) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 4 h at the same temperature, it was filtered through a Celite pad, and to the filtrate was added sat. NaHCO₃ aq. (5 mL). The aqueous layer was extracted with EtOAc (2 × 5 mL), and then the combined extracts were washed with brine (5 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (n-hexane/EtOAc, 1:1) gave 34 (37.3 mg, 16.3 μmol, 75% yield).

White solid; mp 62.0–64.0 °C; [α]_D ²⁶ +16.4 (c 1.0, CHCl₃); R_f = 0.30 (toluene/acetone, 6:1).

¹H NMR (500 MHz, CDCl₃): δ = 7.72–7.67 (m, 4 H), 7.41–7.16 (m, 40 H), 6.83–6.79 (m, 2 H), 6.76–6.71 (m, 2 H), 6.00 (d, J = 10.0 Hz, 1 H), 5.73 (d, J = 8.5 Hz, 1 H), 5.08 (m, 3 H), 4.90–4.84 (m, 4 H), 4.83–4.76 (m, 3 H), 4.75–4.69 (m, 3 H), 4.66 (d, J = 7.5 Hz, 1 H), 4.60–4.52 (m, 4 H), 4.50–4.42 (m, 3 H), 4.40–4.35 (m, 2 H), 4.34 (br s, 1 H), 4.22–4.15 (m, 2 H), 4.08–4.04 (m, 2 H), 4.03–3.98 (m, 2 H), 3.95–3.79 (m, 7 H), 3.77–3.71 (m, 5 H), 3.70 (s, 3 H), 3.67 (s, 3 H), 3.59 (m, 1 H), 3.51–3.35 (m, 6 H), 3.31 (dd, J = 9.5, 9.5 Hz, 1 H), 3.20–3.14 (m, 2 H), 1.97 (s, 3 H), 1.93 (s, 3 H), 1.59–1.34 (m, 6 H), 1.24–1.20 (d, J = 6.0 Hz, 3 H), 1.20–1.16 (d, J = 6.0 Hz, 3 H), 1.06–0.95 (m, 27 H), 0.90–0.85 (m, 9 H), 0.11–0.04 (m, 6 H).

¹³C NMR (125 MHz, CDCl₃): δ = 170.6, 159.2, 159.0, 156.4, 139.0, 138.6, 138.2, 138.1, 138.0, 136.6, 135.6, 135.6, 133.7, 130.5, 130.3, 129.5, 129.3, 129.1, 128.5, 128.3, 128.3, 128.0, 128.0, 127.9, 127.9, 127.6, 127.5, 127.5, 127.1, 127.0, 113.9, 113.6, 103.0, 101.7, 100.6, 100.4, 100.1, 85.4, 80.7, 80.3, 79.1, 78.4, 78.2, 77.5, 76.2, 75.4, 75.0, 74.8, 74.4, 74.2, 74.2, 73.3, 72.6, 71.7, 71.1, 71.0, 69.7, 69.4, 68.7, 68.7, 68.4, 68.2, 67.0, 66.5, 63.7, 56.5, 55.1, 55.0, 49.1, 40.9, 29.6, 29.4, 28.7, 27.4, 27.3, 26.7, 26.2, 23.7, 23.4, 23.3, 20.7, 19.2, 18.5, 18.2, 17.9, –4.1, –4.6.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₁₂₈H₁₇₀N₃O₂₈Si₃: 2281.1273; found: 2281.1262.

5-(Benzyloxycarbonylamino)pentyl 2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-d-mannopyranosyl-(1→2)-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→2)-4-O-benzyl-3-O-(4-methoxybenzyl)-α-l-rhamnopyranosyl-(1→3)-α-d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β-d-glucopyranoside (S6)

To a solution of 34 (9.2 mg, 4.03 μmol) in dry DMF (202 μL) was added TASF (16.7 mg, 60.5 μmol) at room temperature under Ar atmosphere. After the reaction mixture was stirred for 3 h at 80 °C, the reaction was quenched by the addition of phosphate buffer (5 mL). To the resultant mixture was added CHCl₃ (5 mL). The aqueous layer was extracted with CHCl₃ (5 × 5 mL), and then the combined extracts were washed brine (5 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. Purification of the residue by silica gel column chromatography (CHCl₃/MeOH, 15:1) gave S6 (4.9 mg, 2.74 μmol, 68% yield).

White solid; mp 60.0–61.2 °C; [α]_D ²⁶ +3.1 (c 1.0, CH₃OH); R_f = 0.15 (CHCl₃/MeOH, 13:1).

