Total Synthesis of Polysaccharides by Automated Glycan Assembly

Polysaccharides are the most abundant biopolymers on earth that serve various structural and modulatory functions. Pure, completely defined linear and branched polysaccharides are essential to understand carbohydrate structure and function. Polysaccharide isolation provides heterogeneous mixtures, while heroic efforts were required to complete chemical and/or enzymatic syntheses of polysaccharides as long 92-mers. Here, we show that automated glycan assembly (AGA) enables access to a 100-mer polysaccharide via a 201-step synthesis within 188 h. Convergent block coupling of 30- and 31-mer oligosaccharide fragments, prepared by AGA, yielded a multiple-branched 151-mer polymannoside. Quick access to polysaccharides provides the basis for future material science applications of carbohydrates.


General Materials and Methods
All chemicals used were reagent grade and used as supplied unless otherwise noted. All building blocks used were purchased from GlycoUniverse, Germany. Automated syntheses were performed on a home-built synthesizer developed at the Max Planck Institute of Colloids and Interfaces. 1 Merrifield resin LL (100-200 mesh, Novabiochem TM ) was modified and used as solid support. 2 Analytical thin-layer chromatography (TLC) was performed on Merck silica gel 60 F254 plates (0.25 mm). Compounds were visualized by UV irradiation or dipping the plate in a p-anisaldehyde (PAA) solution. Flash column chromatography was carried out by using forced flow of the indicated solvent on Fluka Kieselgel 60 M (0.04 -0.063 mm). Analysis and purification by normal and reverse phase HPLC was performed using an Agilent 1200 series. Products were lyophilized using a Christ Alpha 2-4 LD plus freeze dryer. 1 H, 13 C and HSQC NMR spectra were recorded on a Varian 400-MR (400 MHz), Varian 600-MR (600 MHz), or Bruker Biospin AVANCE700 (700 MHz) spectrometer. Spectra were recorded in CDCl3 by using the solvent residual peak chemical shift as the internal standard (CDCl3: 7.26 ppm 1 H, 77.0 ppm 13 C) or in D2O using the solvent as the internal standard in 1 H NMR (D2O: 4.79 ppm 1 H) and a D6-acetone spike as the internal standard in 13 C NMR (acetone in D2O: 30.89 ppm 13 C) unless otherwise stated. High resolution mass spectra were obtained using a 6210 ESI-TOF mass spectrometer (Agilent) and a MALDI-TOF Autoflex TM (Bruker). MALDI and ESI mass spectra were run on IonSpec Ultima instruments.
Solvents used for dissolving building block and preparing the activator, TMSOTf and capping solutions were taken from an anhydrous solvent system (jcmeyer-solvent systems). Other solvents used were HPLC grade. The building blocks were co-evaporated three times with toluene and dried 2 h under high vacuum before use. Activator, deprotection, acidic wash, capping and building block solutions were freshly prepared and kept under argon during the automation run. All yields of products obtained by AGA were calculated based on resin loading. Resin loading was determined by performing one glycosylation (Module C) with ten equivalents of building block followed by DBU promoted Fmoc-cleavage and determination of dibenzofulvene production by measuring its UV absorbance.
• Activator solution: Recrystallized NIS (1.56 g) was dissolved in 60 mL of a 2:1 mixture of anhydrous DCM and anhydrous dioxane. Then triflic acid (67 μL) was added. The solution is kept at 0°C for the duration of the automation run.

Module A: Resin Preparation for Synthesis (20 min)
All automated syntheses were performed on 19 µmol scale (60 mg). Resin was placed in the reaction vessel and swollen in DCM for 20 min at room temperature prior to synthesis. During this time, all reagent lines required for the synthesis were washed and primed. Before the first glycosylation, the resin was washed with the DMF, tetrahydrofuran (THF), and DCM (three times each with 2 mL for 25 s). This step is conducted as the first step for every synthesis.

Module B: Acidic Wash with TMSOTf Solution (20 min)
The resin was swollen in DCM (2 mL) and the temperature of the reaction vessel was adjusted to -20 °C. Upon reaching the temperature, TMSOTf solution (1 mL) was added drop wise to the reaction vessel. After bubbling for argon 3 min, the acidic solution was drained and the resin was washed with 2 mL DCM for 25 s.

