Polytetrafluoroethylene-Assisted Rapid Preparation of α‑Oligomannosides Containing Mannose-6-Phosphate Residues

Abstract

A Polytetrafluoroethylene (PTFE)-assisted “liquid-phase reaction, solid-phase separation” strategy was used to streamline the assembly of mannose-6-phosphate mannosides isolated from Pichia holstii NRRL Y-2448. Thus, the α-stereoselective tetramannoside and pentamannoside scaffolds were rapidly assembled with an average yield of 90% per step through neighboring group participation (NGP) or remote group participation (RGP), as well as regulation of the reactivities of glycosyl donors or acceptors. Notably, a gram-scale preparation of protected pentamannoside 16 was accomplished, and the average yield per step exceeded 92%. Furthermore, the pentamannoside and tetramannoside were phosphorylated and purified by PTFE-assisted filtration, demonstrating the high efficiency, simplicity, and universality of the purification strategy for well-defined glycan synthesis. The efficient assembly of mannose-6-phosphate mannoside paves the way for uncovering the biological function of phosphorylated glycans.

Introduction

High-mannose glycans bearing mannose-6-phosphate (M6P) residues constitute a class of saccharides, where such residues are present either at the terminal positions or within the glycan chains. These residues typically serve as key targeting signals, mediating the transport of glycoproteins to lysosomes via the two M6P receptors, i.e., a cation-dependent M6P receptors (CD-MPR) and a cation-independent M6P receptors (CI-MPR). − Thus, M6P-based lysosomal targeting has been employed in the treatment of lysosomal storage diseases (LSDs), as well as drug delivery and subcellular imaging.

Mannose-6-Phosphate mannoside A1 (Figure ), the α-1,2/1,3-oligomannan, is the fraction from Pichia holstii NRRL Y-2448 phosphomannan. The pentamannoside A3 (Figure ) constitutes the main component of the aforementioned oligomannan, accounting for approximately 60%. Sulfonation of the oligomannoside mixture A1 provides PI-88, a heparan sulfate mimetic that is a promising heparanase inhibitor and an promising antimetastatic drug candidate. However, the complexity of PI-88 poses challenges for its structural characterization, manufacturing processes, and assessment of structure–activity relationships, which further impact its clinical research and further applications. To acquire an adequate quantity of the oligomannoside in pure form, we proposed chemical synthesis of the glycosides − by PTFE-assisted purification. − We envisioned that the tetra- and pentamannoside can be efficiently prepared via linear assembly by fluorous tag 1, followed by late-state phosphorylation and global deprotection − (Figure ). According to retrosynthetic analyses, building blocks 2–4 were identified as suitable candidates for assembling mannose-6-phosphate oligomannosides. The α-stereoselectivity of the target glycans could be achieved by participation of neighbor or remote acyl groups within the building blocks 2-4. Building block 3 carries a 3-O-levulinate ester (Lev) that can be selectively removed to construct 1,3-linkages, while building block 4 bears a Lev group at the 6-O-position to enable precise phosphorylation.

1.

Structure of Pichia holstii Y-2448 oligosaccharides and their retrosynthetic analysis.

Results and Discussion

We initiated the mannoside synthesis from the installation of tag 1 on imidate donor 2. With trimethylsilyl trifluoromethanesulfonate (TMSOTf) as the catalyst, glycosyl donor 2 was coupled with fluorous tag 1 at −30 °C in CH₂Cl₂, providing the corresponding product 5 in 84% yield (Scheme ). Then, the selective deacetylation of 5 afforded acceptor 6 in 98% yield.

1. Synthesis of Fluorous-Tagged Acceptor 6.

With the acceptor 6 in hand, glycosylation of thioglycoside donor 3a with 6 was carried out for the construction of α-1,3-mannoside (Scheme ). However, disaccharide 7 was only obtained in 59% yield, along with the recovery of acceptor 6 in 30% yield and poor stereoselectivity (3/1, α/β). To enhance the efficiency of the glycosylation and stereoselectivity, imidate donor 3b was synthesized and tested in the glycosylation. Fortunately, when imidate donor 3b was used instead of 3a, the glycosylation with acceptor 6 provided the desired disaccharide 7 as only the α isomer with excellent yield. Thus, after removal of the Lev group in disaccharide 7, the carbohydrate chain was further elongated by reaction with imidate donor 3b again. Unfortunately, a large amount of the disaacharide 8 was recovered, and trisaccharide 9 was only afforded in 31% yield.