¹H NMR (500 MHz, CDCl₃/CD₃OD, 9:1): δ = 7.39–7.22 (m, 32 H), 7.18–7.14 (m, 2 H), 7.09 (d, J = 9.5 Hz, 1 H), 6.87–6.79 (m, 4 H), 5.62 (br s, 1 H), 5.16–5.06 (m, 3 H), 5.02 (d, J = 3.5 Hz, 1 H), 4.98 (br s, 1 H), 4.93–4.87 (m, 3 H), 4.85–4.75 (m, 3 H), 4.71 (d, J = 11.0 Hz, 1 H), 4.67–4.62 (m, 2 H), 4.58–4.52 (m, 3 H), 4.50–4.43 (m, 3 H), 4.41 (d, J = 11.0 Hz, 1 H), 4.39–4.34 (m, 2 H), 4.07 (br s, 1 H), 4.02 (br s, 1 H), 3.94 (dd, J = 3.5, 10.0 Hz, 1 H), 3.92–3.81 (m, 6 H), 3.80–3.67 (m, 11 H), 3.66 (s, 3 H), 3.64–3.55 (m, 4 H), 3.53–3.42 (m, 3 H), 3.36–3.29 (m, 2 H), 3.24 (m, 1 H), 3.18–3.12 (m, 2 H), 1.98 (s, 3 H), 1.97 (s, 3 H), 1.63–1.32 (m, 6 H), 1.31–1.28 (d, J = 6.0 Hz, 3 H), 1.27–1.23 (d, J = 6.0 Hz, 3 H).

¹³C NMR (100 MHz, CDCl₃): δ = 171.7, 170.9, 159.2, 156.6, 156.5, 138.3, 138.2, 138.1, 137.9, 136.5, 132.1, 131.0, 130.4, 130.2, 129.2, 129.1, 128.9, 128.7, 128.5, 128.4, 128.4, 128.3, 128.2, 128.0, 128.0, 127.8, 127.8, 127.7, 113.8, 113.8, 101.4, 100.8, 100.4, 100.2, 99.7, 84.5, 80.4, 80.3, 79.8, 79.2, 76.3, 75.6, 75.2, 75.0, 74.8, 74.5, 74.1, 73.5, 73.2, 72.5, 71.7, 71.5, 71.3, 71.0, 70.6, 69.3, 68.9, 68.7, 68.5, 68.2, 66.5, 63.0, 62.5, 55.4, 55.1, 55.1, 49.2, 40.9, 29.6, 29.3, 23.4, 23.3, 23.0, 18.4, 17.9.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₉₈H₁₂₂N₃O₂₈: 1788.8215; found: 1788.8214.

5-Aminopentyl 2-Acetamido-2-deoxy-β-d-mannopyranosyl-(1→2)-α-l-rhamnopyranosyl-(1→2)-α-l-rhamnopyranosyl-(1→3)-α-d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β-d-glucopyranoside (8)

A solution of S6 (7.1 mg, 3.97 μmol) in dry MeOH (1.98 mL) was stirred under H₂ atmosphere (7 atm) in the presence of 20% Pd(OH)₂/C (21.3 mg) in an autoclave at room temperature for 3 h. After restoring the atmospheric pressure to normal pressure, the resultant mixture was filtered through a disposable membrane filter (DISMIC-13cp), and then the filtrate was concentrated in vacuo. Purification of the residue by reverse-phase silica gel column chromatography (H₂O) gave 8 (3.0 mg, 3.11 μmol, 78% yield).

Colorless syrup; [α]_D ²⁶ –2.6 (c 0.52, H₂O); R_f = 0.33 (n-butanol/MeOH/25% NH₃ aq., 4:4:5).

¹H NMR (500 MHz, D₂O): δ = 8.26 (d, J = 9.5 Hz, 1 H), 7.83 (d, J = 10.0 Hz, 1 H), 5.28 (d, J = 4.0 Hz, 1 H), 5.06 (br s, 1 H), 4.97 (br s, 1 H), 4.72 (s, 1 H), 4.41–4.35 (m, 2 H), 3.96 (br s, 1 H), 3.90–3.85 (m, 2 H), 3.78–3.68 (m, 6 H), 3.68–3.59 (m, 7 H), 3.59–3.50 (m, 5 H), 3.43 (m, 1 H), 3.37 (dd, J = 10.0, 10.0 Hz, 1 H), 3.34–3.25 (m, 2 H), 3.22–3.11 (m, 2 H), 2.83–2.77 (m, 2 H), 1.90 (s, 3 H), 1.87 (s, 3 H), 1.54–1.18 (m, 6 H), 1.14–1.10 (d, J = 6.5 Hz, 3 H), 1.10–1.05 (d, J = 6.0 Hz, 3 H).

¹³C NMR (100 MHz, D₂O): δ = 175.9, 174.5, 101.4, 101.1, 101.0, 100.9, 99.0, 79.4, 79.1, 78.0, 76.7, 76.2, 75.7, 72.4, 72.2, 71.8, 71.2, 70.9, 70.3, 70.2, 69.8, 69.4, 68.9, 68.3, 66.7, 60.7, 60.6, 60.4, 54.2, 53.5, 39.5, 28.3, 26.6, 22.5, 22.4, 22.2, 17.0, 16.9.

