Elucidating the Ordering in Self-Assembled Glycocalyx Mimicking Supramolecular Copolymers in Water

Abstract

Polysaccharides present in the glycocalyx and extracellular matrix are highly important for a multitude of functions. Oligo- and polysaccharides-based biomaterials are being developed to mimic the glycocalyx, but the spatial functionalization of these polysaccharides represents a major challenge. In this paper, a series of benzene-1,3,5-tricarboxamide (BTA) based supramolecular monomers is designed and synthesized with mono- (BTA-β-d-glucose; BTA-Glc and BTA-α-d-mannose; BTA-Man) or disaccharides (BTA-β-d-cellobiose; BTA-Cel) at their periphery or a monosaccharide (BTA-OEG₄-α-d-mannose; BTA-OEG₄-Man) at the end of a tetraethylene glycol linker. These glycosylated BTAs have been used to generate supramolecular assemblies and it is shown that the nature of the carbohydrate appendage is crucial for the supramolecular (co)polymerization behavior. BTA-Glc and BTA-Man are shown to assemble into micrometers long 1D (bundled) fibers with opposite helicities, whereas BTA-Cel and BTA-OEG₄-Man formed small spherical micelles. The latter two monomers are used in a copolymerization approach with BTA-Glc, BTA-Man, or ethylene glycol BTA (BTA-OEG₄) to give 1D fibers with BTA-Cel or BTA-OEG₄-Man incorporated. Consequently, the carbohydrate appendage influences both the assembly behavior and the internal order. Using this approach it is possible to create 1D-fibers with adjustable saccharide densities exhibiting tailored dynamic exchange profiles. Furthermore, hydrogels with tunable mechanical properties can be achieved, opening up possibilities for the development of multicomponent functional biomaterials.

Scheme 1. Synthesis Pathways of the BTA-Saccharides

(A) BTA-Glc, BTA-Man, and BTA-Cel were synthesized by reacting BTA-C12-OH with participating imidate donors followed by deprotection with NaOMe. (B) BTA-OEG₄-Man was synthesized through glycosylation with 2,3,4,6-tetra-O-benzoyl-α-d-mannopyranosyl trichloroacetimidate to BTA-OEG₄ and followed by deprotection with NaOMe.

Figure 1

UV (A–C) and CD (D–F) spectra of the assembled individual molecules in water. (A) BTA-Glc and BTA-Man in water show the typical BTA maxima at 211 and 225 nm, whereas BTA-Cel and BTA-OEG₄-Man show maxima at 196 nm. (B) and (C) Stepwise heating of BTA-Glc and BTA-Man, respectively, from 20 °C to 80 °C showed a change in aggregation between 60 °C and 70 °C for both of BTA-Glc and BTA-Man. (D) BTA-Glc and BTA-Man show a biphasic negative and positive mirror Cotton effect, whereas BTA-Cel and BTA-OEG₄-Man are CD silent. (E) and (F) Stepwise heating of BTA-Glc and BTA-Man, respectively, from 20 to 80 °C showed a drop in ellipticity between 60 °C and 80 °C. Samples were equilibrated for 16 min at the designated temperature to allow the formation of equilibrated aggregates. (c_BTA = 50 μM).

Figure 2

Cryo-TEM of BTA-Glc (A), BTA-Man (B), BTA-Cel (C), and BTA-OEG₄-Man (D). Micrometers long fibrous structures were observed for BTA-Glc and BTA-Man while micellar structures for BTA-Cel and BTA-OEG₄-Man. Scale bars indicate 50 nm, c_BTA = 250 or 500 μM.

Figure 3

UV (top) and CD (bottom) spectra of coassembled BTAs in water at 20 °C. (A) and (D) BTA-Glc coassembled with BTA-Cel. (B) and (E) BTA-Glc coassembled with BTA-OEG₄. (C) and (F) coassembly of BTA-Cel with BTA-OEG₄. CD spectra of BTA-OEG₄ were discarded due to a large LD effect. Note that the 1:2 Glc:OEG₄ mixture displayed a small negative LD effect. (c_BTA, total = 50 μM).

Figure 4

Cryo-TEM of 1:1 mixtures. (A) Glc:Cel, (B) Glc:OEG₄, (C) Cel:OEG₄, (D) OEG₄-Man:OEG₄, (E) Man:OEG₄, and (F) Man:OEG₄-Man. (C) TEM without staining. Fibrous structures and micelles were observed. c_BTA = 500 μM.

Figure 5

HDX-MS curves of homopolymers and copolymers (1:1 ratio) after 100 times dilution into D₂O. The graphs highlight the amount of remaining unexchanged monomers (BTA3NH) as a function of time. (A) BTA-OEG₄-Man coassembled with BTA-OEG₄, (B) BTA-Man with BTA-OEG₄, and (C) BTA-OEG₄-Man and BTA-Man. The data was fitted with a triexponential fit. c_BTA = 5 μM.

Figure 6

Influence of purities on BTA-Glc assembly. A–C indicates UV spectra and D–F CD spectra. (A) and (D) pure BTA-Glc showing a transition between 60 and 70 °C. (B) and (E) 85% BTA-3Glc mixed with 15% BTA-2Glc showing disassembly at 80 °C. (C) and (F) BTA-2Glc revealing a stability beyond 80 °C. Samples were equilibrated for 16 min at the designated temperature to allow the formation of equilibrated aggregates. (c_BTA = 50 μM).

Figure 7

Rheology measurements of 5 wt % hydrogels formed by BTA-Man: BTA-OEG₄ (1:2, HG1, A,D), BTA-OEG₄-Man: BTA-OEG₄ (1:2, HG2, B,E), and BTA-Glc: BTA-OEG₄ (1:2 HG3, C,F) at 37 °C showing the storage and loss moduli (G′, G′′). (A–C) Strain dependent oscillatory rheology (fixed angular frequency of 1 rad/s) with a photograph of the inverted vial containing the corresponding hydrogel in the inset. (D–F) Frequency sweep measurements (fixed applied strain of 1%).