Automated Fluorous-Assisted Solution-Phase Synthesis of β-1,2-, 1,3-, and 1,6-Mannan Oligomers

Abstract

Automated solution-phase syntheses of β-1,2-, 1,3-, and 1,6-mannan oligomers have been accomplished by applying a β-directing C-5 carboxylate strategy. Fluorous-tag-assisted purification after each reaction cycle allowed the synthesis of short β-mannan oligomers with limited loading of glycosyl donor—as low as 3.0 equivalents for each glycosylation cycle. This study showed the capability of the automated solution-phase synthesis protocol for synthesizing various challenging glycosides, including use of a C-6 ester as a protecting group that could be converted under reductive conditions to a hydroxyl group for chain extension.

Graphical Abstract

1. Introduction

β-Mannoside can be found in a wide variety of natural products from mammalian, insect, plant, fungal, and bacterial sources.¹ Many of these β-mannosides have shown potential biological roles that are not yet well understood.^{1, 2} The chemical synthesis of β-mannosides could provide structurally-defined substrates for biological studies; however, the β-mannosidic linkage is one of the most challenging glycosidic linkages to create.³ The challenges of formation of this 1,2-cis linkage are attributed to two major factors. First, the steric hindrance from the axial C-2 substituent blocks the β-face of the pyranose ring and prevents the ready formation of the β-glycosidic bond. Second, unlike α-mannoside, which is stabilized by the anomeric effect, β-mannoside lacks such a stabilizing interaction and its formation is considered thermodynamically disfavored. In order to synthesize this challenging linkage, several methods have been developed, including strategies that can be adapted to automated synthesis of β-mannoside-containing oligosaccharides.^4a,b Previously we have demonstrated the automated solution-phase synthesis^5a of a β-1,4-mannan hexamer^5b and an insect N-glycan structure^5c by applying a β-directing C-5 carboxylate (mannuronate) methodology inspired in part by van der Marel and coworkers.⁶ Unlike the more common 4,6-O-benzylidene-acetal technique for β-mannoside formation invented by Crich and coworkers⁷, the mannuronate glycosyl donor allows a more straightforward deprotection strategy, higher glycosylation temperature, and robustness against acidic conditions.⁶ However, the generality of this approach to making other β-mannoside oligomers such as the β-1,2-, 1,3- and 1,6-mannans was unclear.⁸ Herein we report a series of mannuronate building blocks to test their utility in automated solution-phase syntheses for the production of β-1,2-, 1,3- and 1,6-mannans.

2. Results and discussion

Our automated solution-phase synthesis protocol has shown several advantages compared to solid-phase approaches such as significantly lower donor loading in glycosylation cycles and straightforward off-line reaction monitoring by thin layer chromatography (TLC) or mass spectrometry (MS) due to the homogenous reaction conditions.⁵ Purification is facilitated in the automated synthesis platform by an attached C₈F₁₇ fluorous-tag (F-tag) to the initial sugar building block; this tag has been shown to provide enough of a noncovalent interaction for reliable automated fluorous solid-phase extraction (FSPE) of the growing oligosaccharide chain.⁹ Therefore, a series of mannuronate building block donors were designed for attachment to a fluorous tag at the anomeric center and chain extension via a hydroxyl masked with a readily removable—“temporary”—protecting group. For the automated syntheses of β-1,2- and 1,3-mannans, a p-methoxybenzyl (PMB) group was chosen as the temporary masking group for the acceptor hydroxyl because of its small size, lack of potential for neighboring group participation, stability during glycosylation, and ease of deprotection on the automated synthesis platform. The synthesis of the mannuronate trichloroacetimidate building block 6 (Scheme 1) started from selective opening of a benzylidene acetal on the known precursor 1^7a with borane tetrahydrofuran complex (BH₃·THF) and dibutylboron triflate to afford 2 with a free 6-OH.¹⁰ The oxidation of the primary alcohol by 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO) and (diacetoxyiodo)benzene (BAIB) generated the uronic acid 3.^6a Esterification of the carboxylic acid with methyl iodide (MeI)/K₂CO₃ gave the fully protected methyl mannuronate 4.^6a N-Bromosuccinimide (NBS) removed the anomeric thiophenyl group to produce hemiacetal 5 with a free anomeric hydroxyl group.^5b Lastly, trichloroacetimidate formation under standard conditions^s5b,c produced the desired building block 6 in 44% overall yield from compound 1.

Scheme 1.

Synthesis of building block 6 for the automated solution–phase synthesis of β-1,2-mannan.

This new building block was then tested for its ability to support chain extension to produce a β-1,2-mannan oligosaccharide (Scheme 2). The automated synthesis started from the conjugation of known F-tag 7⁹ and building block 6 (3.0 equiv) catalyzed by TMSOTf (0.11 equiv) at −20 °C. After 30 min, the solvent was removed under reduced pressure and a ceric ammonium nitrate (CAN) solution in MeCN/H₂O = 1/9 was added to the mixture and vortexed for 1 h. After the reaction showed completion based on TLC, the mixture was robotically transferred to the SPE station for the FSPE purification. After the FSPE, the purified product entered another glycosylation-deprotection-FSPE cycle and the crude product was transferred out of the synthesis platform and purified manually to afford the desired β-1,2-mannuronate dimer 8 in 38% over 4 steps (79% per step). Interestingly, subsequent attempts at further chain extension using the standard conditions for glycosylation with the building block 6 proved problematic. The reaction did not proceed as desired; instead, only hydrolyzed donor resulted. Attachment of a bulky saccharide rather than a linear fluorous tag at the anomeric position likely sterically congests the 2-OH of disaccharide 8 enough to complicate subsequent chain extension.

Scheme 2.

Automated solution–phase synthesis of β-1,2-mannan 10.

To complete the synthesis of the β-1,2-mannoside, di-mannuronate 8 was reinjected into the synthesis platform reactor and treated with lithium triethylborohydride (LiTEBH).^5c The esters were reduced quantitatively to afford the β-1,2-mannan disaccharide 9 in quantitative yield. The deprotection of 9 was achieved by hydrogenolysis of the benzyl groups with catalytic amount of Pd/C, and the fully deprotected β-1,2-mannan 10 was thereby obtained in 89% yield (Scheme 2). Clearly, although further chain extension is ruled out at the 2-OH, a mannuronate building block strategy is viable for the production of β-1,2-mannan dimers.