2. Synthesis of Fluorous-Tagged Trisaccharide 9.

We speculated that the multiple electron-withdrawing benzoyl (Bz) groups on either the glycosyl acceptor or the donor may be responsible for the low glycosylation efficiency. , Thus, donor 3c was designed and synthesized with two benzyl groups at the C-2 and C-4 on the mannose ring while retaining the Bz group at C-6 and Lev group at C-3 for realizing high stereoselectivity by RGP (Scheme ). Gratifyingly, acceptor 6 could be smoothly glycosylated with donor 3c in the presence of TMSOTf at −30 °C, providing the fluorous-tagged disaccharide 10 in 82% yield. Selective removal of the Lev group on disaccharide 10 and subsequent glycosylation with donor 3c gave trisaccharide 12 with high stereoselectivity in 84% yield. The repetitive deprotection of the Lev group in compound 12 and glycosylation with donor 3c afforded the tetrasaccharide 14 in good yield and high stereoselectivity. Subsequently, by selective removal of the Lev group in compound 14 using CH₃COO^–N₂H₅ ⁺, followed by PTFE purification, the tetrasaccharide acceptor 15 was afforded in 92% yield.

3. Synthesis of Tetrasaccharide 15.

The same glycosylation condition for compounds 12 and 14 was taken for the coupling reaction between 15 and donor 4. The corresponding pentasaccharide of 16 was obtained with about α/β (10/1) stereoselectivity. The α-configuration of 16 was confirmed by the heteronuclear one-bond C1 constants (J _C1–H1 = 168–169 Hz, see Figure S40 in Supporting Information). Interestingly, the stereoselectivity was remarkably increased with TfOH as the promoter instead of TMSOTf. Consequently, pentasaccharide 16 was synthesized exclusively as the α-anomer in 84% yield (Scheme ). Applying the same coupling condition for 16, the glycosylation of trisaccharide 13 with donor 4 furnished only α-tetrasaccharide 17 in 91% yield.

4. Synthesis of α-Oligomannosides. a) Synthesis of Pentasaccharide 16. b) Synthesis of Tetrasaccharide 17.

To verify the efficiency and simplicity of the PTFE-assisted “solution-phase reaction, solid-phase separation” strategy for oligosaccharide synthesis, we further performed the streamline assembly of the tetra- and pentamannoside starting from acceptor 6 (Scheme ). Tetrasaccharide 17 was obtained in a 61% overall yield over five steps. Notably, the average yield for each step exceeded 90%, and all intermediates were purified solely via PTFE-assisted filtration. Furthermore, the gram-scale preparation of pentasaccharide 16 was conducted. Gratifyingly, the overall yield was 56% over seven steps, with an average step yield of approximately 92%.

5. Iterative and Gram-Scale Assembly of Oligomannoside 16 and 17.

With the completion of the synthesis of pentasaccharide 17 and tetrasaccharide 16, we turned our attention to deprotection and phosphorylation for both oligomannosides (Scheme ). Compound 18 (or 19) was afforded in 99% yield after removal of the Lev protecting group at the C-6 position within 16 (or 17) using CH₃COO^–N₂H₅ ⁺. Phosphitylation of 18 (or 19) with dibenzyl N,N-diisopropylphosphoramidite ((BnO)₂PN ⁱ Pr₂) in the presence of tetrazole (CH₂N₄) for 2 h, followed by oxidation of the intermediate phosphite with m-CPBA ,, to give the phosphate ester 20 (or 21) in 100% (or 99%) yield by PTFE-assisted purification. Subsequently, the Bz groups in compound 20 (or 21) were removed and provided compound 22 (or 23) in 92% (or 70%) yield. Finally, the global deprotection of Bn groups and the fluorous tag was achieved under a hydrogen atmosphere (2 MPa) using 20% Pd­(OH)₂/C as the catalyst, providing phosphorylated oligomannosides 24 and 25 in 89% and 83% yield, respectively.

