Chemical Synthesis and Evaluation of Exopolysaccharide Fragments Produced by Leuconostoc mesenteroides Strain NTM048

Abstract

Exopolysaccharides (EPSs) occur widely in natural products made by bacteria, fungi and algae. Some EPSs have intriguing biological properties such as anticancer and immunomodulatory activities. Our group has recently found that EPSs generated from Leuconostoc mesenteroides ssp. mesenteroides strain NTM048 (NTM048 EPS) enhanced a production of mucosal immunoglobulin A (IgA) of mouse. Herein, we described the synthesis and evaluation of the tetrasaccharide fragments of NTM048 EPS to obtain information about the molecular mechanism responsible for the IgA-inducing activity.

Introduction

Exopolysaccharides (EPSs) are carbohydrate polymers that are secreted by bacteria, fungi and algae.^1,2) EPSs have a variety of biological activities, including immunological effects and antitumor actions. Among them, EPSs produced by lactic acid bacteria have recently attracted attention as potential candidates for therapeutic drugs, such as adjuvants for mucosal vaccination and supplements for human health.^3–5) Recently, our group has reported that EPSs derived from Leuconostoc mesenteroides ssp. mesenteroides strain NTM048 [NTM048 EPS (1), Fig. 1], consisting of 94% glucose polymers and 6% fructose polymers, function as an immunomodulator that can enhance mucosal immunoglobulin A (IgA) production of mouse.⁶⁾ NMR and GC-MS analyses of methylated derivatives of EPSs revealed that NTM048 EPS is a dextran consisting of an α-(1→6)-linked glucan with 4% α-(1→3) branches, and the remaining fructose polymer is composed of β-fructans.⁷⁾ Our group also found that enzymatically synthesized β-fructans exhibited lower IgA-inducing activity than NTM048 EPS, whereas α-glucans synthesized by a GH70 enzyme cloned from strain NTM048 possessed stronger IgA-inducing activity than NTM048 EPS.⁸⁾ These results indicate that α-glucans play an important role in the induction of IgA. Furthermore, additional enzymatic experiments were carried out using the expressed enzymes of EPS-synthesizing genes gtf1 and gtf2 derived from Leuconostoc mesenteroides strain NTM048, which synthesize α-glucan with 1,6- and 1,3-linkages.⁸⁾ These experiments showed that the IgA-inducing activity of the glucan synthesized by Gtf2 was higher than those of the NTM048 EPS and the glucan synthesized by Gtf1. These variations in IgA-inducing activity were likely caused by differences in molecular size, the ratio of the 1,3-linkage/1,6-linkage of the glucose residues or the particle size of the glucans. However, the detailed molecular recognition involved in the regulation of immunological activity remains unclear. Generally, such studies are hampered by the difficulties associated with obtaining homogeneous glycans by means of isolation or enzymatic synthesis because of their inherent microheterogeneity. In this context, chemical synthesis, which allows for the supply of glucans in their pure form, is a promising approach.

Fig. 1. Exopolysaccharide Fragments Produced by Leuconostoc mesenteroides

In this study, we carried out the chemical synthesis of fragments of NTM048 EPS to obtain information about the effects of the fragment structures of glucans on IgA-inducing activity. Furthermore, we examined whether the linear α-(1→6)-linkage or the branched α-(1→3)-linkage of the fragment structure was important for the enhancement of IgA induction.

Results and Discussion

As shown in Fig. 1, we targeted the tetrasaccharide linear fragment 2 and branched fragment 3, both of which have a propyl group at the reducing terminal of the glucan. The pivotal step toward their chemical syntheses is the construction of multiple α-glucosidic linkages including the glucose–glucose α-(1→3)-linkage and α-(1→6)-linkage, which are key moieties of EPS syntheses. In particular, the generation of the α-(1→3)-linkage would be more challenging because of the involvement of a glycosylation step at a less reactive 3-O-position. A variety of glycosylation reactions have been developed to construct the α-linkage and have been applied to the synthesis of α-glucan oligomers.^9,10) Although the synthesis of similar linear α-(1→6)-linked oligomers was reported by several groups,^11–17) the facile synthesis of oligomers containing an α-(1→3)-branched structure is still limited.^14,17)

We synthesized tetrasaccharide 2 (Chart 1), following the strategy reported by Lam and Gervay-Hague^12,13) where the α-(1→6)-linked glucosyl oligomers were synthesized using 6-O-acetyl-2,3,4-tri-O-benzylglucopyranosyl iodide as a glycosyl donor.^18,19) The precursor acetate 5 for glycosyl iodide 6 was prepared from the commercially available methyl α-D-glucopyranoside 4. Treatment of acetate 5 with trimethylsilyl iodide (TMSI) gave the corresponding iodide 6, which was then reacted with n-PrOH in the presence of tetrabutylammonium iodide (TBAI) and N,N-diisopropylethylamine (DIPEA) to afford the desired propyl α-D-glucopyranoside 7 in 63% yield with high stereoselectivity (α : β = >95 : 5). The removal of the Ac group gave the alcohol 8, and subsequent glycosylation using the glycosyl iodide 6 furnished the desired di- saccharide 9 in 64% yield (2 steps) with high α-selectivity. The same two-step sequence (removal of the Ac group and glycosylation with 6) was repeated twice to give the protected tetrasaccharide 13. The tetrasaccharide 13 was converted to fragment 2 by the removal of the Ac group using NaOMe and the Bn groups with Pd(OH)₂/C. The structure of fragment 2 was determined by ¹H- and ¹³C-NMR spectroscopy and mass spectrometry.

Chart 1.