Module C: Thioglycoside Glycosylation (20-60 min)
The building block solution (0.095-0.123 mmol (5-6.5 equivalents) of BB in 1 mL of DCM per glycosylation) was delivered to the reaction vessel. After the set temperature (-20 o C) was reached, the reaction was started by drop wise addition of the activator solution (1.0 mL, excess). The glycosylation was performed by increasing the temperature to 0 o C for 20-60 min (depending on oligosaccharide length). After completion of the reaction, the solution is drained and the resin was washed with DCM, DCM:dioxane (1:2, 3 mL for 20 s) and DCM (twice, each with 2 mL for 25 s). The temperature of the reaction vessel is increased to 25 °C for the next module.

Module D: Capping (30 min)
The resin was washed with DMF (twice with 2 mL for 25 s) and the temperature of the reaction vessel was adjusted to 25 °C. Pyridine solution (2 mL, 10% in DMF) was delivered into the reaction vessel. After 1 min, the reaction solution was drained and the resin washed with DCM (three times with 3 mL for 25 s). The capping solution (4 mL) was delivered into the reaction vessel. After 20 min, the reaction solution was drained and the resin washed with DCM (three times with 3 mL for 25 s).

Module E: Fmoc Deprotection (14 min)
The resin was washed with DMF (three times with 2 mL for 25 s) and the temperature of the reaction vessel was adjusted to 25 °C. Fmoc deprotection solution (2 mL) was delivered into the reaction vessel. After 5 min, the reaction solution was drained and the resin washed with DMF (three times with 3 mL for 25 s) and DCM (five times each with 2 mL for 25 s). The temperature of the reaction vessel is decreased to -20 °C for the next module.

Module F: Lev Deprotection (ca. 100 min)
The resin was washed with DMF (3×30 sec) and DCM (1.3 mL) added to the reaction vessel. Solution F (0.8 mL) was added to the reaction vessel, and the temperature was adjusted to 25 °C. After 30 min, the reaction solution was drained and the entire cycle was repeated twice more. After Lev deprotection was complete, the resin was washed with DMF, THF and DCM.

Cleavage from Solid Support
After automated synthesis, the oligosaccharides were cleaved from the solid support using a continuous-flow photo reactor as described previously. [4] Purification Solvent was evaporated in vacuo and the crude products were dissolved in a 1:1 mixture of hexane and ethyl acetate and analyzed using analytical HPLC (DAD1F, 280 nm). Pure compounds were afforded by preparative HPLC (Agilent 1200 Series spectrometer).

Synthesis of 30-mer glycosyl trichloroacetimidate donor 7:
To a solution of compound 6 (60 mg, 4 mmol) in CH2Cl2 (2 mL) was added DBU (11 µL, 8 mmol) at -10 °C, to this mixture trichloroacetonitrile (1 mL) was added and allowed to stirred for two hours before the reaction mixture was quenched with water. The organic phase was extracted with NaHCO3 solution, dried over MgSO4, filtered, concentrated and purified by preparative HPLC (Method F) to provide compound 7 (33 mg, 55% yield).

S-25
Synthesis of 30 mer glycosyl fluoride donor 8: To a solution of compound 6 (94 mg, 72.8 µmol) in CH2Cl2 (2 mL) was added a solution of Deoxo-Fluor (110 µL, 150 µmol) at -30 °C. The reaction was stirred for 30 minutes. The reaction was quenched with saturated NaHCO3 solution (2 mL  The product was cleaved from the solid support as described in the post-synthesizer manipulations followed by purification by using normal phase preparative HPLC with YMC diol column (Method F) Linear gradient: Hex -35% EtOAc as eluents [isocratic 35% EtOAc (5 min),

Synthesis of Branched 151-mer Polymannoside 11 via Block Coupling
To a solution of glycosyl donor 8 (41 mg, 6 eq) and glycosyl acceptor 10 (7 mg, 1 eq) in 1mL of anhydrous CH2Cl2 was added activated 4Å molecular sieves (20 mg) at room temperature for 5 min protected from light. The reaction was cooled to -40 °C and silver perchlorate (6.2 mg, 10 eq) (WARNING: dry perchlorates are explosive!!) and bis(cyclopentadienyl) hafnium dichloride (11 mg, 10 eq) were added. [5] This mixture was allowed to stir at same temperature for 30 minutes to complete the reaction. This mixture was quenched with Et3N diluted with CH2Cl2 and filtered through Celite. The filtrate was extracted with (10 mL) aqueous NaHCO3 and (10 mL) brine. The organic layer was dried over MgSO4 filtered, concentrated and purified by preparative HPLC (Method H) with a YMC diol column. Pure compound 11 was isolated 27 mg in 78% yield.