6. Synthesis of Phosphorylated Oligomannoside 24 and 25 .

Conclusions

In conclusion, PTFE-assisted “solution-phase reaction, solid-phase separation” strategy was presented to efficiently prepare mannose-6-phosphate oligomannosides. All glycosylation reactions proceeded smoothly and furnished the corresponding α-mannosides in high stereoselectivity via the NGP or RGP strategy. Oligomannosides could be rapidly prepared on a gram scale to achieve the target oligosaccharides with an average yield of over 90% per step. We demonstrated that the PTFE-assisted strategy could be developed as a robust method for rapid assembly of well-defined oligo- and polysaccharides. The successful synthesis of mannose-6-phosphate oligomannosides not only provides a foundation for structural modifications but also facilitates the elucidation of their biological mechanisms, thereby laying the fundamental basis for their applications in fields such as novel drug development and functional foods.

Experimental Section

General Information

Anhydrous dichloromethane (CH₂Cl₂), toluene, and triethylamine (Et₃N) were obtained by refluxing with CaH₂ under argon for more than 5 h. Tetrahydrofuran (THF) was dried by distillation from sodium/benzophenone. For reactions that require heating, the oil (dimethyl silicon oil) bath was used as heat source. Solvents used for column chromatography were analytical grade. TLC was used to detect all reactions, which was achieved by UV absorption (254 nm) and/or by staining with sulfuric acid/ethanol (1/9, v/v). Column chromatography was performed using 300–400 mesh silica gel. Polytetrafluoroethylene (PTFE) powder was purchased from Adamas (>40 μm particle size, CAS: 9002–84–0). Optical rotations (OR) were measured with a Rudolph Research Analytical Autopol IV-T automatic polarimeter. Nuclear magnetic resonance spectra were afforded with JEOL 400 or Bruker 600 MHz for ¹H NMR and 100 or 150 MHz for ¹³C NMR. Chemical shifts (δ) were reported in parts per million (ppm). Coupling constants (J) were reported in Hertz. Multiplicity was reported using the following abbreviations: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublet), and dt (doublet of triplet). ¹H NMR spectra were referenced to 0 (TMS), or to a solvent residual signal (CDCl₃, 7.26 ppm; D₂O, 4.79 ppm). ¹³C NMR spectra were referenced to 77.00 (CDCl₃) ppm. High resolution mass spectra were measured by ESI ionization sources using a Bruker mass spectrometer.

Protocols for PTFE-Assisted Synthesis of Oligosaccharides

Protocol A: General Procedure for Coupling

Coupling

To a mixture of glycosyl donor (1.5–2 equiv), acceptor (1 equiv), and 4 Å MS in anhydrous CH₂Cl₂ (0.03–0.1 mol/L) was added TMSOTf (0.1–0.2 equiv) at −30 °C. Thirty min later, Et₃N (5 ∼ 10 equiv) was added to quench the reaction. The resulting mixture was filtered and concentrated in vacuo, and then the residue was purified by PTFE-assisted purification.

PTFE-Assisted Purification

PTFE (∼10 times to all reactants) was added to the resulting mixture, and the volatile solvent was removed under reduced pressure. Then, acetone aqueous solution (acetone/H₂O = 13/7, v/v) was added (Note: The volume of the acetone aqueous solution should be enough to immerse all PTFE). The suspension was filtered through sand core funnel or qualitative filter paper, washing with additional acetone/H₂O (3×). At last, the crude product was detached by washing with acetone (4×), and the resultant solution was concentrated under reduced pressure to give the crude product.

Protocol B: General Procedure for Removal of Lev Group

Deprotection

To a solution of glycosides (1 equiv) in CH₂Cl₂ (∼0.1 mol/L) was added CH₃COO^–N₂H₅ ⁺ (0.1 mol/L in MeOH, 1.5 equiv) at room temperature. Three hour later, the reaction mixture was purified by the following PTFE-assisted purification.

PTFE-Assisted Purification

Following the above-mentioned PTFE-assisted purification in Protocol A, an acetone aqueous solution acetone/H₂O (1/1) was used instead of acetone/H₂O (13/7).