We went on to synthesize the branched fragment 3 including the Glc(α1-3)Glc structure (Chart 2). Initially, we examined the construction of the Glc(α1-3)Glc structure, and selected the 6-O-benzoyl-2,4-O-benzyl-protected glucose 16 as an acceptor for glycosylation. The acceptor 16 was prepared starting from the glucose derivative 15 following a literature procedure.²⁰⁾ Next, we investigated the α-selective glycosylation of acceptor 16 using two glycosyl donors. First, the reaction of acceptor 16 with the imidate 17 in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) at −20 °C provided the desired product 19 in 83% yield. However, the selectivity was very low (α : β = 6 : 4), and separation of the diastereomixture was difficult. We next attempted the glycosylation reaction using glycosyl iodide 18, which was easily prepared from benzyl-protected glucose. The reaction of acceptor 16 with glycosyl iodide 18 in the presence of TBAI and DIPEA in benzene favored production of the α-anomer 19 (α : β = >95 : 5). With the Glc(α1-3)Glc structure in hand, we proceeded to examine the construction of the Glc(α1-6)Glc structure. Removing the 4-methoxyphenyl (PMP) group with cerium ammonium nitrate (CAN) gave compound 20 along with unidentified side products. Compound 20 was converted to the corresponding imidate²¹⁾ with ClC(NPh)CF₃, DIPEA and N,N-4-dimethylaminopyridine (DMAP). The imidate was treated with acceptor 8 and TMSOTf in Et₂O to give the desired trisaccharide 21 in 27% yield (3 steps) as the sole diastereomer (α : β = >95 : 5). In general, it is known that the use of Et₂O as a solvent leads to preferential formation of α-glucosides.²²⁾ The α-selectivity might also be resulted from the long range participation of the 6-O-benzoyl protecting group of compound 20.^23–25) Gao and colleagues also reported that the use of a 6-O-benzoyl-protected glycosyl donor favored α-glycosylation via remote anchimeric assistance.²⁵⁾ The removal of the Bz group with NaOMe afforded alcohol 22, which was reacted with glycosyl iodide 18 to provide the protected tetrasaccharide 23 (23% yield, α : β = >95 : 5). Finally, all protective groups were removed from 23 with Pd(OH)₂/C to afford the desired fragment 3 in quantitative yield. The structure of fragment 3 was determined by ¹H- and ¹³C-NMR spectroscopy and mass spectrometry. The characteristic peak at the branching point of the Glc(3,6-Glc) of fragment 3 (H-1 of branched-Glc: 5.32 ppm, J_1,2 = 4.0 Hz) was consistent with that of NTM048 EPS (H-1 of branched-Glc: 5.32 ppm, J_1,2 = 3.9 Hz).⁷⁾

Chart 2.

With fragments 2 and 3 in hand, we next evaluated their IgA-inducing ability using murine Payer’s patch cells.⁸⁾ In this assay, α-glucans synthesized by Gtf1 and Gtf2 were also used as controls in addition to NTM048 EPS. As shown in Fig. 2, 250 µg of fragments 2 and 3 slightly induced the IgA-production levels without a significant difference in activity; however, their IgA-inducing activities were lower than those of NTM048 EPS and the glucans synthesized by Gtf1 and Gtf2. These results suggested that molecular recognition of the polymer structure of glucans might be required for promoting IgA-inducing activity.

Fig. 2. Evaluation of IgA-Inducing Activity in Vitro

Comparison of IgA-inducing activities of fragments 2, 3, NTM048 exopolysaccharide (EPS) and glucans synthesized by Gtf1 and Gtf2. Lipopolysaccharide (LPS; 5 µg/mL) was used as the positive control. Each value represents the mean ± standard error of the mean (S.E.M.) (n = 6). ** p < 0.01 (versus the saline group).

In conclusion, we accomplished the chemical synthesis of the exopolysaccharide fragments produced by Leuconostoc mesenteroides and evaluated their IgA-inducing activity. The α-selective glycosylation for the synthesis was performed by using the glycosyl iodides or the imidate. The developed synthetic routes will contribute to the comprehensive synthesis of various EPS fragments and also to the analysis of their biological functions. Fragments 2 and 3 showed slight IgA-inducing activity, but the levels were not as high as those of the EPS of NTM048 or the glucan synthesized by Gtf1 and Gtf2, implying that the observed IgA induction by EPS might result from the recognition of larger fragments or whole glucans, rather than the recognition portions of glycan linkages. Further studies are needed to understand the detailed mechanism underlying the IgA induction activity by NTM048 EPS. To this end, we are currently undertaking additional research into NTM048 EPS, including structure activity relationship studies.

Experimental

General Methods

All moisture-sensitive reactions were performed using syringe-septum cap techniques under an argon atmosphere. Analytical TLC was performed on Silica gel 60 F₂₅₄ Plates (Merck, 0.25 mm thickness). Column chromatography was performed using a forced flow (flash chromatography) of the indicated solvent system on Wakogel C-300E (Wako, Osaka, Japan) or Biotage Isolera flash purification system on Presep^® Silica Gel (Wako) or Biotage^® KP-Sil Flash Cartridges (Biotage). ¹H-NMR spectra were recorded using a JEOL ECA-500 spectrometer at 500 MHz frequency. Chemical shifts are reported in δ (ppm) relative to Me₄Si (in CDCl₃ or CD₃OD) and MeCN (in D₂O) as internal standard. ¹³C-NMR spectra were recorded using a JEOL ECA-500 and referenced to the residual CHCl₃ signal (in CDCl₃) and MeCN (in D₂O). ¹H- and ¹³C-NMR spectra using D₂O as solvent were recorded at 25 °C. Exact mass [high resolution (HR)MS] spectra were recorded on a Shimadzu LC-ESI-IT-TOF-MS equipment [electrospray ionization (ESI)] or JMS-700 mass spectrometer.

Compound (7)