Given the challenges in formation of β-1,2-mannans, the ability of the mannuronate building blocks to support chain extension at the next position over—the 3-OH—was explored. The automated synthesis of β-1,3-mannans began with the glycosylation of the previously reported building block 11 (3.5 equiv)^5c and F-tag 7 with TMSOTf (0.11 equiv) at −20 °C for 45 min. After this initial glycosylation of the tag, the cycle was completed by the removal of the PMB group by CAN and FSPE to give a mixture of anomers (α/β = 1/3.6), which were then separated manually on bench top to afford the β-monosaccharide 12. Although efforts in the lab are ongoing to develop standard operating protocols for the purification of products from the automation platform, a significant advantage of this solution-phase-based automation platform over solid-phase-based syntheses is the ability to remove any intermediate from the platform for analysis and/or purification without precluding the ability of the compound to undergo further automated synthesis cycles. Pure 12 was just reinjected into the automated synthesis platform for three additional cycles of glycosylation/deprotection. The 2^nd cycle gave complete conversion of monosaccharide 12 to the disaccharide based on TLC. However, TLC observation of the 3^rd cycle showed a mixture of disaccharide and trisaccharide. After the 4^th cycle, the mixture was transferred out of the synthesis platform and manually purified. Yet, instead of having the desired tetrasaccharide, the major product was the trisaccharide 13. This was possibly due to the steric hindrance from the 2,3-cis conformation of the growing β-1,3-mannan chain, which gives a lower nucleophilicity to the acceptor 3-OH. Automated synthesis, especially via this solution-phase approach, allows the rapid testing of hypotheses of the utility of various building blocks to support syntheses and can thereby also quickly identify limitations of approach as evidenced here. To prove that the mannuronate approach was viable for the production of at least shorter β-1,3-mannan oligomers, β-1,3-mannuronate trimer 13 was reinjected to the synthesis platform for the reduction of the methyl esters by LiTEBH to form β-1,3-mannan trisaccharide 14. Hydrogenolysis of 14 then delivered the fully deprotected trisaccharide 15 in 95% yield (Scheme 3).

Scheme 3.

Automated solution–phase synthesis of β-1,3-mannan 15.

In order to design a building block for the automated solution-phase synthesis of β-1,6-mannans, we decided to test the utility of the β-directing C5-ester group as a temporary protecting group for chain extension (Scheme 4). In other words, could a reduction cycle be effectively incorporated after each glycosylation step as a deprotection step or would reaction byproducts cause problems in subsequent steps? To test this strategy, the trichloroacetimidate building block 17 was synthesized from the previously reported 2,3,4-O-benzylated lactol 16.^6b The automated synthesis started with glycosylation of the trichloroacetimidate building block 17 (3.5 equiv.) with the F-tag 7 catalyzed by TMSOTf (0.11 equiv.) at −20 °C. The following deprotection of the 6-position was furnished by a hydride reduction of the methyl ester by LiTEBH followed by FSPE to afford a mixture (α/β = 1/3) of F-tag-modified anomers, which was transferred out of the automation platform and manually purified to obtain the β-anomer 18 in 67% yield over 2 steps. Pure 18 was then reinjected into the synthesis platform for the next three repeating cycles of glycosylation and deprotection. The TLC of the 2^nd and 3^rd glycosylation and deprotection steps showed completed reactions. However, after the 4^th cycle the TLC showed that the only product has the same R_f value as the starting material trisaccharide. The mixture was transferred out of the synthesis platform and purified manually, and the isolated product was the trimannoside 19 instead of the expected tetramannoside. The production of this truncated sequence could be due to the lack of reactivity of the growing glycosyl acceptor because of the possible steric hindrance from the more freely rotated β-1,6-mannan chain. Methods to predict such chain extension issues, however, remain a frontier in carbohydrate synthesis. The isolated trisaccharide 19 was deprotected by hydrogenolysis to afford the fully deprotected β-1,6-mannan trisaccharide 20 in 74% yield (Scheme 4), thereby demonstrating the utility of the approach to make at least smaller β-1,6-mannan oligomers.

Scheme 4.

Synthesis of the trichloroacetimidate building block 17 and automated solution-phase synthesis of β-1,6-mannan 20.

Clearly, the β-directing mannuronate method is general enough to support the synthesis of not only β-1,4-mannopyranoside linkages, but also the synthesis of β-1,2-, 1,3- and 1,6-mannan linkages. We have also demonstrated effective automated solution-phase synthesis protocols to synthesize short oligomers of β-1,2-, 1,3- and 1,6-mannans and identified limitations of the approach for the synthesis of large homo-oligomers. Besides the flexibility to remove fluorous-tagged intermediates for analysis and/or purification from the automation platform, the solution-phase reaction conditions require less than 4 equiv of precious glycosyl donors in each glycosylation cycle. Compared to automated solid-phase oligosaccharide synthesis protocols, which usually demands 9.0 to 10 equiv for each glycosylation step^4b,c this solution-phase strategy can greatly reduce the tremendous cost of oligosaccharide synthesis by limiting the donor loading. The ability to easily monitor reaction progress by TLC was also shown to be extremely helpful for the rapid development of automated synthesis procedures. Moreover, the fluorous tag-modified fully deprotected β-mannans 10, 15 and 20 can be directly applied to fluorous-based carbohydrate microarrays for biological activity assays as needed.^{5c, 11} We are currently investigating the potential of this β-directing mannuronate strategy to be incorporated into automated synthesis protocols for more complex β-mannoside-containing oligosaccharides.

3. Experimental section

3.1. General materials and methods

Dichloromethane (DCM) for glycosylation reactions was distilled from calcium hydride. Tetrahydrofuran (THF) was collected from PureSolv Micro solvent purification system (Innovative Technology, Inc., Amesbury, MA) before reactions. All other commercial solvents and reagents were reagent grade and used as received without further purification. Reactions were monitored by thin layer chromatography (TLC) with 250 μm Sorbent Technologies silica gel HL TLC plates. Hydrogenation reaction under 1000 psi hydrogen was operated in the Parr model 4766 general purpose vessel high pressure reactor (Parr Instrument Company, Moline, IL). Developed TLC plates were visualized by stain with p-anisaldehyde solution followed by heating on a hot plate. Flash column chromatography was performed with Zeochem ZEOprep 60 silica gel, 40–63 μm particle size. Preparative TLC was performed with Dynamic Adsorbents Prep TLC, Silica Gel, HLO, 20 cm × 20 cm F-254, 1000 micron layer. Fluorous solid-phase extraction was performed with SPE cartridges containing 2.0 g of silica gel bonded with perfluorooctylethylsilyl chains (Fluorous Technologies, Inc., Pittsburgh, PA). Automated solution phase synthesis was performed in a modified Chemspeed ASW1000 (Chemspeed, Augst, Switzerland) synthesis platform with hood, 16 reactor vials (13-mL capacity each) and heating/cooling unit (200 °C to −20 °C) machined to hold the SPE cartridges at the Iowa State University Machine Shop. ¹H and ¹³C NMR spectra were obtained at 600 MHz and 150 MHz on a Bruker Avance III 600 spectrometer, and 700 MHz on a Bruker Avance II 700 spectrometer. The C-H coupling constants were measured by the ¹H coupled ¹³C NMR spectra. Chemical shifts (δ) were reported in parts per million (ppm) relative to CDCl₃ and CD₃OD as internal references. Mass spectra were obtained on a Finnigan TSQ700 triple quadrupole mass spectrometer (Finnigan MAT, San Jose, CA) fitted with a Finnigan ESI interface.

3.2. General procedure for bench-top fluorous solid-phase extraction (FSPE)

Crude residue (less than 0.30 g) was dissolved in 1.0 mL of dimethyl sulfoxide (DMSO) and loaded onto a 80% MeOH preconditioned 2 g FSPE cartridge. The cartridge was washed with 80% MeOH (4.0 mL × 3 times). Finally, the product was eluted with acetone (12 mL). The solvent was then removed under reduce pressure to obtain the purified product.