(4-(1,3-Bis­(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyloxy)­propan-2-yloxy)­phenyl) methyl 2-O-acetyl-3,4,6-tribenzyl-α-d-mannopyranoside 5

A solution of compound 1 (299 g, 0.34 mmol), 2 (480 mg, 0.75 mmol) and powdered 4 Å MS in anhydrous CH₂Cl₂ (3 mL) was cooled at −30 °C for 10 min, then TMSOTf (10 μL in 1 mL CH₂Cl₂, 0.6 mL) was added dropwise. After 30 min, Et₃N was added to quench the reaction, and the resulting mixture was filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 4/1) to give 5 (395 mg, 0.29 mmol, 84%) as colorless syrup. ${[\alpha ]}_D^{18}$ +4 (c 0.2, CH₂Cl₂); ¹ H NMR (400 MHz, CDCl₃) δ 7.41 – 7.19 (m, 15H), 7.18 – 7.12 (m, 2H), 6.92 (d, J = 8.6 Hz, 2H), 5.40 (dd, J = 3.2, 1.8 Hz, 1H), 4.91 (d, J = 1.6 Hz, 1H), 4.85 (d, J = 10.7 Hz, 1H), 4.71 (d, J = 4.2 Hz, 1H), 4.68 (d, J = 3.1 Hz, 1H),4.63 (d, J = 11.5 Hz, 1H), 4.56 – 4.40 (m, 5H), 4.01 (dd, J = 8.8, 3.3 Hz, 1H), 3.93 – 3.64 (m, 12H), 2.50 – 2.28 (m, 4H), 2.14 (s, 3H); ¹³ C NMR (100 MHz, CDCl₃) δ 170.45, 157.60, 138.32, 138.23, 137.98, 129.88, 128.37, 128.32, 128.04, 127.93, 127.78, 127.70, 127.66, 127.59, 116.11, 96.79, 78.35, 77.20, 76.25, 75.25, 74.38, 73.47, 71.83, 71.58, 69.66, 68.89, 68.79, 68.77, 63.47 (t), 31.43 (t), 21.09. HRMS (ESI) m/z Calcd for C₆₁H₄₈F₂₆NaO₁₃ (M + Na)⁺ 1387.2887, Found 1387.2901.

(4-(1,3-Bis­(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyloxy)­propan-2-yloxy)­phenyl) Methyl 3,4,6-tribenzyl-α-d-mannopyranoside 6

To a solution of compound 5 (1.21 g, 0.89 mmol) in anhydrous THF (12 mL) was added MeONa/MeOH­(1.1 mL, 0.16 mol/L) at room temperature. After stirring at room temperature for 3.5 h, the reaction mixture was diluted with CH₂Cl₂ and washed with NH₄Cl, The organic layer was dried over MgSO₄, filtered and concentrated under reduced pressure to give 6 (1.15 g, 0.87 mmol, 98%) as a colorless syrup. ¹ H NMR (400 MHz, CDCl₃) δ 7.41 – 7.21 (m, 15H), 7.20 – 7.14 (m, 2H), 6.92 (d, J = 7.3 Hz, 2H), 4.97 (s, 1H), 4.82 (d, J = 10.7 Hz, 1H), 4.72 – 4.61 (m, 4H), 4.59 – 4.38 (m, 4H), 4.05 (s, 1H), 3.96 – 3.64 (m, 13H), 2.49 (s, 1H), 2.48 – 2.25 (m, 4H); ¹³ C NMR (100 MHz, CDCl₃) δ 157.57, 138.27, 137.94, 130.23, 129.85, 128.51, 128.35, 128.32, 127.96, 127.89, 127.82, 127.78, 127.70, 127.56, 116.11, 98.10, 80.34, 77.20, 76.30, 75.19, 74.39, 73.48, 72.02, 71.29, 69.68, 69.00, 68.54, 68.45, 63.47, 31.45 (t).