To a stirred solution of 5 (3.01 g, 5.64 mmol) in CH₂Cl₂ (24 mL) was added TMSI (0.90 mL, 6.5 mmol) at 0 °C under Ar. After the mixture was stirred for 2.0 h at 0 °C, toluene was added and the resulting mixture was concentrated in vacuo. The residue was azeotroped with toluene three times, and was diluted with benzene (16 mL) to give the solution of compound 6. To a stirred solution of n-PrOH (0.253 mL, 3.42 mmol), TBAI (1.80 g, 4.86 mmol), DIPEA (0.412 mL, 2.41 mmol) and MS3A (1.04 g) in benzene (8.0 mL) was added the above solution of compound 6 (8.0 mL) at room temperature under Ar. After being stirred for 4.0 h under reflux, the mixture was filtered with Celite, and the filtrate was diluted with EtOAc. The organic layer was washed with saturated Na₂S₂O₃ aq. and brine, and was dried over Na₂SO₄. The filtrate was concentrated under reduced pressure to give a residue, which was purified by flash chromatography over silica gel with hexane-EtOAc (10 : 1 to 5 : 1) to give 7 as a colorless oil (960 mg, 63% yield): ¹H-NMR (500 MHz, CDCl₃) δ: 0.94 (t, J = 7.5 Hz, 3H), 1.62–1.69 (m, 2H), 2.02 (s, 3H), 3.39 (dt, J = 9.5, 7.0 Hz, 1H), 3.48 (dd, J = 10.9, 10.9 Hz, 1H), 3.52–3.58 (m, 2H), 3.83–3.87 (m, 1H), 4.00–4.04 (m, 1H), 4.23 (dd, J = 12.0, 2.3 Hz, 1H), 4.28 (dd, J = 12.0, 4.6 Hz, 1H), 4.56 (d, J = 10.9 Hz, 1H), 4.65 (d, J = 10.9 Hz, 1H), 4.72 (d, J = 3.4 Hz, 1H), 4.78 (d, J = 10.9 Hz, 1H), 4.82 (d, J = 10.9 Hz, 1H), 4.88 (d, J = 10.9 Hz, 1H), 5.02 (d, J = 10.9 Hz, 1H), 7.25–7.37 (m, 15H); ¹³C-NMR (125 MHz, CDCl₃) δ: 10.6, 20.7, 22.5, 63.0, 68.4, 69.8, 73.0, 74.9, 75.6, 77.2, 80.0, 81.9, 96.7, 127.6, 127.79, 127.83, 127.87, 127.92, 128.1, 128.3, 128.4, 137.7, 138.1, 138.6, 170.6; HRMS [ESI-time-of-flight (TOF)] m/z: [M + Na]⁺ Calcd for C₃₂H₃₈NaO₇, 557.2510; Found, 557.2496.

Compound (9)

To a stirred solution of 7 (479 mg, 0.895 mmol) in MeOH (5.0 mL) was added NaOMe (5 M in MeOH solution, 0.358 mL, 1.79 mmol) at room temperature. After the mixture was stirred for 0.5 h at room temperature, Dowex (50W × 8 200–400 mesh, 350 mg) was added, and the mixture was filtered. The filtrate was concentrated under reduced pressure to give a residue, which was purified by flash chromatography over silica gel with toluene–EtOAc (10 : 1) to give 8 as a white solid (401 mg), which was used without further purification. The preparation of the benzene solution of 6 was carried out by a procedure identical with that described for synthesis of 7 from 5. To a stirred solution of 8 (446 mg), TBAI (1.69 g, 4.58 mmol), DIPEA (0.400 mL, 2.34 mmol) and MS4A (1.03 g) in benzene (7.0 mL) was added the solution of compound 6 (7.0 mL) at room temperature under Ar. After being stirred for 4.0 h under reflux, the mixture was filtered with Celite, and the filtrate was diluted with EtOAc. The organic layer was washed with saturated Na₂S₂O₃ aq. and brine, and was dried over Na₂SO₄. The filtrate was concentrated under reduced pressure to give a residue, which was purified by flash chromatography over silica gel with toluene-EtOAc (10 : 1) to give 9 as a colorless oil (609 mg, 64% yield): ¹H-NMR (500 MHz, CDCl₃) δ: 0.92 (t, J = 7.5 Hz, 3H), 1.61–1.68 (m, 2H), 1.97 (s, 3H), 3.38 (dt, J = 9.8, 6.6 Hz, 1H), 3.44 (dd, J = 9.8, 3.5 Hz, 1H), 3.46 (dd, J = 9.8, 8.6 Hz, 1H), 3.50 (dd, J = 9.8, 3.5 Hz, 1H), 3.58 (dt, J = 9.8, 6.9 Hz, 1H), 3.64 (dd, J = 9.2, 9.2 Hz, 1H), 3.68–3.70 (m, 1H), 3.80 (dd, J = 11.5, 4.5 Hz, 1H), 3.82–3.85 (m, 1H), 3.86–3.88 (m, 1H), 3.97 (dd, J = 9.3, 9.3 Hz, 1H), 4.00 (dd, J = 9.5, 9.5 Hz, 1H), 4.16–4.22 (m, 2H), 4.54 (d, J = 11.5 Hz, 1H), 4.57 (d, J = 11.5 Hz, 1H), 4.64 (d, J = 11.5 Hz, 1H), 4.65 (d, J = 11.5 Hz, 1H), 4.67 (d, J = 11.5 Hz, 1H), 4.69 (d, J = 3.5 Hz, 1H), 4.70 (d, J = 11.5 Hz, 1H), 4.77 (d, J = 11.5 Hz, 1H), 4.81 (d, J = 11.5 Hz, 1H), 4.86 (d, J = 11.5 Hz, 1H), 4.93 (d, J = 11.5 Hz, 1H), 4.95 (d, J = 3.5 Hz, 1H), 4.96 (d, J = 11.5 Hz, 1H), 4.98 (d, J = 11.5 Hz, 1H), 7.23–7.34 (m, 30H); ¹³C-NMR (125 MHz, CDCl₃) δ: 10.6, 20.8, 22.6, 63.0, 66.1, 68.6, 69.8, 70.4, 72.3, 73.1, 74.9, 75.0, 75.6, 75.7, 77.1, 77.8, 79.9, 80.3, 81.7, 82.1, 96.5, 97.0, 127.5, 127.59, 127.63, 127.7, 127.76, 127.80, 127.90, 127.94, 128.1, 128.3, 128.36, 128.39, 138.0, 138.27, 138.33, 138.6, 138.9, 170.7; HRMS (ESI-TOF) m/z: [M + Na]⁺ Calcd for C₅₉H₆₆NaO₁₂, 989.4446; Found, 989.4442.

