Supramolecular Assembly and Chirality of Synthetic Carbohydrate Materials

Abstract Hierarchical carbohydrate architectures serve multiple roles in nature. Hardly any correlations between the carbohydrate chemical structures and the material properties are available due to the lack of standards and suitable analytic techniques. Therefore, designer carbohydrate materials remain highly unexplored, as compared to peptides and nucleic acids. A synthetic d‐glucose disaccharide, DD, was chosen as a model to explore carbohydrate materials. Microcrystal electron diffraction (MicroED), optimized for oligosaccharides, revealed that DD assembled into highly crystalline left‐handed helical fibers. The supramolecular architecture was correlated to the local crystal organization, allowing for the design of the enantiomeric right‐handed fibers, based on the l‐glucose disaccharide, LL, or flat lamellae, based on the racemic mixture. Tunable morphologies and mechanical properties suggest the potential of carbohydrate materials for nanotechnology applications.


General materials and methods
All chemicals used were reagent grade and used as supplied unless otherwise noted. Analytical thinlayer 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 staining solution (sugar stain: 10% H2SO4 in EtOH; CAM: 48 g/L ammonium molybdate, 60 g/L ceric ammonium molybdate in 6% H2SO4 aqueous 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, HSQC, and COSY 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), in D2O using the solvent as the internal standard (D2O: 4.79 ppm 1 H) or in MeOD using the solvent as the internal standard (MeOD: 4.87 ppm 1 H, 49.0 ppm 13 C). The 13 C cross-polaization/ magic angle spinning (CP/MAS) NMR spectrum was measured with a Bruker Avance II spectrometer operating at 100 MHz for 13 C. The dry powder sample was packed in a zirconia rotor with a diameter of 3 mm. The measurement was performed with a spinning speed of 12 kHz, a sweep width of 29761 Hz, a recycle delay of 2 s and a cross-polarization contact of 2 ms. The 13 C chemical shifts were calibrated with the glycine carboxyl group at 176.03 ppm. High resolution mass spectra were obtained using a 6210 ESI-TOF mass spectrometer (Agilent). ESI mass spectra were run on IonSpec Ultima instruments. IR spectra were recorded on a Perkin-Elmer 1600 ATR-FTIR spectrometer. Optical rotations were measured by using a Perkin-Elmer 241 and Unipol L1000 polarimeter. Scanning electron microscopy (SEM) images were obtained with a Gemini SEM, LEO 1550 system with cold field emission gun operation at 3 kV. All the samples were coated with Au/Pd. Atomic force microscopy (AFM) was carried out with a Multimode Nanoscope IIIa AFM. Images were attained with conventional AC mode and flattened without further modification. Qualitative imaging (QI) mode was applied for nanoindentation with a silicon cantilever. With JPK data processing software, force-distance curves were fit to the Hertz model and manipulated to obtain Young's modulus. Transition in ordered status was verified via polarized optical microscope (POM), an Olympus BX41, and X-ray diffractometer (XRD), a Bruker D8 with Cu Kα radiation. Circular dichroism (CD) spectra were acquired with a Chrascan qCD spectrometer (Applied Photophysics Ltd. Leatherhead, UK) using a quartz cuvette (Hellma GmbH & Co. KG, Mullheim, Germany) at RT with a band width of 1 nm. FT-IR spectra were recorded using a Perkin Elmer FT-IR spectrometer, Spectrum 100. Transmission electron microscopy was performed using a JEM-2100Plus (JEOL Ltd., Japan) equipped with a GATAN Rio16 CMOS camera, operated at an accelerating voltage at 200 kV. Drops of aqueous suspensions of crystallites were deposited on glow-discharged carbon-coated copper grids. All the measurements were carried out at a cryogenic temperature with an Elsa cryo-transfer holder (Gatan Inc., USA) to protect the electron sensitive DD crystals. All electron micrographs and electron diffraction patterns were recorded on a Gatan Rio 16 camera (Gatan Inc., USA). Low-dose bright-field imaging and microED (µED) measurements were achieved using the SerialEM program. The tilt-series experiments were performed with an increment angle of 0.1° and an overall rotation of about 40°. A selected area aperture with a diameter of 200 nm was inserted. A focused electron probe with a diameter of about 100 nm was used to follow the twist geometry of DD crystals. The camera length was calibrated using a powder ED pattern of evaporated aluminum. The µED patterns were analyzed using the Fiji program and in-house scripts. The tilt-series ED patterns were remapped in reciprocal space to determine the unit cell parameters (Fig. S2) The 3D cartoons in Figure 3A were made with Blender v2.82.

