Systematic Structural Characterization of Chitooligosaccharides Enabled by Automated Glycan Assembly

Abstract Chitin, a polymer composed of β(1–4)‐linked N‐acetyl‐glucosamine monomers, and its partially deacetylated analogue chitosan, are abundant biopolymers with outstanding mechanical as well as elastic properties. Their degradation products, chitooligosaccharides (COS), can trigger the innate immune response in humans and plants. Both material and biological properties are dependent on polymer length, acetylation, as well as the pH. Without well‐defined samples, a complete molecular description of these factors is still missing. Automated glycan assembly (AGA) enabled rapid access to synthetic well‐defined COS. Chitin‐cellulose hybrid oligomers were prepared as important tools for a systematic structural analysis. Intramolecular interactions, identified by molecular dynamics simulations and NMR analysis, underscore the importance of the chitosan amino group for the stabilization of specific geometries.


General Materials and Methods
All chemicals used were reagent grade and used as supplied unless otherwise noted. The automated syntheses were performed on a home-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 reverse 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 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) or in CD3OD using the solvent as the internal standard in 1 H NMR (CD3OD: 4.87 ppm 1 H, 49.0 ppm 13 C). 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. IR spectra were recorded on a Perkin-Elmer 1600 FTIR spectrometer. Optical rotations were measured by using a Perkin-Elmer 241 and Unipol L1000 polarimeter. For XRD measurements, a Bruker D8 Advanced X-ray diffractometer with Cu Kα radiation was used.

Synthesis of Building Blocks
BB1a and BB3 were obtained from commercial sources, while photocleavable linkers 4 and 6 were prepared according to literature. [1]

General Materials and Methods for AGA
All solvents used were HPLC-grade. The solvents used for the building block, activator, TMSOTf and capping solutions were taken from an anhydrous solvent system (jcmeyer-solvent systems). The building blocks were co-evaporated three times with toluene and dried for 1 h under high vacuum before use. Activator, capping, deprotection, acidic wash and building block solutions were freshly prepared and kept under argon during the automation run. All yields of products obtained by AGA were calculated on the basis of resin loading. Resin loading was determined following previously established procedures. [2] 3.2 Preparation of stock solutions  Building Block: 0.06 mmol (5 equiv.) of glycosyl phosphate or 0.08 mmol (6.5 equiv.) of thioglycoside donor was dissolved in DCM (1 mL).
 Activator solution: 1.35 g of recrystallized NIS was dissolved in 40 mL of a 2:1 mixture of anhydrous DCM and anhydrous dioxane. Then triflic acid (55 μL) was added. The solution is kept at 0°C for the duration of the automation run.
 Fmoc deprotection solution: A solution of 20% piperidine in DMF (v/v) was prepared.
 Capping solution: A solution of 10% acetic anhydride and 2% methanesulfunic acid in DCM (v/v) was prepared.

Modules for automated synthesis Module A: Resin Preparation for Synthesis (20 min)
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. During this time, all reagent lines needed for the synthesis were washed and primed. Before the first glycosylation, the resin was washed with the DMF, THF, and DCM (three times each with 2 mL for 25 s).

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

Module C: Thioglycoside Glycosylation (35 min)
The building block solution (0.08 mmol of BB in 1 mL of DCM per glycosylation) was delivered to the reaction vessel. After the set temperature was reached, the reaction was started by drop wise addition of the activator solution (1.0 mL, excess). The glycosylation conditions are building block dependent (we report the most common set of conditions). After completion of the reaction, the solution is drained and the resin washed with DCM (2 mL), DCM:dioxane (1:2, 3 mL for 20 s) and DCM (two times, each with 2 mL for 25 s). The temperature of the reaction vessel is increased to 25 °C for the next module.

Action Cycles Solution Amount T (°C) Incubation time
Cooling ----20 -Deliver The building block solution (0.06 mmol of BB in 1 mL of DCM per glycosylation) was delivered to the reaction vessel. After the set temperature was reached, the reaction was started by drop wise addition of the TMSOTf solution (1.0 mL, excess). The glycosylation conditions are building block dependent (we report the most common set of conditions). After completion of the reaction, the solution is drained and the resin washed with 2 mL DCE (2 mL) and with DCM (2 x 2 mL for 20 s). The temperature of the reaction vessel is increased to 25 °C for the next module. *Time required to reach the desired temperature.

Module D: Capping (30 min)
The resin was washed with DMF (two times with 2 mL for 25 s) and the temperature of the reaction vessel was adjusted to 25 °C. 2 mL of Pyridine solution (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). 4 mL of capping solution 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 (9 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. 2 mL of Fmoc deprotection solution 1 was delivered to 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.

Post-synthesizer manipulations Module F: On-resin Methanolysis
The resin was suspended THF (5 mL). 0.5 mL of NaOMe in MeOH (0.5 M) was added and the suspension was gently shaken at room temperature. After micro-cleavage (see Module G1) indicated the complete removal of benzoyl groups (generally around 4 hours), the resin was repeatedly washed with MeOH (2mL x 3) and DCM (2mL x 3).

Module G: Cleavage from Solid Support
The oligosaccharides were cleaved from the solid support using a continuous-flow photoreactor as described previously. [1b] Module G*: Micro-cleavage from Solid Support Trace amount of resin (around 20 beads) was dispersed in DCM (0.1 mL) and irradiated with a UV lamp (6 watt, 356 nm) for 10 minutes. ACN (10 µL) was then added to the resin and the resulting solution analyzed by MALDI.

Module H: Hydrogenolysis at ambient pressure
The crude compound obtained from Module H was dissolved in 2 mL of EA:tBuOH:H2O (1:0.5:0.5). 100% by weight Pd-C (10%) was added and the reaction was stirred under H2-atmosphere overnight. The reaction was filtered through celite and washed with EA, tBuOH and H2O with a 0.1% of formic acid. The filtrates were concentrated in vacuo.
Following final purification, all deprotected products were lyophilized on a Christ Alpha 2-4 LD plus freeze dryer prior to characterization.

XRD Analysis
X-ray diffraction experiments were carried out using a D8 Avance diffractometer (Bruker) in reflection mode with monochromatic Cu Kα radiation (λ= 1.5418 Å) generated at 40 kV and 40 mA (Siemens X-ray tube KFL CU 2K). The scans were performed in the scattering angle range between 4°and 40°with a step of 0.02°and an accumulation time of 6 s. The oligosaccharide samples were lyophilized prior to XRD measurement. Raw XRD profiles were corrected by subtraction of the sample holder signal, smoothing and baseline correction. Figure S6: Powder XRD profiles of synthetic COS and hybrid chitin-cellulose oligomers. The XRD profiles obtained for the polysaccharide α-chitin and cellulose II are reported for comparison. XRD profiles for A6 and N6 were previously reported. [4] *Crystalline carbonate peak resulting from the prolonged exposure of the polyamino compound to air. [5] Definition of dihedrals exemplified for a β-1,4-glucose disaccharide (The atoms in red belong to the following residue) Figure S7: MD characterization of the hexaglucoside A6 (for comparison). [4] Representative snapshot Radius of gyration Ramachandran plot End-to-End Distance Omega dihedrals superimposed R1 R2 R3 R4 R5 R6 71 Figure S8: End-to-end distance as a function of time.