Compound (10)

By a procedure identical with that described for synthesis of 8 from 7, the ester 9 (657 mg, 0.679 mmol) was converted into the alcohol 10 as a colorless oil (503 mg, 79% yield): ¹H-NMR (500 MHz, CDCl₃) δ: 0.92 (t, J = 7.5 Hz, 3H), 1.60–1.68 (m, 2H), 3.38 (dt, J = 9.7, 6.3 Hz, 1H), 3.44 (dd, J = 9.8, 3.5 Hz, 1H), 3.47 (dd, J = 9.8, 3.5 Hz, 1H), 3.51 (dd, J = 9.2, 9.2 Hz, 1H), 3.56–3.72 (m, 6H), 3.79–3.84 (m, 2H), 3.97 (dd, J = 9.2, 9.2 Hz, 1H), 4.00 (dd, J = 9.2, 9.2 Hz, 1H), 4.56 (d, J = 10.9 Hz, 1H), 4.62 (d, J = 10.9 Hz, 1H), 4.66 (d, J = 10.9 Hz, 1H), 4.67 (d, J = 10.9 Hz, 1H), 4.68 (d, J = 10.9 Hz, 1H), 4.69 (d, J = 3.5 Hz, 1H), 4.69 (d, J = 10.9 Hz, 1H), 4.78 (d, J = 10.9 Hz, 1H), 4.81 (d, J = 10.9 Hz, 1H), 4.88 (d, J = 10.9 Hz, 1H), 4.91–4.95 (m, 3H), 4.98 (d, J = 10.9 Hz, 1H), 7.23–7.35 (m, 30H); ¹³C-NMR (125 MHz, CDCl₃) δ: 10.7, 22.6, 61.9, 66.1, 69.8, 70.5, 70.8, 72.3, 73.1, 74.9, 75.0, 75.5, 75.6, 77.3, 77.8, 80.1, 80.3, 81.6, 82.1, 96.6, 97.1, 127.51, 127.53, 127.57, 127.59, 127.7, 127.8, 127.85, 127.90, 127.93, 128.0, 128.3, 128.36, 128.38, 138.27, 138.32, 138.36, 138.38, 138.7, 138.9; HRMS (ESI-TOF) m/z: [M + NH₄]⁺ Calcd for C₅₇H₆₈NO₁₁, 942.4787; Found, 942.4765.

Compound (11)

By a procedure identical with that described for synthesis of 9 from 8, the alcohol 10 (455 mg, 0.492 mmol) was converted into the ester 11 as a colorless oil (269 mg, 39% yield): ¹H-NMR (500 MHz, CDCl₃) δ: 0.92 (t, J = 7.5 Hz, 3H), 1.58–1.68 (m, 2H), 1.96 (s, 3H), 3.36–3.48 (m, 4H), 3.49 (dd, J = 9.7, 3.5 Hz, 1H), 3.55–3.62 (m, 1H), 3.62–3.84 (m, 9H), 3.94–4.01 (m, 3H), 4.10–4.20 (m, 2H), 4.54 (d, J = 10.9 Hz, 1H), 4.54 (d, J = 10.9 Hz, 1H), 4.56 (d, J = 10.9 Hz, 1H), 4.58 (d, J = 10.9 Hz, 1H), 4.59 (d, J = 10.9 Hz, 1H), 4.64–4.70 (m, 5H), 4.76 (d, J = 10.9 Hz, 1H), 4.77 (d, J = 10.9 Hz, 1H), 4.81 (d, J = 10.9 Hz, 1H), 4.86 (d, J = 10.9 Hz, 1H), 4.91–4.99 (m, 6H), 5.01 (d, J = 3.5 Hz, 1H), 7.18–7.33 (m, 45H); ¹³C-NMR (125 MHz, CDCl₃) δ: 10.7, 20.8, 22.6, 63.0, 65.9, 65.6, 68.6, 69.7, 70.5, 70.6, 72.1, 72.3, 73.1, 74.9, 75.0, 75.5, 75.56, 75.61, 77.1, 77.2, 77.4, 77.9, 80.0, 80.3, 80.4, 81.6, 81.7, 82.1, 96.6, 97.0, 97.1, 127.4, 127.48, 127.52, 127.54, 127.59, 127.61, 127.64, 127.7, 127.79, 127.82, 127.87, 127.92, 127.99, 128.1, 128.27, 128.31, 128.35, 128.38, 138.0, 138.30, 138.34, 138.45, 138.6, 138.8, 138.9, 170.7; HRMS (ESI-TOF) m/z: [M + Na]⁺ Calcd for C₈₆H₉₄NaO₁₇, 1421.6383; Found, 1421.6383.

Compound (12)

By a procedure identical with that described for synthesis of 8 from 7, the ester 11 (245 mg, 0.175 mmol) was converted into the alcohol 12 as a colorless oil (149 mg, 63% yield): ¹H-NMR (500 MHz, CDCl₃) δ: 1.24 (t, J = 6.9 Hz, 3H), 1.60–1.67 (m, 2H), 3.35–3.85 (m, 18H), 3.95–4.02 (m, 3H), 4.53–4.70 (m, 10H), 4.76 (d, J = 10.9 Hz, 1H), 4.78 (d, J = 10.9 Hz, 1H), 4.81 (d, J = 10.9 Hz, 1H), 4.87 (d, J = 10.9 Hz, 1H), 4.91–4.99 (m, 7H), 7.11–7.33 (m, 45H); ¹³C-NMR (125 MHz, CDCl₃) δ: 10.7, 22.6, 61.9, 65.6, 66.0, 69.7, 70.5, 70.6, 70.8, 72.2, 72.3, 73.1, 74.88, 74.93, 75.0, 75.5, 75.6, 77.2, 77.3, 77.4, 77.9, 80.1, 80.3, 80.4, 81.5, 81.7, 82.1, 96.6, 97.0, 97.1, 125.3, 127.4, 127.48, 127.51, 127.62, 127.64, 127.7, 127.75, 127.79, 127.87, 127.89, 127.92, 127.93, 127.98, 128.03, 128.27, 128.2, 128.29, 128.31, 128.34, 128.38, 128.40, 128.44, 128.6, 129.0, 138.2, 138.44, 138.36, 138.3, 138.5, 138.6, 138.7, 138.8, 138.9; HRMS (ESI-TOF) m/z: [M + Na]⁺ Calcd for C₈₄H₉₂NaO₁₆, 1379.6278; Found, 1379.6281.