Synthesis
Compound DD was synthesized according to a previously reported procedure. [1] 3.1. Synthesis of L-Glc building block Scheme S1. Synthetic steps for the synthesis of L-Glc building block L-glucose 1 (4.95 g, 27.5 mmol) was slowly added (over 10 min) to a stirred solution of sodium acetate (1.9 g, 13.8 mmol) in acetic anhydride (45 mL) at 120°C. The mixture was stirred for 1 h and then cooled to RT. The reaction was quenched with ice (100 g) and diluted with EtOAc. The reaction mixture was washed five times with saturated aqueous solution of NaHCO3 and one time with brine. The crude was passed through a short plug of silica with EtOAc, dried over Na2SO4, and concentrated in vacuo. 2,3,4,6tetra-O-acetyl-1-O-acetyl-L-glucopyranoside 2 was obtained as a white solid (11.3 g, quantitative yield, α:β ratio 1:4).  3.83 (ddd, J = 9.9, 4.6, 2.2 Hz, 0.8H), 2.14 -1.98 (m, 15H). 13

Synthesis of ethyl 4,6-O-benzylidene-1-thio-β-L-glucopyranoside, 4
2,3,4,6-tetra-O-acetyl-1-O-acetyl-L-glucopyranoside 2 (6.78 g, 17.3 mmol) was dissolved in MeOH (60 mL) and a 0.5 M solution of MeONa in MeOH (7.0 mL, 3.5 mmol) was slowly added. The mixture was stirred at RT for 1 h. The reaction was neutralized with Amberlite IR-120 (H + form), filtered, and concentrated under reduced pressure. The product was used in the next step without any further purification assuming quantitative conversion. The crude product was dissolved in DMF and a catalytic amount of p-toluensulfonic acid monohydrate (356 mg, 1.7 mmol) was added to the mixture. Benzaldehyde dimethyl acetal (5.2 mL, 34.6 mmol) was then added dropwise at RT to the stirred solution. The reaction was heated to 45°C overnight, after which time it was quenched with triethylamine (2 mL) at 0 °C. The reaction mixture was diluted with EtOAc and washed three times with saturated aqueous solution of NaHCO3 and once with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The sticky oil was recrystallized from hexane:EtOAc to yield 4 as a white solid (4.65 g, 86% over 2 steps).

Self-Assembly
A) Solvent-switch method (s): Stock solutions of the disaccharides (20, 40 and 100 mg mL -1 ) in HFIP were diluted with water to reach a final concentration of 2 mg mL -1 with different ratio of water and HFIP. The temperature was controlled with an oil bath. When not mentioned, the conditions are 2 % HFIP in water at room temperature. The samples were incubated for 3 days without agitation before the measurement. Table S1. Summary of the samples prepared by the solvent-switch method (s). ) were drop casted and dried on the substrate for 12 h. The dried film was transferred into a humidity chamber and observed at different time scale. When not mentioned, the concentration is 100 mg mL -1 . Table S2. Summary of the samples prepared by the film-rehydration method (fr).   Figure S4. Energy minimized computational model of DD. The initial conformation of the structure was constructed with tleap. The topology was converted to gromacs format using the glycam2gmx.pl script and solvated with 2100 water molecules (TIP5P [2] ) using gromacs tools. [3] 7. Morphological analysis at different T

2D self-assembly
The samples were prepare as described in TableS2 (Section 3). Figure S14. SEM images showing morphology transition on a thin film of Compound DD. Compound DD in HFIP generated a continuous film by simple evaporation (left). The film is highly hydrophobic. Upon direct contact with water, a fibrous structure is generated (middle). Similarly, the film incubated in a humidity chamber shows the transition to a crystalline structure (right, DD(fr)). Figure S15. XRD profiles of DD(s) (red), DD(fr) (t = 0, black) and DD(fr) (t = 3 h, blue). The sample was directly prepared on the XRD sample holder, zero diffraction silicon. The DD(fr) (t = 0) is amorphous, showing a low intensity broad peak between 5° and 25°. Upon rehydration DD(fr) (t = 3 h) develops sharp peaks, identical to the peaks observed for the sample obtained upon solvent switch (DD(s)).     showing HFIP depletion upon incubation in water vapor. [4] The spectrum recorded between 998 and 1100 cm -1 in (D), highlighted with a red box, develop sharp peaks as an indication of molecular reorganization. Upon incubation of DD(fr) in HFIP vapor, the crystallized film returned to the amorphous state. The IR spectra (E) is identical to the one for DD(fr) at time 0, (C), showing the reversibility of the process.