3.3. Synthetic procedures

3.3.1. Phenyl 2-O-p-methoxybenzyl-3,4-di-O-benzyl-thio-α-D-mannopyranoside (2)

To the phenyl 2-O-p-methoxybenzyl-3-O-benzyl-4,6-O-benzylidene-thio-α-D-mannopyranoside 1 (1.4 mmol) was added BH₃·THF 1.0 M solution in THF (14 mL, 14.0 mmol) under argon atmosphere at 0 °C and stirred until the starting material was dissolved. Then the dibutylboryl trifluoromethanesulfonate 1.0 M solution in DCM (1.7 mL, 1.7 mmol) was added dropwise and the reaction mixture was stirred under argon atmosphere at 0 °C for 3 h. Triethylamine (TEA) (0.30 mL, 2.2 mmol) was added dropwise to the reaction then methanol was added slowly to quench the reaction under 0 °C. The solvent was removed under reduced pressure and the crude mixture was coevaporated with methanol twice. The crude product was purified by flash column chromatography on silica gel using EtOAc/petroleum ether (1/2) as eluent. The product was obtained as a light yellow syrup (0.69 g, 1.2 mmol, 86%). R_f: 0.14 (EtOAc/petroleum ether: 1/3). ¹H NMR (CDCl₃, 400 MHz): δ (ppm) 7.40-7.25 (m, 17H, H_arom), 6.85 (dd, 2H, J = 6.8, 2.0 Hz, H_arom), 5.47 (d, 1H, J = 1.6 Hz, H-1), 4.97 (d, 1H, J = 10.8 Hz), 4.67-4.57 (m, 5H), 4.13-4.09 (m, 1H, H-5), 4.04 (d, 1H, J = 9.2Hz), 3.99 (dd, 1H, J = 3.2, 2.0 Hz, H-2), 3.89 (dd, 1H, J = 9.2, 2.8 Hz, H-3), 3.83-3.79 (m, 5H). ¹³C NMR (CDCl₃, 100 MHz): δ (ppm) 159.4, 138.4, 138.2, 134.1, 131.8, 129.8, 129.7, 129.1, 128.4, 128.1, 127.8, 127.7, 125.6, 113.8, 86.1, 80.0, 75.8, 75.3, 74.7, 73.4, 72.1, 72.0, 62.0, 55.2. HRMS (ESI): [M + Na]⁺ calcd for C₃₄H₃₆NaO₆S⁺ 595.2125, found 595.2083

3.3.2. Phenyl 2-O-p-methoxybenzyl-3,4-di-O-benzyl-thio-α-D-mannopyranosiduronic acid (3)

To a solution of phenyl 2-O-p-methoxybenzyl-3,4-di-O-benzyl-thio-α-D-mannopyranoside 2 (0.50 g, 0.87 mmol) in DCM/water (5.8 mL/2.9 mL) were added TEMPO (30 mg, 0.19 mmol) and BAIB (0.70 g, 2.17 mmol) and stirred at room temperature. After 45 min, the mixture was diluted with DCM (10 mL) and washed with a 10% Na₂S₂O₃ solution (10 mL) and water (10 mL). The organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography on silica gel using EtOAc/petroleum ether (1/1 → 1/0) as eluent. The product was obtained as a light yellow syrup (0.39 g, 0.66 mmol, 76%). R_f: 0.11 (EtOAc/petroleum ether: 1/1). ¹H NMR (CDCl₃, 400 MHz): δ (ppm) 7.50 (s, 1H, H_arom), 7.48 (s, 1H, H_arom), 7.35-7.21 (m, 15 H, H_arom), 6.85 (d, 2H, J = 12.0 Hz, H_arom), 5.63 (d, 1H, J = 4.8 Hz, H-1), 4.74-4.46 (m, 7H), 4.22 (t, 1H, J = 7.2 Hz), 3.91 (dd, 1H, J = 4.4, 2.8 Hz, H-2), 3.81 (d, 1H, J = 3.2 Hz), 3.79 (s, 1H). ¹³C NMR (CDCl₃, 100 MHz): δ (ppm) 174.5, 159.4, 137.8, 137.7, 133.8, 131.4, 129.9, 129.8, 129.1, 128.5, 128.5, 128,0, 128.0, 127.9, 127.9, 127.4, 113.9, 84.1, 75.7, 74.3, 73.8, 72.5, 72.4, 72.0, 55.3, 29.8. HRMS (ESI): [M + Na]⁺ calcd for C₃₄H₃₄NaO₇S⁺ 609.1917, found 609.1912

3.3.3. Methyl (phenyl 2-O-p-methoxylbenzyl-3,4-di-O-benzyl-thio-α-D-mannopyranoside) uronate (4)

To a solution of phenyl 2-O-p-methoxybenzyl-3,4-di-O-benzyl-thio-α-D-mannopyranosiduronic acid 3 (0.50 g, 0.85 mmol) in anhydrous DMF (4.0 mL) were added K₂CO₃ (0.12 g, 0.85 mmol) and MeI (0.30 g, 2.1 mmol). The reaction mixture was stirred at room temperature under argon atmosphere for 6 h. The mixture was diluted with EtOAc (10 mL) and washed with water (10 mL). The aqueous portion was separated and extracted with EtOAc (2 x 10 mL). The combined organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography on silica gel using EtOAc/petroleum ether (1/3) as eluent. The product was obtained as a light yellow syrup (0.44 g, 0.74 mmol, 87%). R_f: 0.80 (EtOAc/petroleum ether: 1/1). ¹H NMR (CDCl₃, 400 MHz): δ (ppm) 7.54 (d, 2H, J = 6.4 Hz, H_arom), 7.34-7.21 (m, 15H, H_arom), 6.84 (d, 2H, J = 8.4 Hz, H_arom), 5.65 (d, 1H, J = 5.2 Hz, H-1), 4.66-4.59 (m, 4H), 4.55 (s, 2H), 4.48 (d, 1H, J = 11.6 Hz), 4.26 (t, 1H, J = 6.4 Hz, H-3), 3.90 (dd, 1H, J = 5.6, 3.2 Hz, H-2), 3.79-3.77 (m, 4H), 3.65 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ (ppm) 169.7, 159.4, 138.0, 137.9, 134.0, 131.4, 129.8, 129.8, 129.0, 128.4, 128.4, 127.9, 127.8, 127.8, 127.3, 113.8, 84.1, 76.0, 74.3, 73.7, 73.0, 72.4, 72.0, 55.5, 55.3, 55.2, 55.0, 52.2. HRMS (ESI): [M + Na]⁺ calcd for C₃₅H₃₆NaO₇S⁺ 623.2074, found 623.2069

3.3.4. Methyl (2-O-p-methoxylbenzyl-3,4-di-O-benzyl-α-D-mannopyranose) uronate (5)

To a solution of methyl (phenyl 2-O-p-methoxylbenzyl-3,4-di-O-benzyl-thio-α-D-mannopyranoside) uronate 4 (0.50 g, 0.83 mmol) in 10% water/acetone (13 mL) were added NBS (1.3 g, 7.5 mmol) and NaHCO₃ (1.4 g, 16 mmol). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was dilute with EtOAc (30 mL) and washed with a saturated NaHCO₃ solution (30 mL). The organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography on silica gel using EtOAc/petroleum ether (2/3) as eluent. The product was obtained as a pale yellow syrup (0.37 g, 0.72 mmol, 87%). R_f: 0.52 (EtOAc/petroleum ether: 1/1). ¹H NMR (CDCl₃, 400MHz): δ 7.25–7.33 (m, 12H, H_arom), 6.85 (d, 2H, J = 8.4 Hz, H_arom), 5.39 (s, 1H, H-1), 4.56–4.73 (m, 6H), 4.46 (d, 1H, J = 6.4 Hz, H-5), 4.21 (t, 1H, J = 6.8 Hz, H-4), 3.87–3.90 (dd, 1H, J = 6.4, 2.8 Hz, H-3), 3.79 (s, 3H), 3.71–3.73 (dd, 1H, J = 4.0, 3.2 Hz, H-2), 3.65 (s, 3H), 3.09 (s, 1H). ¹³C NMR (CDCl₃, 100 MHz): δ 170.3, 159.2, 138.2, 138.1, 130.3, 130.0, 129.6, 128.6, 128.4, 128.4, 128.0, 127.9, 127.8, 127.7, 127.6, 114.0, 113.8, 92.8 (J_C1-H1 = 167.6 Hz, C-1), 77.8, 75.9, 74.8, 74.0, 72.4, 72.3, 72.1, 55.3, 52.3. HRMS (ESI): [M + Na]⁺ calcd for C₂₉H₃₂NaO₈⁺ 531.1989, found 531.1988