Compound (13)

By a procedure identical with that described for synthesis of 9 from 8, the alcohol 12 (131 mg, 0.0964 mmol) was converted into the ester 13 as a colorless oil (35.5 mg, 20% yield): ¹H-NMR (500 MHz, CDCl₃) δ: 0.91 (t, J = 7.5 Hz, 3H), 1.59–1.66 (m, 2H), 1.96 (s, 3H), 3.34–3.50 (m, 7H), 3.56–3.85 (m, 14H), 3.93–4.00 (m, 3H), 4.13–4.19 (m, 2H), 4.48–4.60 (m, 7H), 4.62–4.70 (m, 7H), 4.73–4.87 (m, 5H), 4.88–4.98 (m, 8H), 5.03 (d, J = 3.5 Hz, 1H), 7.15–7.37 (m, 60H); ¹³C-NMR (125 MHz, CDCl₃) δ: 10.7, 20.8, 22.6, 63.0, 65.3, 65.45, 65.50, 65.8, 68.7, 69.7, 70.6, 70.7, 70.8, 72.1, 72.2, 72.3, 73.1, 73.4, 74.88, 74.90, 74.93, 75.0, 75.4, 75.5, 75.6, 77.1, 77.2, 77.4, 77.8, 80.0, 80.3, 80.4, 81.5, 81.56, 81.64, 82.1, 96.6, 97.0, 97.1 (2C), 127.0, 127.34, 127.40, 127.44, 127.47, 127.51, 127.53, 127.59, 127.61, 127.64, 127.7, 127.80, 127.83, 127.85, 127.88, 127.91, 127.97, 128.00, 128.02, 128.05, 128.11, 128.18, 128.20, 128.25, 128.30, 128.34, 128.37, 128.39, 128.42, 128.46, 128.54, 128.6, 138.0, 138.3, 138.4, 138.46, 138.48, 138.50, 138.54, 138.6, 138.7, 138.80, 138.83, 139.0, 170.7; HRMS (FAB) m/z: [M + H + Na]⁺ Calcd for C₁₁₃H₁₂₃NaO₂₂, 1854.8398; Found, 1854.8416.

Compound (14)

By a procedure identical with that described for synthesis of 8 from 7, the ester 13 (25 mg, 0.0136 mmol) was converted into the alcohol 14 as a colorless oil (14.0 mg, 57% yield): ¹H-NMR (500 MHz, CDCl₃) δ: 0.91 (t, J = 7.5 Hz, 3H), 1.60–1.65 (m, 2H), 3.34–3.50 (m, 6H), 3.55–3.84 (m, 16H), 3.93–4.00 (m, 4H), 4.48–4.68 (m, 12H), 4.75 (d, J = 10.9 Hz, 1H), 4.75 (d, J = 10.9 Hz, 1H), 4.77 (d, J = 10.9 Hz, 1H), 4.80 (d, J = 10.9 Hz, 1H), 4.85–5.43 (m, 11H), 4.99 (d, J = 3.5 Hz, 1H), 7.36–7.16 (m, 60H); ¹³C-NMR (125 MHz, CDCl₃) δ: 10.7, 22.6, 61.8, 65.4, 65.7, 65.8, 69.7, 70.6, 70.7, 70.77, 70.84, 72.19, 72.23, 72.3, 73.1, 74.86, 74.92, 74.94, 75.0, 75.4, 75.45, 75.47, 75.6, 77.3, 77.36, 77.41, 77.8, 80.1, 80.28, 80.30, 80.4, 81.4, 81.6, 81.7, 82.1, 96.6, 97.0, 97.1, 97.2, 127.36, 127.38, 127.42, 127.46, 127.48, 127.52, 127.60, 127.63, 127.68, 127.72, 127.77, 127.84, 127.86, 127.92, 127.94, 127.99, 128.03, 128.26, 128.31, 128.34, 128.38, 128.40, 138.2, 138.4, 138.46, 138.48, 138.6, 138.65, 138.69, 138.81, 138.83, 138.9; HRMS (FAB) m/z: [M + H + Na]⁺ Calcd for C₁₁₁H₁₂₁NaO₂₁, 1812.8293; Found, 1812.8280.

Compound (2)

To a stirred solution of 14 (7.0 mg, 0.0039 mmol) in EtOAc/EtOH (2 : 1, 2.0 mL) was added 20% w/w Pd(OH)₂/C (13.4 mg, 0.0191 mmol) at room temperature. After being stirred for 19 h at this temperature under H₂ atmosphere (5.0 atm), the mixture was filtered with membrane filter. The filtrate was concentrated under reduced pressure to give 2 as a white solid (2.3 mg, 83% yield): ¹H-NMR (500 MHz, CD₃OD) δ: 0.97 (t, J = 7.5 Hz, 3H), 1.62–1.70 (m, 2H), 3.33–3.46 (m, 8H), 3.57–3.82 (m, 13H), 3.85–3.99 (m, 5H), 4.78 (d, J = 4.0 Hz, 1H), 4.83–4.90 (m, 3H); ¹³C-NMR (125 MHz, CD₃OD) δ: 11.1, 23.9, 62.6, 67.5, 67.55, 67.61, 71.0, 71.7, 71.88, 71.93, 71.95, 71.98, 72.00 (2C), 73.5, 73.6, 73.7, 73.75, 73.81, 75.2, 75.3, 75.36, 75.43, 99.6, 99.73, 99.76, 100.25; HRMS (ESI-TOF) m/z: [M + Na]⁺ Calcd for C₂₇H₄₈NaO₂₁, 731.2580; Found, 731.2577.