HRMS (ESI-TOF): m/z [M + H]⁺ calcd for C₃₉H₇₀N₃O₂₄: 964.4349; found: 964.4382.

Synthesis of Glycoconjugate 9

To a solution of 7 (5.7 mg, 5.9 μmol) in dry DMF (148 μL) were added linker 35 [12] (16.1 mg, 41 μmol) and Et₃N (8.2 μL, 59 μmol) under Ar atmosphere. After the reaction mixture was stirred for 5 h when TLC indicated almost complete conversion of 7, the reaction mixture was purified by size exclusion chromatography (MeOH) to afford corresponding crude 36 (5.7 mg). The crude 36 was used for the next reaction without further purification.

To a solution of the crude 36 (5.7 mg) in DMF (45 μL) was added a solution of BSA in phosphate buffer (pH 7.5, 70 μM, 1.12 mL). After the reaction mixture was incubated for 18 h at 25 °C, it was diluted with deionized water and dialyzed against 5 changes of deionized water (1.0 L). Lyophilization of the solution gave white solid 9 (6.0 mg).

Synthesis of Glycoconjugate 10

To a solution of 8 (3.0 mg, 3.1 μmol) in dry DMF (78 μL) were added linker 35 (8.5 mg, 22 μmol) and Et₃N (4.3 μL, 31 μmol) under Ar atmosphere. After the reaction mixture was stirred for 5 h when TLC indicated almost complete conversion of 8, the reaction mixture was purified by size exclusion chromatography (MeOH) to afford corresponding crude 37 (3.0 mg). The crude 37 was used for the next reaction without further purification.

To a solution of the crude 37 (3.0 mg) in DMF (24 μL) was added a solution of BSA in phosphate buffer (pH 7.5, 70 μM, 589 μL). After the reaction mixture was incubated for 18 h at 25 °C, it was diluted with deionized water and dialyzed against 5 changes of deionized water (1.0 L). Lyophilization of the solution gave white solid 10 (3.5 mg).

Synthesis of Glycoconjugate 11

To a solution of 1 (3.7 mg, 3.9 μmol) in dry DMF (98 μL) were added linker 35 (10.6 mg, 27 μmol) and Et₃N (5.4 μL, 39 μmol) under Ar atmosphere. After the reaction mixture was stirred for 5 h when TLC indicated almost complete conversion of 1, the reaction mixture was purified by size exclusion chromatography (MeOH) to afford corresponding crude 38 (3.0 mg). The crude 38 was used for the next reaction without further purification.

To a solution of the crude 38 (1.2 mg) in DMF/phosphate buffer (1:9, v/v, 100 μL) was added a solution of KLH in phosphate buffer (pH 7.5, 2.0 mg/mL, 3.25 mL). After the reaction mixture was incubated for 18 h at 25 °C, it was diluted with deionized water and dialyzed against 5 changes of deionized water (1.0 L). Lyophilization of the solution gave white solid 11 (4.4 mg).

Conflict of Interest

The authors declare no conflict of interest.

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Corresponding Authors

Publication History

Received: 27 July 2023

Accepted after revision: 10 August 2023

Accepted Manuscript online:
10 August 2023

Article published online:
21 September 2023

© 2023. Thieme. All rights reserved

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References


For selected reviews, see:
• 1a Guabiraba R, Schouler C. FEMS Microbiol. Lett. 2015; 362: fnv118
  CrossrefPubMedSearch in Google Scholar
• 1b Dho-Moulin M, Fairbrother JM. Vet. Res. 1999; 30: 299
  PubMedSearch in Google Scholar