3.3.5. Methyl (2-O-p-methoxylbenzyl-3,4-di-O-benzyl-α/β-D-mannopyranose) uronate trichloroacetimidate (6)

To a solution of methyl (2-O-p-methoxylbenzyl-3,4-di-O-benzyl-α-D-mannopyranose) uronate (0.50 g, 0.98 mmol) in DCM (35 mL) was added trichloroacetonitrile 5 (0.85 g, 5.9 mmol) at 0 °C under argon atmosphere. 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) (30 mg, 0.20 mmol) was then added and the reaction mixture was stirred at 0 °C under argon atmosphere for 3 h. The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography on silica gel using EtOAc/petroleum ether/TEA (1/1/0.1) as eluent. The product was obtained as a mixture of anomers (α/β = 4/1) (0.57 g, 0.87 mmol, 89%). R_f: 0.40 (EtOAc/petroleum ether: 1/3). ¹H NMR (CDCl₃, 400MHz): δ (ppm) 9.32 (s, 1H, NH), 8.62 (s, 1H, NH), 7.35-7.22 (m, 12H), 6.87 (d, 2H, J = 2.1 Hz), 6.39 (d, 1H, J = 2.4 Hz, Ha-1), 5.96 (d, 1H, J = 8.0 Hz, Hb-1), 4.83 (d, 1H, J = 10.8 Hz), 4.75 (d, 1H, J = 12.0 Hz), 4.66 (s, 1H), 4.63-4.48 (m, 6H), 4.39 (d, 1H, J = 8.4 Hz, Ha-4), 4.29 (t, 1H, J = 8.8, 8.0 Hz, Ha-3), 4.18 (dd, 1H, J = 4.4, 2.0 Hz), 3.91-3.84 (m, 2H), 3.81 (s, 3H), 3.71 (s, 3H), 3.62 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ (ppm) 169.4, 169.1, 160.5, 159.5, 159.0, 138.0, 138.0, 137.4, 137.2, 130.1, 129.9, 129.8, 128.7, 128.6, 128.5, 128.3, 128.1, 128.0, 127.9, 113.9, 95.8, 95.2, 90.9, 77.8, 75.9, 75.7, 75.5, 75.0, 74.3, 74.1, 73.2, 73.2, 73.1, 72.9, 72.6, 55.4, 52.7, 52.6. HRMS (ESI): [M + Na]⁺ calcd for C₃₁H₃₂Cl₃NNaO₈⁺ 674.1086, found 674.1085

3.3.6. cis-4-(1H,1H,2H,2H,3H,3H-Perfluoroundecyloxy)-2-butenyl (methyl 3,4-di-O-benzyl-2-O-(methyl 3,4-di-O-benzyl-β-D-mannopyranosyl uronate)-β-D-mannopyranoside uronate) (8)

After the second cycle (14^th step of the automated synthesis, Table S1), the mixture was transferred out of the synthesis platform and purified by flash column chromatography on silica gel using diethyl ether/benzene (1/4) as eluent. The product was obtained as a colorless syrup (25 mg, 19 μmol, 38% over 4 steps, 79% per step). R_f: 0.19 (diethyl ether/benzene = 1/4 developed for 3 times). ¹H NMR (CDCl₃, 400MHz): δ (ppm) 7.41-7.22 (m, 20H, H_arom), 5.77-5.57 (m, 2H, HC=CH), 4.95 (s, 1H, CHHPh), 4.92 (s, 1H, H-1), 4.88-4.82 (m, 3H, CHHPh), 4.69-4.66 (m, 2H, CHHPh), 4.61 (d, 1H, J = 10.8 Hz, CHHPh) 4.48-4.43 (m, 2H, CHHPh, H-2), 4.46 (s, 1H, H-1), 4.37-4.33 (m, 2H, H-2, O-CHHC=C), 4.30 (t, 1H, J = 9.2 Hz, H-4), 4.21 (dd, 1H, J = 12.8, 7.6 Hz, O-C-HHC=C), 4.11 (t, 1H, J = 9.6 Hz, H-4), 4.01 (d, 2H, J = 6.4 Hz, C=CCH₂-O), 3.87 (d, J = 9.6 Hz, H-5), 3.80 (d, J = 9.6 Hz, H-5), 3.73 (s, 3H, CO₂CH₃), 3.61 (s, 3H, CO₂CH₃), 3.58 (m, 1H, H-3), 3.54 (m, 1H, H-3), 3.49 (m, 2H, OCH₂CH₂), 2.76 (s, 1H, OH), 2.25 (m, 2H, CH₂CF₂), 1.91 (m, 2H, OCH₂CH₂). ¹³C NMR (CDCl₃, 100 MHz): δ (ppm) 168.8, 168.7, 138.4, 138.2, 138.0, 130.8, 128.7, 128.6, 128.5, 128.5, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.9, 127.7, 100.0 (J_C1-H1 = 156.0 Hz), 99.8 (J_C1-H1 = 162.9 Hz), 80.5, 79.6, 76.9, 75.4, 75.3, 75.1, 75.1, 71.0, 70.8, 70.8, 69.1, 67.7, 66.6, 64.9, 52.6, 52.4, 28.3 (t, J_C-F = 21.8 Hz), 21.0. HRMS (ESI): [M + Na]⁺ calcd for C₅₇H₅₇F₁₇NaO₁₄⁺ 1311.3369, found 1311.3357

3.3.7. cis-4-(1H,1H,2H,2H,3H,3H-Perfluoroundecyloxy)-2-butenyl (3,4-di-O-benzyl-2-O-(3,4-di-O-benzyl-β-D-mannopyranosyl)-β-D-mannopyranoside (9)