Compound (19)

The preparation of the benzene solution of 18 (2.5 mL) from the corresponding acetate (514 mg, 0.882) was carried out by a procedure identical with that described for the preparation of 6. To a stirred solution of 16 (136 mg, 0.239 mmol), TBAI (439 mg, 1.18 mmol), DIPEA (0.102 mL, 0.600 mmol) and MS4A (0.75 g) in benzene (2.5 mL) was added the above solution of compound 18 (2.5 mL) at room temperature under Ar. After being stirred for 3.0 h under reflux, the mixture was filtered with Celite, and the filtrate was diluted with EtOAc. The organic layer was washed with saturated Na₂S₂O₃ aq. and brine, and was dried over Na₂SO₄. The filtrate was concentrated under reduced pressure to give a residue, which was purified by flash chromatography over silica gel with hexane–EtOAc (16 : 1 to 1 : 1) to give 19 as a colorless oil (121 mg, 46% yield): ¹H-NMR (500 MHz, CDCl₃) δ: 3.30 (dd, J = 10.9, 1.8 Hz, 1H), 3.33 (dd, J = 10.9, 2.9 Hz, 1H), 3.62 (dd, J = 9.7, 3.4 Hz, 1H), 3.84–3.68 (m, 4H), 3.72 (s, 3H), 4.06–4.11 (m, 2H), 4.24–4.28 (m, 2H), 4.36 (dd, J = 11.5, 6.0 Hz, 1H), 4.43 (d, J = 10.9 Hz, 1H), 4.50–4.52 (m, 2H), 4.60–4.62 (m, 2H), 4.74 (d, J = 10.9 Hz, 1H), 4.76 (d, J = 10.9 Hz, 1H), 4.82 (d, J = 10.9 Hz, 1H), 4.92 (d, J = 10.9 Hz, 1H), 4.92 (d, J = 7.5 Hz, 1H), 4.97 (d, J = 10.9 Hz, 1H), 5.02 (d, J = 10.9 Hz, 1H), 5.04 (d, J = 10.9 Hz, 1H), 5.59 (d, J = 4.1 Hz, 1H), 6.68–6.72 (m, 2H), 6.95–6.99 (m, 2H), 7.07–7.36 (m, 30H), 7.43–7.46 (m, 2H), 7.56–7.59 (m, 1H), 8.01–8.03 (m, 2H); ¹³C-NMR (125 MHz, CDCl₃) δ: 55.5, 63.4, 67.9, 70.2, 72.9, 73.3, 74.0, 74.1, 74.7, 75.0, 75.5, 78.0, 79.0, 79.1, 79.4, 79.8, 82.4, 97.6, 102.8, 114.4, 118.2, 127.3, 127.5, 127.56, 127.62, 127.66, 127.68, 127.71, 127.8, 127.9, 128.0, 128.2, 128.25, 128.33, 128.34, 128.4, 128.8, 129.7, 129.8, 133.1, 137.4, 137.6, 137.7, 137.8, 138.5, 138.6, 151.2, 155.2, 166.1; HRMS (ESI-TOF) m/z: [M + NH₄]⁺ Calcd for C₆₈H₇₂NO₁₃, 1110.4998; Found, 1110.5021.

Compound (21)

To a stirred solution of 19 (1.30 g, 1.19 mmol) in MeCN (83 mL) and H₂O (20 mL) was added CAN (3.03 g, 5.52 mmol) at 0 °C. After being stirred for 2.0 h at 0 °C, the mixture was diluted with EtOAc. The organic layer was washed with saturated NaHCO₃ aq. and H₂O, and was dried over Na₂SO₄. The filtrate was concentrated under reduced pressure to give a residue, which was purified by flash chromatography over silica gel with hexane–EtOAc (92 : 8 to 43 : 57) to give 20 as a colorless oil (677 mg), which was used without further purification. To a stirred solution of 20 (677 mg) and ClC(NPh)CF₃ (427 mg, 2.06 mmol) in CH₂Cl₂ (23 mL) were added DIPEA (0.226 mL, 1.30 mmol) and DMAP (17.1 mg, 0.140 mmol) at room temperature under Ar. After being stirred for 1 h at room temperature, the mixture was concentrated under reduced pressure to give a residue. The residue was purified by flash chromatography over silica gel with hexane–EtOAc (98 : 2 to 41 : 59) to give the corresponding imidate as a colorless oil (498 mg), which was used without further purification. To a stirred solution of the imidate, 8 (304 mg, 0.616 mmol) and MS4A (1.1 g) in Et₂O (7.8 mL) was added TMSOTf (0.078 mL, 0.43 mmol) at −20 °C under Ar. After the mixture was stirred for 2.5 h at −20 °C, the reaction was quenched with Et₃N (0.12 mL). The mixture was filtered with Celite, and the filtrate was concentrated under reduced pressure to give a residue, which was purified by flash chromatography over silica gel with hexane–EtOAc (95 : 5 to 0 : 100) to give 21 as a colorless oil (467 mg, 27% yield): ¹H-NMR (500 MHz, CDCl₃) δ: 0.92 (t, J = 7.4 Hz, 3H), 1.58–1.66 (m, 2H), 3.31–3.32 (m, 2H), 3.32–3.39 (m, 2H), 3.52–3.84 (m, 9H), 3.97–4.03 (m, 3H), 4.15 (d, J = 10.9 Hz, 1H), 4.27–4.33 (m, 3H), 4.37 (d, J = 10.9 Hz, 1H), 4.44–4.59 (m, 7H), 4.63–4.75 (m, 5H), 4.82–4.94 (m, 4H), 4.98–5.01 (m, 2H), 5.02 (d, J = 3.5 Hz, 1H), 5.56 (d, J = 4.0 Hz, 1H), 7.03–7.37 (m, 45H), 7.41–7.45 (m, 2H), 7.55–7.59 (m, 1H), 8.01–8.02 (m, 2H); ¹³C-NMR (125 MHz, CDCl₃) δ: 10.8, 22.6, 63.3, 65.5, 68.0, 68.4, 69.7, 70.0, 70.4, 72.2, 73.1, 73.4, 73.7, 73.9, 74.7, 75.3, 75.5, 75.7, 75.8, 78.0, 78.1, 78.2, 79.2, 79.3, 80.4, 82.1, 82.5, 96.1, 96.5, 97.6, 120.0, 127.4, 127.47, 127.53, 127.56, 127.63, 127.71, 127.73, 127.78, 127.80, 127.88, 127.91, 128.0, 128.09, 128.14, 128.19, 128.22, 128.3, 128.35, 128.38, 128.40, 128.5, 129.7, 129.9, 133.1, 137.70, 137.71, 137.8, 137.9, 138.3, 138.7, 138.8, 139.0, 166.2; HRMS (ESI-TOF) m/z: [M + Na]⁺ Calcd for C₉₁H₉₆NaO₁₇, 1483.6540; Found, 1483.6542.