For selected reviews, see:
• 2a Mellata M. Foodborne Pathog. Dis. 2013; 10: 916
  CrossrefPubMedSearch in Google Scholar
• 2b Nhung NT, Chansiripornchai N, Carrique-Mas JJ. Front. Vet. Sci. 2017; 4, 1; DOI:
  CrossrefPubMed
• 3a Johnson TJ, Kariyawasam S, Wannemuehler Y, Mangiamele P, Johnson SJ, Doetkott C, Skyberg JA, Lynne AM, Johnson JR, Nolan LK. J. Bacteriol. 2007; 189: 3228
  CrossrefPubMedSearch in Google Scholar
• 3b Moulin-Schouleur M, Répérant M, Laurent S, Brée A, Mignon-Grasteau S, Germon P, Rasschaert D, Schouler C. J. Clin. Microbiol. 2007; 45: 3366
  CrossrefPubMedSearch in Google Scholar
• 4a Ghunaim H, Abu-Madi MA, Kariyawasam S. Vet. Microbiol. 2014; 172: 13
  CrossrefPubMedSearch in Google Scholar
• 4b Han Y, Liu Q, Yi J, Liang K, Wei Y, Kong Q. Pathog. Dis. 2017; 75: ftx102
  PubMedSearch in Google Scholar
• 4c Han Y, Liu Q, Willias S, Liang K, Li P, Cheng A, Kong Q. Vaccine 2018; 36: 1038
  CrossrefPubMedSearch in Google Scholar
• 5a Jann B, Shashkov AS, Gupta DS, Panasenko SM, Jann K. Carbohydr. Polym. 1992; 18: 51
  CrossrefSearch in Google Scholar
• 5b Gupta DS, Shashkov AS, Jann B, Jann K. J. Bacteriol. 1992; 174: 7963
  CrossrefPubMedSearch in Google Scholar
• 6 Nishi N, Seki K, Takahashi D, Toshima K. Angew. Chem. Int. Ed. 2021; 60: 1789
  CrossrefPubMedSearch in Google Scholar
• 7a Nakagawa A, Tanaka M, Hanamura S, Takahashi D, Toshima K. Angew. Chem. Int. Ed. 2015; 54: 10935
  CrossrefPubMedSearch in Google Scholar
• 7b Tanaka M, Nashida J, Takahashi D, Toshima K. Org. Lett. 2016; 18: 2288
  CrossrefPubMedSearch in Google Scholar
• 7c Tanaka M, Takahashi D, Toshima K. Org. Lett. 2016; 18: 5030
  CrossrefPubMedSearch in Google Scholar
• 7d Nishi N, Nashida J, Kaji E, Takahashi D, Toshima K. Chem. Commun. 2017; 53: 3018
  CrossrefPubMedSearch in Google Scholar
• 7e Nashida J, Nishi N, Takahashi Y, Igarashi M, Hayashi C, Takahashi D, Toshima K. J. Org. Chem. 2018; 83: 7281
  CrossrefPubMedSearch in Google Scholar
• 7f Tanaka M, Nakagawa A, Nishi N, Iijima K, Sawa R, Takahashi D, Toshima K. J. Am. Chem. Soc. 2018; 140: 3644
  CrossrefPubMedSearch in Google Scholar
• 7g Nishi N, Sueoka K, Iijima K, Sawa R, Takahashi D, Toshima K. Angew. Chem. Int. Ed. 2018; 57: 13858
  CrossrefPubMedSearch in Google Scholar
• 7h Tanaka M, Sato K, Yoshida R, Nishi N, Oyamada R, Inaba K, Takahashi D, Toshima K. Nat. Commun. 2020; 11: 2431
  CrossrefPubMedSearch in Google Scholar
• 7i Inaba K, Endo M, Iibuchi N, Takahashi D, Toshima K. Chem. Eur. J. 2020; 26: 10222
  CrossrefPubMedSearch in Google Scholar
• 7j Tomita S, Tanaka M, Inoue M, Inaba K, Takahashi D, Toshima K. J. Org. Chem. 2020; 85: 16254
  CrossrefPubMedSearch in Google Scholar
• 7k Inaba K, Naito Y, Tachibana M, Toshima K, Takahashi D. Angew. Chem. Int. Ed. 2023; in press
  CrossrefPubMed
• 8 Halcomb RL, Danishefsky SJ. J. Am. Chem. Soc. 1989; 111: 6661
  CrossrefSearch in Google Scholar
• 9 Hargreaves JM, Guen YL, Guerreiro C, Descroix K, Mulard LA. Org. Biomol. Chem. 2014; 12: 7728
  CrossrefPubMedSearch in Google Scholar
• 10 Bock K, Pedersen C. J. Chem. Soc., Perkin Trans. 2 1974; 293
  Crossref
• 11 Park TK, Kim IJ, Hu S, Bilodeau MT, Randolph JT, Kwon O, Danishefsky SJ. J. Am. Chem. Soc. 1996; 118: 11488
  CrossrefPubMedSearch in Google Scholar
• 12 Wu X, Ling C.-C, Bundle DR. Org. Lett. 2004; 6: 4407
  CrossrefPubMedSearch in Google Scholar
• 13 Xiong C, Feng S, Qiao Y, Guo Z, Gu G. Chem. Eur. J. 2018; 24: 8205
  CrossrefPubMedSearch in Google Scholar
• 14 Microbiologically Safe Foods . Stein E. ED-Tech Press; Essex: 2018: 53
  Search in Google Scholar
• 15 Davis MR. Jr, Goldberg JB. J. Visualized Exp. 2012; 63: e3916
  CrossrefPubMedSearch in Google Scholar
• 16 Leyva A, Quintana A, Sánchez M, Rodríguez EN, Cremata J, Sánchez JC. Biologicals 2008; 36: 134
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material