Gram-Scale Assembly of Pentamannoside 16

Following protocols A and B, we started from 6 (1.07 g, 0.81 mmol) and after 4 times coupling and 3 times deprotection to afford the crude product 16 (2.14 g). Furthermore, the crude product could be purified by silica gel column chromatography (petroleum ether/EtOAc, 3/1–2/1) to afford pure product 16 (1.46 g, 0.46 mmol, 56% over 7 steps) as a porous white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.03 (d, J = 7.8 Hz, 4H), 7.95 (d, J = 7.8 Hz, 4H), 7.59 (t, J = 7.5 Hz, 2H), 7.54–7.47 (m, 2H), 7.43 (d, J = 7.7 Hz, 2H), 7.35 – 7.28 (m, 14H), 7.28 – 7.15 (m, 39H), 7.16 – 7.10 (m, 6H), 7.07 (s, 4H), 6.95 – 6.83 (m, 2H), 5.60 (dd, J = 9.6, 2.9 Hz, 1H), 5.31 (s, 2H), 5.26 (s, 1H), 5.18 (s, 1H), 4.97 (s, 1H), 4.88 (d, J = 10.8 Hz, 1H), 4.83 (d, J = 11.3 Hz, 2H), 4.81 – 4.73 (m, 1H), 4.71 (s, 1H), 4.68 (s, 1H), 4.65 – 4.51 (m, 10H), 4.53 – 4.46 (m, 2H), 4.44 – 4.34 (m, 6H), 4.33 – 4.27 (m, 4H), 4.27 – 4.17 (m, 10H), 4.17 – 4.10 (m, 3H), 3.99 – 3.88 (m, 10H), 3.84 (d, J = 5.8 Hz, 2H), 3.82–3.73 (m, 4H), 3.73 – 3.67 (m, 4H), 2.57 – 2.51 (m, 2H), 2.51 – 2.46 (m, 2H), 2.45 – 2.31 (m, 4H), 2.03 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 206.37, 172.20, 166.24, 166.07, 165.52, 157.39, 138.28, 138.17, 138.11, 138.06, 137.90, 137.78, 137.73, 137.63, 133.15, 132.92, 132.87, 129.66, 128.50, 128.43, 128.36, 128.27, 128.20, 128.16, 128.09, 128.00, 127.80, 127.67, 127.56, 127.52, 127.48, 127.44, 127.37, 127.29, 127.20, 127.09, 126.99, 115.93, 100.09, 99.07, 98.11, 80.44, 79.99, 79.85, 78.08, 77.86, 76.25, 75.98, 75.29, 75.11, 74.67, 74.28, 74.22, 73.95, 73.68, 73.18, 72.63, 72.55, 71.82, 71.43, 70.73, 70.44, 70.24, 69.42, 69.15, 68.56, 63.37, 62.98, 37.63, 31.25 (t), 29.67, 27.73; HRMS (ESI) m/z calcd for C₁₆₆H₁₅₈F₂₆Na₂O₃₅ ²⁺ (M + 2Na)²⁺ 1625.4977; found: 1625.5006.

Iterative Assembly of Tetramannoside 17

Following protocols A and B, starting from 6 (316 mg, 0.24 mmol), after 3 times coupling and 2 times deprotection to afford the crude product 17, which was further purified by silica gel cholumn chromatography (petroleum ether/EtOAc, 3/1–2/1) to afford pure product 17 (402 mg, 0.15 mmol, 61% over 5 steps) as a pale yellow syrup. ${\left[\alpha \right]}_D^{18}$ + 4 (c 0.2, CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 7.96 (d, J = 7.8 Hz, 4H), 7.85 (d, J = 7.7 Hz, 2H), 7.50 (t, J = 7.5 Hz, 1H), 7.45 – 7.38 (m, 2H), 7.36 (t, J = 7.7 Hz, 2H), 7.28 – 7.21 (m, 5H), 7.20 – 7.05 (m, 41H), 6.99 – 6.97 (m, 4H), 6.82 (d, J = 8.1 Hz, 2H), 5.54 (d, J = 9.5 Hz, 1H), 5.26 (s, 1H), 5.16 (d, J = 16.8 Hz, 2H), 4.90 (s, 1H), 4.79 (dd, J = 16.9, 11.1 Hz, 2H), 4.72 (d, J = 10.6 Hz, 1H), 4.62 (d, J = 10.8 Hz, 2H), 4.53 (t, J = 11.5 Hz, 5H), 4.48 – 4.43 (m, 3H), 4.42 – 4.36 (m, 4H), 4.34 – 4.28 (m, 3H), 4.27 – 4.13 (m, 9H), 4.09 (d, J = 11.2 Hz, 1H), 4.06 – 4.02 (m, 2H), 3.96 – 3.89 (s, 3H), 3.89 – 3.82 (m, 5H), 3.77 – 3.73 (m, 2H), 3.73 – 3.67 (m, 4H), 3.66 – 3.57 (m, 6H), 2.48 (t, J = 6.6 Hz, 2H), 2.42 (t, J = 6.6 Hz, 2H), 2.38 – 2.26 (m, 4H), 1.98 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 206.39, 172.27, 166.25, 166.11, 165.57, 157.40, 138.29, 138.21, 138.15, 138.11, 137.80, 137.70, 133.15, 132.92, 132.85, 130.19, 129.94, 129.85, 129.74, 129.67, 129.64, 128.49, 128.44, 128.37, 128.32, 128.27, 128.24, 128.19, 128.10, 128.01, 127.83, 127.79, 127.73, 127.69, 127.59, 127.56, 127.47, 127.38, 127.29, 127.16, 115.96, 100.03, 98.18, 80.00, 77.85, 77.65, 76.39, 76.04, 75.30, 75.15, 74.77, 74.67, 74.29, 73.99, 73.21, 72.84, 72.62, 72.57, 71.85, 71.63, 70.60, 70.28, 69.47, 69.44, 69.15, 68.59, 63.39, 63.04, 63.01, 37.67, 31.30 (t), 29.69, 27.78; HRMS (ESI) m/z calcd for C₁₃₉H₁₃₂F₂₆Na₂O₂₉ ²⁺(M + 2Na)²⁺ 1402.4112; found: 1402.4127.