Compound (22)

By a procedure identical with that described for synthesis of 8 from 7, the ester 21 (105 mg, 0.0720 mmol) was converted into the alcohol 22 as a colorless oil (70.0 mg, 72% yield): ¹H-NMR (500 MHz, CDCl₃) δ: 0.93 (t, J = 7.5 Hz, 3H), 1.58–1.69 (m, 2H), 3.26–3.31 (m, 2H), 3.33–3.39 (m, 1H), 3.42 (dd, J = 9.5, 3.5 Hz, 1H), 3.49 (dd, J = 9.8, 3.5 Hz, 1H), 3.53–3.60 (m, 3H), 3.64–3.71 (m, 6H), 3.77–3.83 (m, 2H), 3.98–4.03 (m, 2H), 4.14 (d, J = 11.5 Hz, 1H), 4.26–4.30 (m, 2H), 4.36 (d, J = 11.5 Hz, 1H), 4.46 (d, J = 11.5 Hz, 1H), 4.49–4.58 (m, 5H), 4.66 (d, J = 3.5 Hz, 1H), 4.67 (d, J = 11.5 Hz, 1H), 4.69 (d, J = 11.5 Hz, 1H), 4.69 (d, J = 11.5 Hz, 1H), 4.74 (d, J = 11.5 Hz, 1H), 4.84 (d, J = 11.5 Hz, 1H), 4.84 (d, J = 11.5 Hz, 1H), 4.90 (d, J = 11.5 Hz, 1H), 4.94 (d, J = 11.5 Hz, 1H), 4.95 (d, J = 11.5 Hz, 1H), 5.00 (d, J = 11.5 Hz, 1H), 5.03 (d, J = 3.5 Hz, 1H), 5.56 (d, J = 3.5 Hz, 1H), 7.03–7.38 (m, 45H); ¹³C-NMR (125 MHz, CDCl₃) δ: 10.9, 22.5, 61.5, 65.5, 68.0, 69.7, 69.9, 70.5, 72.1, 73.1, 73.3, 73.5, 73.8, 74.6, 75.2, 75.4, 75.6, 77.9, 78.0, 78.2, 78.4, 79.4, 80.3, 82.1, 82.4, 96.3, 96.6, 97.4, 126.7, 127.26, 127.28, 127.4, 127.5, 127.55, 127.63, 127.72, 127.74, 127.8, 127.9, 128.0, 128.06, 128.12, 128.15, 128.22, 128.25, 128.29, 128.33, 128.36, 128.41, 137.7, 137.8, 137.9, 138.25, 138.27, 138.7, 138.9; HRMS (ESI-TOF) m/z: [M + Na]⁺ Calcd for C₈₄H₉₂NaO₁₆, 1379.6278; Found, 1379.6273.

Compound (23)

To a stirred solution of 22 (65.0 mg, 0.0479 mmol), TBAI (91.4 mg, 0.246 mmol), DIPEA (0.0212 mL, 0.125 mmol) and MS4A (0.2 g) in benzene (0.40 mL) was added a benzene solution of compound 18 (0.50 mL) at room temperature under Ar. After being stirred for 3.0 h under reflux, the mixture was filtered with Celite, and the filtrate was diluted with EtOAc. The organic layer was washed with saturated Na₂S₂O₃ aq. and brine, and was dried over Na₂SO₄. The filtrate was concentrated under reduced pressure to give a residue, which was purified by flash chromatography over silica gel with hexane–EtOAc (97 : 3 to 35 : 65), NH silica gel with hexane–EtOAc (94 : 6 to 50 : 50) and silica gel with toluene-EtOAc (100 : 0 to 80 : 20) to give 23 as a colorless oil (20.4 mg, 23% yield): ¹H-NMR (500 MHz, CDCl₃) δ: 0.92 (t, J = 7.5 Hz, 3H), 1.58–1.66 (m, 2H), 3.24–3.27 (m, 2H), 3.33–3.42 (m, 3H), 3.46–3.70 (m, 10H), 3.72–3.83 (m, 4H), 3.89 (dd, J = 9.5, 9.5 Hz, 1H), 3.94 (dd, J = 9.2, 9.2 Hz, 1H), 3.98–4.03 (m, 2H), 4.10 (d, J = 11.5 Hz, 1H), 4.24–4.29 (m, 2H), 4.33–4.54 (m, 9H), 4.59 (d, J = 11.5 Hz, 1H), 4.62–4.69 (m, 7H), 4.72 (d, J = 11.5 Hz, 1H), 4.78 (d, J = 11.5 Hz, 1H), 4.82–4.85 (m, 3H), 4.89 (d, J = 11.5 Hz, 1H), 4.92 (d, J = 11.5 Hz, 1H), 4.96–5.00 (m, 4H), 5.03 (d, J = 11.5 Hz, 1H), 5.56 (d, J = 4.0 Hz, 1H), 6.98–7.39 (m, 65 H); ¹³C-NMR (125 MHz, CDCl₃) δ: 10.8, 22.6, 65.3, 65.7, 68.0, 68.3, 69.7, 69.8, 70.1, 70.2, 70.6, 72.1, 72.2, 73.1, 73.3, 73.4, 73.7, 74.6, 75.0, 75.3, 75.5, 75.65, 75.72, 77.5, 78.0, 78.1, 78.5, 78.7, 79.4, 80.0, 80.4, 81.9, 82.2, 82.5, 96.3, 96.6, 97.50, 97.53, 126.5, 127.0, 127.25, 127.30, 127.4, 127.45, 127.47, 127.54, 127.57, 127.61, 127.71, 127.74, 127.85, 127.91, 128.0, 128.05, 128.07, 128.14, 128.16, 128.24, 128.27, 128.30, 128.34, 128.36, 128.44, 128.6, 137.8, 137.87, 137.89, 138.0, 138.3, 138.4, 138.46, 138.53, 138.6, 138.79, 138.81, 138.9, 139.0; HRMS (FAB) m/z: [M + H + Na]⁺ Calcd for C₁₁₈H₁₂₇NaO₂₁, 1902.8762; Found, 1902.8761.

