Automated glycan assembly of arabinomannan oligosaccharides from Mycobacterium tuberculosis

Arabinomannan (AM) polysaccharides are clinical biomarkers for Mycobacterium tuberculosis (MTB) infections due to their roles in the interaction with host cells and interference with macrophage activation. Collections of defined AM oligosaccharides can help to improve the understanding of these polysaccharides and the development of novel therapeutical and diagnostic agents. Automated glycan assembly (AGA) was employed to prepare the core structure of AM from MTB, containing α-(1,6)-Man, α-(1,5)-Ara, and α-(1,2)-Man linkages. The introduction of a capping step after each glycosylation and further optimized reaction conditions allowed for the synthesis of a series of oligosaccharides, ranging from hexa- to branched dodecasaccharides.


General methods
All chemicals were reagent grade and used as supplied unless otherwise noted. The automated syntheses were performed on a self-built synthesizer developed at the Max Planck Institute of Colloids and Interfaces. 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 reversed-phase HPLC was performed by 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 chemical shifts () are reported in ppm (relative to the resonance of the solvent) on a Varian 400-MR (400 MHz), Varian 600-MR (600 MHz), or Bruker Biospin AVANCE700 (700 MHz) spectrometer. Residual solvent peaks were used as internal standard (CDCl3: 7.26 ppm for 1 H and 77.1 ppm for 13 C; D2O: 4.79 ppm for 1 H). Highresolution mass spectra were obtained using a 6210 ESI-TOF mass spectrometer (Agilent) and MALDI-TOF autoflexTM (Bruker) instruments. IR spectra were recorded on a PerkinElmer 1600 FTIR spectrometer. Optical rotations were measured by using a PerkinElmer 241 and Unipol L1000 polarimter, with concentrations expressed in g/100 mL. The loading determination of functionalized resins was obtained using a Shimadzu UV-MINI-1240 spectrometer.

Materials and measurements
Solvents used for dissolving all building blocks and making AGA solutions were taken from an anhydrous solvent system (jcmeyer phoenix solvent drying system). Solvents for washing were HPLC grade. The building blocks were coevaporated three times with anhydrous toluene and dried for 1 h under high vacuum prior to use. All solution bottles were freshly prepared and kept under argon during the automation process.
Isolated yields of products were calculated on the basis of resin loading. Resin loading was determined following a reported protocol [1]. Briefly, functionalized resin (40 mg) was treated with one glycosylation cycle using 65 mg of BB A (excess), followed by DBU-promoted Fmoc cleavage and determination of dibenzofulvene concentration by UV absorbance. All automated syntheses were performed on a 0.0125 mmol scale.
Resin was placed in the reaction vessel and was swollen in DCM for 20 min at room temperature before starting the first module. During this time, all reagent lines involved in the synthesis were washed and primed.

Modules for automated synthesis Module A: Preparation of resin for synthesis
All automated syntheses were performed on 0.0125 mmol scale. Resin was placed in the reaction vessel and swollen in DCM for 20 min at room temperature prior to synthesis. Before the first glycosylation, the resin was washed with the DMF, THF, and DCM (three times with 2 mL for 25 s each).

Module B: Acidic wash
The resin was swollen in 2 mL DCM and 1 mL of solution B (TMSOTf) was delivered to the reaction vessel. After 3 min at −20 °C, the solution was drained and the resin was washed with 2 mL DCM for 25 s.

Module C1: Glycosylation
The thioglycoside building block solution (6.5 equiv, 0.08 mmol in 1.0 mL DCM) was delivered to the reaction vessel and the temperature was adjusted to −20 °C. After the set temperature was reached, the reaction was started by the addition of the activator solution C. The glycosylation was performed for 5 min at −20 °C and for 20 min at 0 °C. After the glycosylation, the solution was drained and the resin was washed with DCM, DCM:dioxane (1:2, v/v, 3 mL for 20 s) and DCM (2 × 2 mL for 25 s).

Module C2: Glycosylation
The thioglycoside building block solution (6.5 equiv, 0.08 mmol in 1.0 mL DCM) was delivered to the reaction vessel and the temperature was adjusted to −40 °C. After the set temperature was reached, the reaction was started by the addition of the activator solution C. The glycosylation was performed for 5 min at −40 °C and for 30 min at −20 °C. After the glycosylation, the solution was drained and the resin was washed with DCM, DCM:dioxane (1:2, v/v, 3 mL for 20 s) and DCM (two times, each with 2 mL for 25 s).

Module D: Fmoc deprotection
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).

Module E: Lev deprotection
The resin was washed with DMF (3 × 30 min) and 1.3 mL DCM was added to the reaction vessel. Solution E (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 whole cycle was repeated two more times. After Lev deprotection was complete, the resin was washed with DMF, THF, and DCM.

Module F: Capping
The resin was washed with DMF (two times with 2 mL for 25 s) and the temperature was adjusted to 25 °C. 2 mL of solution G was delivered into the reaction vessel. After 1 min, the reaction solution was drained and the resin was washed with DCM (three times with 3 mL for 25 s). 4 mL of capping solution F 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) [6].

Post-synthesizer manipulations
Cleavage from the solid support: The oligosaccharides were cleaved from the solid support using the Vapourtec E-Series UV-150 photoreactor as reported previously [7].
Purification: The crude products were analyzed and purified using analytical and preparative HPLC (Agilent 1200 Series spectrometer).
 resin, filtered and concentrated in vacuo. The crude compound was used for hydrogenolysis without further purification.

Module H: Hydrogenolysis with Pd/C
The crude compound obtained from module G was dissolved in 2 mL of DCM:t-BuOH:H2O (2:1:1, v/v/v), and Pd/C (10%) was added. The reaction was stirred in a H2 bomb with 60 psi pressure for 16 h. The reaction was filtered, washed with DCM, t-BuOH, and H2O. The filtrates were concentrated in vacuo.