After the third cycle (19^th step of the automated synthesis, Table S2), the mixture was transferred out of the synthesis platform and washed with sat. NH₄Cl solution. The organic layer was concentrated and the purified by FSPE. The product was obtained as a colorless syrup (23 mg, 19 μmol, quant.). R_f: 0.78 (MeOH/DCM = 1/9). ¹H NMR (CDCl₃, 400MHz): δ (ppm) 7.41-7.28(m, 20H, H_arom), 5.77-5.61 (m, 2H, HC=CH), 4.92-4.80 (m, 4H, CHHPh), 4.87 (s, 1H, H-1), 4.67-4.59 (m, 4H, CHHPh), 4.47 (s, 1H, H-1), 4.39 (dd, 1H, J = 12.8, 5.6 Hz, O-CHHC=C), 4.23-4.19 (m, 3H, O-CHHC=C, 2 × H-2), 4.00 (m, 2H, C=CHCH₂O), 3.97 (t, 1H, J = 8.4 Hz, H-4), 3.89 (t, 1H, J = 9.6 Hz, H-4), 3.85 (m, 2H, 2 × H-6), 3.71 (m, 3H, H-3, 2 × H-6), 3.60 (dd, J = 9.2, 2.8 Hz), 3.50-3.44 (m, 3H, H-5, OCH₂CH₂), 3.31 (m, 1H, H-3), 3.16 (d, 1H, J = 2.4 Hz, OH), 2.52 (s, 1H, OH), 2.23 (m, 1H, CH₂CF₂), 1.99 (s, 1H, OH), 1.89 (m, 2H, OCH₂CH₂). ¹³C NMR (CDCl₃, 100 MHz): δ (ppm) 138.4, 138.3, 137.7, 130.4, 128.7, 128.6, 128.5, 128.5, 128.3, 128.2, 128.2, 128.1, 128.1, 128.0, 127.9, 127.9, 127.9, 127.8, 101.2 (J_C1-H1 = 162.0 Hz), 99.5 (J_C1-H1 = 157.2 Hz), 80.8, 80.7, 76.1, 76.0, 75.4, 74.9, 74.4, 74.2, 74.1, 71.7, 71.3, 69.0, 67.9, 66.6, 64.8, 62.3, 62.1, 28.3 (t, J_C-F = 22.3 Hz), 20.9. HRMS (ESI): [M + Na]⁺ calcd for C₅₅H₅₇F₁₇NaO₁₂⁺ 1255.3471, found 1255.3471

3.3.8. 3-(Per uorooctyl)propanyloxybutanyl (2-O-(β-D-mannopyranosyl)-β-D-mannopyranoside) (10)

cis-4-(1H,1H,2H,2H,3H,3H-perfluoroundecyloxy)-2-butenyl (3,4-di-O-benzyl-2-O-(3,4-di-O-benzyl-β-D-mannopyranosyl)-β-D-mannopyranoside 9 (30 mg, 24 μmol) was dissolved in MeOH (2.0 mL) and 10% Pd/C (10 mg) was added and stirred 20 °C under 1000 psi H₂ atmosphere. After 20 h, the mixture was filter through a small pad of silica gel and elute with MeOH. The collected solution was concentrated under reduced pressure and the product was obtained as a colorless syrup (19 mg, 22 μmol, 89%). R_f: 0.45 (MeOH/DCM = 1/3). ¹H NMR (CD₃OD, 400MHz): δ (ppm) 4.79 (s, 1H, H-1), 4.59 (s, 1H, H-1), 4.13 (d, 1H, J = 3.2 Hz, H-2), 3.99 (d, 1H, J = 3.2 Hz, H-2), 3.97 (m, 1H), 3.90 (dd, 1H, J = 7.6, 2.0 Hz, H-6), 3.88 (dd, 1H, J = 8.0, 2.4 Hz, H-6), 3.74 (t, 1H, J = 6.0 Hz, H-6), 3.69 (t, 1H, J = 6.4 Hz, H-6), 3.58-3.46 (m, 8H), 3.41 (dd, 1H, J = 9.2, 3.2 Hz, H-3), 3.23-3.15 (m, 2H, 2 × H-5), 2.33 (m, 2H, CH₂CF₂), 1.90 (m, 2H, O-CH₂CH₂), 1.68 (t, 4H). ¹³C NMR (CD₃OD, 100 MHz): δ (ppm) 102.3, 102.1, 79.1, 78.7, 78.6, 75.4, 74.6, 72.2, 71.8, 70.3, 69.1, 68.6, 62.9, 62.8, 29.2 (t, J_C-F = 22.4 Hz), 27.7, 27.6, 22.0. HRMS (ESI): [M +Na]⁺ calcd for C₂₇H₃₅F₁₇NaO₁₂⁺ 897.1749, found 897.1756

3.3.9. Methyl (cis-4-(1H,1H,2H,2H,3H,3H-Perfluoroundecyloxy)-2-butenyl 2,4-di-O-benzyl-β-D-mannopyranoside) uronate (12)

The crude product solution was transferred out of the synthesis platform after the FSPE step (29^th step of automated synthesis, Table S3) of the 1^st cycle. The solvent was removed under reduced pressure and the product was purified by flash column chromatography on silica gel using EtOAc/petroleum ether (1/3.5) as eluent. The product was obtained as a colorless syrup (72 mg, 0.078 mmol, 78% over 2 steps). R_f: 0.57 (EtOAc/petroleum ether: 1/2). ¹H NMR (CDCl₃, 400 MHz): δ (ppm) 7.35-7.23 (m, 10H, H_arom), 5.74 (m, 2H, HC=CH), 4.96 (d, 1H, J = 12.0 Hz, CHHPh), 4.77 (d, 1H, J = 11.2 Hz, CHHPh), 4.63 (s, 1H, H-1), 4.61 (d, J = 12.0 Hz, CHHPh), 4.60 (d, J = 11.2 Hz, CHHPh), 4.47 (dd, J = 12.8, 4.8 Hz, O-CHHC=C), 4.24 (dd, J = 12.8, 6.4 Hz, O-CHHC=C), 4.04 (m, 2H, C=CCH₂-O), 3.99 (t, J = 7.6 Hz, H-4), 3.90 (d, J = 8.0 Hz, H-5), 3.81 (d, J = 2.4 Hz, H-2), 3.78 (m, 1H, H-3), 3.71 (s, 3H, CO₂CH₃), 3.49 (m, 2H, O-CH₂CH₂), 2.69 (d, 1H, J = 9.6 Hz, 4-OH), 2.22 (m, 2H, CH₂CF₂), 1.88 (m, 2H, O-CH₂CH₂). ¹³C NMR (CDCl₃, 100 MHz): δ (ppm) 169.4, 138.2, 138.2, 130.6, 128.7, 128.6, 128.4, 128.1, 128.1, 128.0, 100.3 (J_C1-H1 = 154.7 Hz, C-1), 78.0, 76.0, 74.5, 74.3, 72.3, 69.0, 66.7, 65.1, 52.5, 28.4 (t, J_C-F = 22.0 Hz), 21.0. HRMS (ESI): [M + Na]⁺ calcd for C₃₆H₃₅F₁₇NaO₈⁺ 941.1953, found 941.1959

3.3.10. cis-4-(1H,1H,2H,2H,3H,3H-Perfluoroundecyloxy)-2-butenyl (methyl 2,4-di-O-benzyl-3-O-(methyl 2,4-di-O-benzyl-3-O-(methyl 2,4-di-O-benzyl-β-D-mannopyranosyl uronate)-β-D-mannopyranoside uronate)-β-D-mannopyranoside uronate) (13)