Compound (3)

By a procedure identical with that described for synthesis of 2 from 14, the compound 23 (10 mg, 0.0053 mmol) was converted into the alcohol 3 as a colorless oil (3.8 mg, quant.): ¹H-NMR (500 MHz, D₂O) δ: 0.93 (t, J = 7.2 Hz, 3H), 1.60–1.68 (m, 2H), 3.37–3.60 (m, 7H), 3.60–4.07 (m, 19H), 4.94 (d, J = 4.0 Hz, 1H), 4.95–4.97 (m, 2H), 5.32 (d, J = 4.0 Hz, 1H); ¹³C-NMR (125 MHz, D₂O) δ: 10.6, 22.7, 60.8, 61.1, 66.0, 66.1, 69.9, 70.1, 70.2, 70.4, 70.6, 70.7, 70.8, 71.9, 72.1, 72.30, 72.32, 72.5, 73.5, 73.7, 74.1, 80.9, 98.4, 98.5, 98.7, 99.9; HRMS (ESI-TOF) m/z: [M + Na]⁺ Calcd for C₂₇H₄₈NaO₂₁, 731.2580; Found, 731.2577.

IgA Induction Assay

IgA inducing-ability was evaluated using Peyer’s patch (PP) cells from six-week-old male BALB/cA mice. PP cells were prepared according to our previous study⁶⁾ and resuspended at 2.5 × 10⁶ cells/mL in complete RPMI 1640 medium [RPMI 1640 (Gibco BRL, Grand Island, NY, U.S.A.) containing 100 U/mL penicillin, 100 µg/mL streptomycin, 55 µmol/L 2-mercaptoethanol, and 10% fetal bovine serum (Gibco BRL)]. Various concentrations of saccharides were added to PP cells (final concentration of 1.25 × 10⁶ cells/mL) and incubated in 96-well T-cell activation plates (Becton Dickinson, Franklin Lakes, NJ, U.S.A.) at 37 °C in a humidified atmosphere of 5% CO₂ in air. After 5 d, the IgA levels in the culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) using a mouse IgA ELISA kit (Bethyl Laboratories Inc, Montgomery, TX, U.S.A.). Lipopolysaccharides (LPS; 5 µg/mL) was used as a positive control. Animals were handled in accordance with the guidelines for the proper conduct of animal experiments issued by the Science Council of Japan (2006). The animal experimentation ethics committee of Ishikawa prefectural University approved this study (approval ID: 31-14-12).

Statistics

The results were compared with one-way ANOVA, followed by Dunnett’s post hoc test. All statistical analyses were conducted with the Ekuseru-Toukei software (SSRI, Tokyo, Japan).

Acknowledgments

This work was supported by AMED under Grant Number JP20gm1010007, and the JSPS KAKENHI (Grant Numbers 20H04773 and 20K06938).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials (details of computations).

References

• 1) Schmid J., Curr. Opin. Biotechnol., 53, 130–136 (2018).
• 2) Shetty P. R., Batchu U. R., Buddana S. K., Sambasiva Rao K., Penna S., Carbohydr. Res., 503, 108297 (2021).
• 3) Zannini E., Waters D. M., Coffey A., Arendt E. K., Appl. Microbiol. Biotechnol., 100, 1121–1135 (2016).
• 4) Saadat Y. R., Khosroushahi A. Y., Gargari B. P., Carbohydr. Polym., 217, 79–89 (2019).
• 5) Zhou Y., Cui Y., Qu X., Carbohydr. Polym., 207, 317–332 (2019).
• 6) Matsuzaki C., Hayakawa A., Matsumoto K., Katoh T., Yamamoto K., Hisa K., J. Agric. Food Chem., 63, 7009–7015 (2015).
• 7) Matsuzaki C., Takagaki C., Tomabechi Y., Forsberg L. S., Heiss C., Azadi P., Matsumoto K., Katoh T., Hosomi K., Kunisawa J., Yamamoto K., Hisa K., Carbohydr. Res., 448, 95–102 (2017).
• 8) Matsuzaki C., Nakashima Y., Endo I., Tomabechi Y., Higashimura Y., Itonori S., Hosomi K., Kunisawa J., Yamamoto K., Hisa K., Gut Microbes, 13, 1949097 (2021).
• 9) Zhu X., Schmidt R. R., Angew. Chem. Int. Ed., 48, 1900–1934 (2009).
• 10) Manabe S., Heterocycles, 102, 177–201 (2021).
• 11) Kováč P., Lerner L., Carbohydr. Res., 184, 87–112 (1988).
• 12) Lam S. N., Gervay-Hague J., Org. Lett., 4, 2039–2042 (2002).
• 13) Lam S. N., Gervay-Hague J., Carbohydr. Res., 337, 1953–1965 (2002).
• 14) Boltje T. J., Kim J. H., Park J., Boons G. J., Nat. Chem., 2, 552–557 (2010).
• 15) Chu A. H., Nguyen S. H., Sisel J. A., Minciunescu A., Bennett C. S., Org. Lett., 15, 2566–2569 (2013).
• 16) Komarova B. S., Dorokhova V. S., Tsvetkov Y. E., Nifantiev N. E., Org. Chem. Front., 5, 909–928 (2018).
• 17) Ding F., Ishiwata A., Zhou S., Zhong X., Ito Y., J. Org. Chem., 85, 5536–5558 (2020).
• 18) Meloncelli P. J., Martin A. D., Lowary T. L., Carbohydr. Res., 344, 1110–1122 (2009).
• 19) Gervay-Hague J., Acc. Chem. Res., 49, 35–47 (2016).
• 20) Komarova B. S., Orekhova M. V., Tsvetkov Y. E., Nifantiev N. E., Carbohydr. Res., 384, 70–86 (2014).
• 21) The information about the stereochemistry of the imidate anomeric position was not obtained, because the reaction produced the imidate along with inseparable byproducts, making it difficult to determine its stereochemistry.
• 22) Demchenko A. V., “Handbook of Chemical Glycosylation,” ed. by Demchenko A. V., Wiley-VCH, Weinheim, 2008.
• 23) Demchenko A. V., Synlett, 2003, 1225–1240 (2003).
• 24) Komarova B. S., Tsvetkov Y. E., Nifantiev N. E., Chem. Rec., 16, 488–506 (2016).
• 25) Zhang Y., Zhou S., Wang X., Zhang H., Guo Z., Gao J., Org. Chem. Front., 6, 762–772 (2019).