6-O-Phosphate-α-d-mannopyranosyl-(1→3)-α-d-mannopyranosyl-(1→3)-α-d-mannopyranosyl-(1→3)-α-d-mannopyranosyl-(1→2)-d-mannopyranose (24)

Step 1: To a solution of the compound 16 (241.8 mg, 0.075 mmol) in anhydrous CH₂Cl₂ (0.95 mL), 1 M CH₃COO-N₂H₅ ⁺/MeOH (0.12 mL) was added at room temperature. After stirring at room temperature for 3 h, the resulting was concentrated in vacuo, then the residue was purified by PTFE-assisted filtration (see the general procedure in protocol B) to give 18 (230.0 mg, 0.074 mmol, 98%).

Step 2: To a solution of compound 18 (0.46 g, 0.15 mmol) and powdered 4 Å MS in anhydrous CH₂Cl₂ (16 mL), CH₂N₄ (51.10 mg, 0.73 mmol) and (BnO)₂PNPr₂ (0.2 mL) was added. After the mixture was stirred at room temperature for 2 h, TLC indicated the reaction was complete. The reaction mixture was cooled at −30 °C for 10 min, m-CPBA (134 mg, 0.58 mmol) was added. After 1 h, the mixture was filtered and concentrated in vacuo, and the reaction was diluted with CH₂Cl₂ and washed with NaHCO₃ aqueous. The organic layer was dried over MgSO₄, filtered and concentrated under reduced pressure, then the residue was purified by PTFE-assisted filtration (see the general procedure in protocol A) to give 20 (0.51 g, 0.15 mmol, 100%).

Step 3: To a solution of compound 20 (0.51 g, 0.15 mmol) in anhydrous MeOH (1.5 mL) and CH₂Cl₂ (1.5 mL) was added MeONa (0.15 mol/L, 1 mL MeOH) at room temperature. After stirring at room temperature for 12 h, the solution was neutralized with Amberlite (R) IR 120 H⁺ form ion-exchange resin, and then the residue was purified by PTFE-assisted filtration (see the general procedure in protocol B) to give 22 (0.41 g, 0.14 mmol, 92%) as a colorless syrup.

Step 4: To a solution of compound 22 (55.8 mg, 0.019 mmol) in MeOH:H₂O:EtOAc = 4:1:3 (4.4 mL:1.1 mL: 3.3 mL), 20% Pd­(OH)₂/C was added at 0 °C. The mixture was stirred under H₂ atmosphere at room temperature for 72 h. Then, the reaction mixture was poured into a separatory funnel with about 8 mL of EtOAc, the organic layer was washed with H₂O, and the aqueous layer was combined and concentrated under reduced pressure to afford 24 (15.2 mg, 0.017 mmol, 89%). ¹ H NMR (400 MHz, D₂O) δ 5.37 (s, 1H), 5.11 (s, 2H), 5.08 (s, 1H), 5.02 (s, 1H), 4.32 (s, 1H), 4.21 (s, 3H), 4.06 (s, 2H), 4.01 – 3.89 (m, 6H), 3.89 – 3.82 (m, 6H), 3.82 – 3.67 (m, 12H); ¹³ C NMR (150 MHz, D₂O) δ 196.41, 102.84, 102.56, 102.04, 102.00, 92.47, 79.46, 79.04, 78.24, 77.83, 73.60, 73.41, 73.32, 72.47, 70.22, 70.08, 69.94, 69.79, 69.52, 69.23, 66.99, 66.78, 66.16, 65.82, 65.78, 61.12, 60.92; ³¹ P NMR (243 MHz, D₂O) δ 4.16; HRMS (ESI) m/z C₃₀H₅₂O₂₉P^–(M-H)⁻ calcd for 907.2337; found 907.2334.