The product solution was transferred out of the synthesis platform after the FSPE step of the third cycle (42^nd step of automated synthesis, Table S4). The solvent was removed under reduced pressure and the product was purified by flash chromatography by using EtOAc/petroleum ether (1/2). Then the crude product was purified by a preparative TLC by using diethyl ether/benzene (1/5). The product was collected from the preparative TLC plate and obtained as a colorless syrup (14 mg, 11 μmol, 22% over 6 steps). R_f: 0.35 (diethyl ether/benzene: 1/5). ¹H NMR (CDCl₃, 600MHz): δ (ppm) 7.43-7.41 (m, 2H, H_arom), 7.34-7.18 (m, 28H, H_arom), 5.76 (m, 2H, HC=CH), 4.98-4.96 (m, 3H, CHHPh), 4.93 (d, 1H, J = 10.8 Hz, CHHPh), 4.90 (d, 1H, J = 12.0 Hz, CHHPh), 4.86 (d, 1H, J = 10.8 Hz, CHHPh), 4.75 (d, 1H, J = 12.6 Hz, CHHPh), 4.72 (d, 1H, J = 12.0 Hz, CHHPh), 4.61 (s, 1H, H-1), 4.59 (d, 1H, J = 4.2 Hz, CHHPh), 4.51-4.47 (m, 3H, OHHCHC=C, 2 × CHHPh), 4.44 (d, 1H, J = 10.2 Hz, CHHPh), 4.28-4.24 (m, 2H, H-5, OHHCHC=C), 4.19 (s, 1H, H-1), 4.14 (s, 1H, H-1), 4.12 (d, 1H, J = 9.6 Hz, H-4), 4.07 (d, 2H, J = 3.6 Hz, C=CHCH₂O), 3.99 (dd, 1H, J = 8.4, 3.0 Hz, H-3), 3.92-3.87 (m, 3H, H-2, 2 × H-4), 3.74-3.67 (m, 10H, H-5, 9 × OCH₃), 3.63-3.61 (m, 2H, H-2, H-5), 3.50-3.45 (m, 3H, H-3, OCH₂CH₂), 3.37 (d, 1H, J = 3.6 Hz, H-2), 2.34 (d, 1H, J = 10.2 Hz, 3-OH), 2.21 (m, 2H, CH₂CF₂), 1.90 (m, 2H, OCH₂CH₂). ¹³C NMR (CDCl₃, 150 MHz): δ (ppm) 168.9, 168.8, 168.6, 138.6, 138.4, 138.4, 138.3, 138.1, 130.7, 128.8, 128.8, 128.7, 128.7, 128.6, 128.6, 128.6, 128.6, 128.6, 128.5, 128.5, 128.5, 128.4, 128.4, 128.3, 128.3, 128.2, 128.2, 128.1, 128.1, 128.1, 128.0, 128.0, 127.8, 100.4 (J_C1-H1 = 156.3 Hz), 98.6 (J_C1-H1 = 155.3 Hz), 98.2 (J_C1-H1 = 152.9 Hz), 78.4, 77.8, 77.4, 75.3, 75.3, 75.2, 75.2, 75.2, 75.1, 74.9, 74.9, 74.6, 73.9, 73.6, 73.0, 72.4, 69.0, 66.7, 65.0, 52.6, 52.6, 52.5, 28.3 (t, J_C-F = 21.8 Hz), 21.0. HRMS (ESI): [M + Na]⁺ calcd for C₇₈H₇₉F₁₇NaO₂₀⁺ 1681.4785, found 1681.4762

3.3.11. cis-4-(1H,1H,2H,2H,3H,3H-Perfluoroundecyloxy)-2-butenyl 2,4-di-O-benzyl-3-O-(2,4-di-O-benzyl-3-O-(2,4-di-O-benzyl-β-D-mannopyranosyl)-β-D-mannopyranosyl)-β-D-mannopyranoside (14)

The product solution was transferred out of the synthesis platform after the FSPE step of the first cycle (53^rd step of the automated synthesis, Table S5). The solvent was removed under reduced pressure and the product was purified by flash chromatography by using EtOAc/petroleum ether (3/1 to 1/0). The product was obtained as a colorless syrup (10 mg, 6.3 μmol, 57%). R_f: 0.23 (EtOAc/benzene: 4/1). ¹H NMR (CDCl₃, 700 MHz): δ (ppm) 7.44 (d, 2H, J = 7.7 Hz, H_arom), 7.35-7.21 (m, 28H, H_arom), 5.77 (m, 2H, HC=CH), 5.05-4.99 (m, 4H, CHHPh), 4.95 (d, 1H, J = 11.9 Hz, CHHPh), 4.91 (d, 1H, J = 11.2 Hz, CHHPh), 4.77 (d, 2H, J = 11.2 Hz, CHHPh), 4.63 (d, 1H, J = 11.2 Hz, CHHPh), 4.56-4.48 (m, 4H, H-1, 3 × CHHPh), 4.47 (dd, 1H, J = 12.6, 3.5 Hz, OHHCHC=C), 4.34 (s, 1H, H-1), 4.26 (dd, 1H, J = 12.6, 4.9 Hz, OHHCHC=C), 4.22 (s, 1H, H-1), 4.07 (m, 2H, C=CHCH₂O), 3.97-3.89 (m, 5H, H-6), 3.86-3.76 (m, 6H, 3 × H-3), 3.70 (dd, 1H, J = 9.8, 2.8 Hz), 3.65-3.60 (m, 3H), 3.59-3.56 (m, 3H, H-6), 3.53 (s, 1H), 3.50 (m, 2H, OCH₂CH₂), 3.38 (m, 1H, H-5), 3.21 (s, 1H, H-5), 3.16 (m, 1H, H-5), 2.31 (d, 1H, J = 8.4 Hz, OH), 2.21 (m, 2H, CH₂CF₂), 2.13 (s, 1H, OH), 1.90 (m, 2H, OCH₂CH₂), 1.82 (s, 1H, OH). ¹³C NMR (CDCl₃, 150 MHz): δ (ppm) 138.8, 138.5, 138.5, 138.3, 138.2, 130.2, 128.8, 128.7, 128.7, 128.6, 128.6, 128.5, 128.4, 128.4, 128.3, 128.3, 128.3, 128.2, 128.1, 128.1, 128.0, 127.9, 127.9, 127.9, 100.5, 98.4, 97.8, 79.8, 79.1, 78.3, 76.7, 75.9, 75.6, 75.6, 75.4, 75.2, 75.0, 75.0, 75.0, 74.8, 74.6, 74.5, 74.3, 74.0, 73.8, 73.7, 69.1, 66.7, 65.1, 62.7, 62.4, 62.3, 28.3 (t, J_C-F = 22.2 Hz), 21.0. HRMS (ESI): [M + Na]⁺ calcd for C₇₅H₇₉F₁₇NaO₁₇⁺ 1597.4938, found 1597.4925

3.3.12. 3-(Per uorooctyl)propanyloxybutanyl 3-O-(3-O-(β-D-mannopyranosyl)-β-D-mannopyranosyl)-β-D-mannopyranoside (15)

cis-4-(1H,1H,2H,2H,3H,3H-perfluoroundecyloxy)-2-butenyl 2,4-di-O-benzyl-3-O-(2,4-di-O-benzyl-3-O-(2,4-di-O-benzyl-β-D-mannopyranosyl)-β-D-mannopyranosyl)-β-D-mannopyranoside 14 (10 mg, 6.3 μmol) was dissolved in MeOH/AcOH = 10/1 (2.5 mL) and 10% Pd/C (5.0 mg) and Pd black (5.0 mg) were added. The mixture was stirred under 1000 psi H₂ atmosphere at 20 °C. After 48 h, the mixture was filtered through a short pad of Celite and the solvent was removed under reduced pressure. The desired product was collected as a white foam (6.2 mg, 6.0 μmol, 95%). ¹H NMR (CD₃OD, 700 MHz): δ (ppm) 4.74 (s, 1H, H-1), 4.73 (s, 1H, H-1), 4.52 (s, 1H, H-1), 4.15 (d, 1H, J = 2.8 Hz, H-2), 4.06 (s, 1H, J = 2.8 Hz, H-2), 3.98 (s, 1H, J = 2.8 Hz, H-2), 3.96 (dd, 1H, J = 6.3, 2.8 Hz, -OCH₂CH₂CH₂CH₂O-), 3.93-3.89 (m, 3H, 3 × H-6), 3.84-3.81 (m, 2H, 2 × H-3), 3.75-3.72 (m, 2H, 2 × H-6), 3.71-3.66 (m, 3H, 2 × H-4, H-6), 3.59 (dd, 1H, J = 5.6, 3.5 Hz, -OCH₂CH₂CH₂CH₂O-), 3.56 (t, 1H, J = 9.8 Hz, H-4), 3.52 (t, 2H, J = 6.3 Hz, -OCH₂CH₂CH₂CF₂), 3.49 (m, 3H, H-3, CH₂CH₂O-), 3.28-3.24 (m, 2H, 2 × H-5), 2.29 (m, 2H, CH₂CF₂), 1.87 (m, 2H, OCH₂CH₂CH₂CF₂), 1.69 (m, 4H, - OCH₂CH₂CH₂CH₂O-). ¹³C NMR (CD₃OD, 150 MHz): δ (ppm) 101.7, 98.5, 98.1, 81.2, 81.2, 78.7, 78.3, 78.1, 75.3, 72.7, 71.9, 70.6, 70.2, 69.5, 69.3, 68.8, 67.0, 66.9, 63.1, 62.9, 29.1 (t, J_C-F = 21.9 Hz), 27.6, 27.5, 22.0. HRMS (ESI): [M + Na]⁺ calcd for C₃₃H₄₅F₁₇NaO₁₇⁺ 1059.2278, found 1059.2292

3.3.13. Methyl (2,3,4-tri-O-benzyl-α/β-D-mannopyranose) uronate trichloroacetimidate (17)

To a solution of methyl (2,3,4-tri-O-benzyl-α/β-D-mannopyranose) uronate 16 (0.50 g, 1.0 mmol) in DCM (35 mL) was added trichloroacetonitrile (0.85 g, 5.9 mmol) at 0 °C under argon atmosphere. DBU (30 μg, 0.20 mmol) was then added and the reaction mixture was stirred at 0 °C under argon atmosphere for 3 h. The solvent was removed under reduced pressure and the crude product was purified by flash column chromatography on silica gel using EtOAc/petroleum ether/TEA (1/2/0.1) as eluent. The product was obtained as a colorless syrup and a mixture of anomers (α/β = 4/1) (0.59 g, 0.93 mmol, 95%). R_f: 0.66 (EtOAc/petroleum ether: 1/2). ¹H NMR (CDCl₃, 600MHz): δ (ppm) 9.34 (s, 1H, NH_β), 8.62 (s, 1H, NH_α), 7.4 (d, 2H, J = 7.2 Hz, H_arom), 7.35-7.21 (m, 13H, H_arom), 6.41 (d, 1H, J = 2.4 Hz, H_α-1), 5.98 (d, 1H, J = 7.8 Hz, H_β-1), 4.86-4.79 (m, 2H, CHHPh), 4.74 (m, 1H, CHHPh), 4.66-4.56 (m, 3H, CHHPh), 4.54 (d, 1H, J = 11.4 Hz, H_β-4), 4.52 (d, 1H, J = 12.0 Hz CHHPh), 4.40 (d, 1H, J = 9.0 Hz, H_α-3), 4.30 (t, 1H, J = 8.4 Hz, H_α-3), 4.20 (dd, 1H, J = 4.2, 1.8 Hz, H_β-3), 3.92-3.88 (m, 3H, H_α-5, H_β-3, H_β-2), 3.87 (t, 1H, J = 3.0 Hz, H_α-2), 3.71 (s, 3H, CO₂CH_α3), 3.63 (s, 3H, CO₂CH_β3). ¹³C NMR (CDCl₃, 150 MHz): δ (ppm) 169.3, 169.0, 160.4, 158.9, 138.0, 137.9, 137.9, 137.8, 137.4, 137.1, 128.7, 128.5, 128.5, 128.5, 128.2, 128.2, 128.1, 128.1, 128.0, 128.0, 128.0, 128.0, 128.0, 127.9, 95.7, 95.1, 92.3, 90.9, 77.8, 75.8, 75.6, 75.5, 75.0, 74.3, 74.0, 73.6, 73.5, 73.2, 73.0, 72.6, 72.5, 52.6, 52.5. HRMS (ESI): [M + H]⁺ calcd for C₃₀H₃₁Cl₃NO₇⁺ 622.1161, found 622.1145

3.3.14. cis-4-(1H,1H,2H,2H,3H,3H-Perfluoroundecyloxy)-2-butenyl 2,3,4-tri-O-benzyl- β-D-mannopyranoside (18)

The product solution was transferred out of the synthesis platform after the FSPE step of the first cycle (66^th step of automated synthesis, Table S6). The solvent was removed under reduced pressure and the product was purified by preparative TLC by using diethyl ether/DCM (0.75/9). The product was obtained as a colorless syrup (66 mg, 0.067 mmol, 67% over 2 steps). R_f: 0.35 (diethyl ether/DCM: 0.75/9). ¹H NMR (CDCl₃, 600MHz): δ (ppm) 7.45 (d, 2H, J = 7.2 Hz, H_arom), 7.34-7.26 (m, 10H, H_arom), 5.75 (m, 2H, HC=CH), 4.96 (d, 2H, J = 12.0 Hz, CHHPh), 4.85 (d, 1H, J = 12.6 Hz, CHHPh), 4.65 (d, 1H, J = 10.8 Hz, CHHPh), 4.54 (d, 1H, J = 12.0 Hz, CHHPh), 4.49 (d, 1H, J = 12.0 Hz, CHHPh), 4.45 (s, 1H, H-1), 4.44 (dd, 1H, J = 12.6, 3.6 Hz, OHHCHC=C), 4.21 (dd, 1H, J = 12.6, 4.2 Hz, OHHCHC=C), 4.07 (m, 2H, C=CHCH₂O), 3.94-3.89 (m, 3H, H-2, H-4, H-6), 3.79 (m, 1H, H-6′), 3.53 (dd, 1H, J = 9.6, 3.0 Hz, H-3), 3.48 (t, 2H, J = 5.4 Hz, OCH₂CH₂), 3.33 (m, 1H, H-5), 2.22 (m, 2H, CH₂CF₂), 2.09 (t, 1H, J = 6.6 Hz, 6-OH), 1.89 (m, 2H, OCH₂CH₂). ¹³C NMR (CDCl₃, 150 MHz): δ (ppm) 138.8, 138.4, 138.3, 130.0, 128.6, 128.6, 128.5, 128.3, 128.0, 127.8, 127.7, 127.7, 100.8 (J_C1-H1 = 153.2 Hz, C-1), 82.5, 76.2, 75.5, 75.0, 74.4, 74.3, 71.8, 69.0, 66.7, 65.1, 62.7, 28.3 (t, J_C-F = 22.2 Hz), 21.0. HRMS (ESI): [M + Na]⁺ calcd for C₄₂H₄₁F₁₇NaO₇⁺ 1003.2473, found 1003.2476