6-O-Phosphate-α-d-mannopyranosyl-(1→3)-α-d-mannopyranosyl-(1→3)-α-d-mannopyranosyl-(1→2)-d-mannopyranose (25)

Step 1: To a solution of compound 17 (169 mg, 0.061 mmol) in anhydrous CH₂Cl₂ (0.8 mL), 1 M CH₃COO^–N₂H₅ ⁺/MeOH (0.09 mL) was added at room temperature. After stirring at room temperature for 3 h, the resulting product was concentrated in vacuo, and then the residue was purified by PTFE-assisted filtration (see the general procedure in protocol B) to give 19 (160 mg, 0.060 mmol, 99%).

Step 2: To a solution of compound 19 (77.2 mg, 0.029 mmol) and powdered 4 Å MS in anhydrous CH₂Cl₂ (2.9 mL), CH₂N₄ (10.2 mg, 0.073 mmol) and (BnO)₂PNPr₂ (39 μL) was added. After stirring at room temperature for 2 h, the reaction mixture was cooled at −30 °C for 10 min, m-CPBA (26.7 mg, 0.12 mmol) was added. One hour later, the mixture was filtered and concentrated in vacuo, the reaction was diluted with CH₂Cl₂ and washed with saturated NaHCO₃ aqueous. The organic layer was dried over MgSO₄, filtered and concentrated under reduced pressure, then the residue was purified by PTFE-assisted filtration (see the general procedure in protocol A) to give 21 (81.78 mg, 0.028 mmol, 99%).

Step 3: To a solution of compound 21 (95.9 mg, 0.033 mmol) in anhydrous MeOH (0.3 mL) was added MeONa (0.8 mg of Na in 0.1 mL of MeOH) at room temperature. After stirring at room temperature for 12 h, the solution was neutralized with Amberlite (R) IR 120 H⁺ to form ion-exchange resin, and then the residue was purified by PTFE-assisted filtration (see the general procedure in protocol B) to give 23 (60.3 mg, 0.023 mmol, 70%) as colorless syrup.

Step 4: To a solution of compound 23 (60.3 mg, 0.023 mmol) in MeOH: H₂O: EtOAc = 4:1:3 (4.4 mL:1.1 mL: 3.3 mL), 20% Pd­(OH)₂/C was added at 0 °C, and then the reaction mixture was stirred in H₂ atmosphere at room temperature for 72 h. The reaction mixture was poured into a separatory funnel with about 8 mL of EtOAc, the organic layer was washed with H₂O, and the aqueous layer was combined and concentrated under reduced pressure to afford 25 (14.1 mg, 0.019 mmol, 83%). ¹ H NMR (400 MHz, D₂O) δ 5.36 (s, 1H), 5.10 (s, 1H), 5.06 (s, 1H), 5.00 (s, 1H), 4.30 (s, 1H), 4.23 – 4.10 (m, 2H), 4.05 (s, 1H), 3.94 – 3.81 (m, 10H), 3.78 – 3.64 (m, 10H); ¹³ C NMR (150 MHz, D₂O) δ 102.79, 102.38, 102.01, 92.46, 79.04, 78.06, 77.83, 73.43, 73.33, 72.56, 72.52, 72.47, 70.22, 70.03, 69.95, 69.70, 66.98, 66.68, 65.88, 65.86, 63.84, 61.06, 60.94; ¹³ P NMR (243 MHz, D₂O) δ 2.80; HRMS (ESI) m/z calcd for C₂₄H₄₂O₂₄P^–(MH)⁻, 745.1809; found 745.1814.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c11554.

• Synthesis of monosaccharide building block and NMR spectra of compounds (PDF)

The authors declare no competing financial interest.