3.3.15. cis- 4-(1H,1H,2H,2H,3H,3H-Perfluoroundecyloxy)-2-butenyl 2,3,4-tri- O-benzyl-6-O-(2,3,4-tri-O-benzyl-6-O-(2,3,4-tri-O-benzyl-β-D-mannopyranosyl) -β-D-mannopyranosyl) -β-D-mannopyranoside (19)

The product solution was transferred out of the synthesis platform after the FSPE step of the thrid cycle (82^nd step of automated synthesis, Table S7). The solvent was removed under reduced pressure and the product was purified by flash chromatography by using EtOAc/petroleum ether (1/1). Then the crude product was purified by a preparative TLC by using diethyl ether/DCM (1/10 developed for 3 times). The product was collected from the preparative TLC plate and obtained as a colorless syrup (11 mg, 5.7 μmol, 11% over 6 steps). R_f: 0.48 (diethyl ether/DCM: 1.5/8, TLC developed with the solvent twice). ¹H NMR (CDCl₃, 600MHz): δ (ppm) 7.43 (m, 6H, H_arom), 7.31-7.19 (m, 39H, H_arom), 5.74 (m, 2H, HC=CH), 4.92-4.87 (m, 5H, CHHPh), 4.83-4.78 (m, 4H, CHHPh), 4.61 (d, 1H, J = 10.8 Hz, CHHPh), 4.55 (d, 1H, J = 11.4 Hz, CHHPh), 4.50-4.46 (m, 2H, CHHPh), 4.43-4.34 (m, 7H, 5 × CHHPh, H-1, OHHCHC=C), 4.30 (s, 1H, H-1), 4.26 (s, 1H, H-1), 4.22 (d, 1H, J = 10.2 Hz, H-4), 4.10 (m, 2H, H-2, OHHCHC=C), 4.00 (m, 2H, C=CHCH₂O), 3.88-3.83 (m, 3H, H-2, 2 × H-6), 3.81-3.71 (m, 5H, 2 × H-2, 4 × H-6), 3.67 (m, 2H, 2 × H-3), 3.53 (dd, 1H, J = 9.0, 2.4 Hz, H-5), 3.49-3.43 (m, 3H, H-3, 2 × H-4), 3.39 (dd, 1H, J = 9.6, 3.0 Hz, H-5), 3.35 (m, 2H, OCH₂CH₂), 3.26 (m, 1H, H-5), 2.34 (t, 1H, J = 6.6 Hz, 6-OH), 2.13 (m, 2H, CH₂CF₂), 1.79 (m, 2H, OCH₂CH₂). ¹³C NMR (CDCl₃, 150 MHz): δ (ppm) 138.9, 138.8, 138.6, 138.5, 138.5, 138.3, 130.0, 128.8, 128.7, 128.6, 128.6, 128.5, 128.5, 128.3, 128.3, 128.3, 128.0, 127.8, 127.7, 127.7, 127.6, 127.5, 102.6 (J_C1-H1 = 154.2 Hz), 102.5 (J_C1-H1 = 154.2 Hz), 101.0 (J_C1-H1 = 153.6 Hz), 82.5, 82.5, 82.3, 76.1, 75.7, 75.6, 75.4, 75.1, 75.1, 75.0, 74.8, 74.1, 74.1, 74.0, 73.9, 73.7, 73.4, 71.6, 71.5, 71.5, 69.7, 69.6, 68.9, 66.8, 65.1, 62.6, 28.3 (t, J_C-F = 22.5 Hz), 21.0. HRMS (ESI): [M + Na]⁺ calcd for C₉₆H₉₇F₁₇NaO₁₇⁺ 1867.6347, found 1867.6322

3.3.16. 3-(Per uorooctyl)propanyloxybutanyl 6-O-(6-O-(β-D-mannopyranosyl)-β-D-mannopyranosyl)-β-D-mannopyranoside (20)

cis-4-(1H,1H,2H,2H,3H,3H-perfluoroundecyloxy)-2-butenyl 2,3,4-tri-O-benzyl-6-O-(2,3,4-tri-O-benzyl-6-O-(2,3,4-tri-O-benzyl-β-D-mannopyranosyl)-β-D-mannopyranosyl)-β-D-mannopyranoside 19 (11 mg, 5.7 μmol) was dissolved in MeOH/AcOH = 10/1 (2.5 mL) and 10% Pd/C (5.0 mg) and Pd black (5.0 mg) were added. The mixture was stirred under 1000 psi H₂ atmosphere at 20 °C. After 48 h, the mixture was filtered through a short pad of Celite and the solvent was removed under reduced pressure. The desired product was collected as a white foam (4.4 mg, 4.2 μmol, 74%). ¹H NMR (CD₃OD, 700 MHz): δ (ppm) 4.64 (s, 1H, H-1), 4.62 (s, 1H, H-1), 4.50 (s, 1H, H-1), 4.21 (dd, 1H, J = 11.2, 2.1 Hz, H-6), 4.17 (dd, 1H, J = 11.2, 1.4 Hz, H-6), 3.93 (d, 1H, J = 3.5 Hz, H-2), 3.92 (d, 1H, J = 2.8 Hz, H-2), 3.89-3.86 (m, 3H, -OCH₂CH₂CH₂CH₂O-, H-2, H-6), 3.83 (dd, 1H, J = 11.9, 6.3 Hz, H-6), 3.79 (dd, 1H, J = 11.2, 5.6 Hz, H-6), 3.74 (dd, 1H, J = 11.2, 5.6 Hz, H-6), 3.62-3.55 (m, 4H, 3 × H-4, - OCH₂CH₂CH₂CH₂O-, 3.53 (t, 2H, J = 6.3 Hz, -OCH₂CH₂CH₂CF₂), 3.45 (m, 2H, CH₂CH₂O-), 3.45 (m, 3H, 3 × H-3), 3.38 (m, 2H, 2 × H-5), 3.23 (m, 1H, H-5), 2.29 (m, 2H, CH₂CF₂), 1.87 (m, 2H, OCH₂CH₂CH₂CF₂), 1.68 (m, 4H, -OCH₂CH₂CH₂CH₂O-). ¹³C NMR (CD₃OD, 150 MHz): δ (ppm) 102.6, 102.3, 102.0, 78.4, 77.3, 77.3, 75.3, 75.2, 75.2, 72.5, 72.5, 72.4, 71.9, 70.7, 70.2, 70.0, 69.4, 68.6, 68.5, 68.4, 62.8, 29.1 (t, J_C-F = 22.2 Hz), 27.6, 27.6, 22.0. HRMS (ESI): [M + Na]⁺ calcd for C₃₃H₄₅F₁₇NaO₁₇⁺ 1059.2278, found 1059.2291

Highlights.

• Automated solution-phase synthesis of β-1,2-, 1,3-, and 1,6-mannopyranoside linkages with mannuronate donors

• Use of the β-directing C-5 ester of mannuronate donors as a protecting group that can support the automated synthesis of short β-1,6-mannan oligomers

• Demonstration of the removal of the soluble fluorous-tagged intermediates for analysis and/or purification and subsequent return to the automated solution-phase synthesis platform for additional reactions using fewer equivalents of glycosyl donors than related solid-phase-based protocols