Introduction: Orthogonal ligation strategies to post-functionalize material surfaces in order to immobilize compounds have gained interest in recent years due to versatile applications in biomedical engineering and materials science[1]-[4]. In particular, the catalyst-free inverse electron demanding Diels-Alder (iEDDA) reaction between 1,2,4,5-tetrazines as electron deficient dienes and trans-cyclooctenes (TCO) as strained electron rich dienophiles has emerged as a compelling advancement in the field[5]-[10]. Our group designs and synthesizes supramolecular materials based on the 2-ureido-4[1H]-pyrimidinone (UPy) quadruple hydrogen bonding motif, where UPy-modified guest molecules can be easily incorporated via simply mixing the UPy-host polymer and UPy-guest molecules in solution. These are proposed to co-assemble into supramolecular nanofibers via a modular approach and can be processed into drop cast biomaterial films[11],[12]. Here, we propose a new strategy to covalently immobilize biomolecules at the surface of supramolecular materials via orthogonal tetrazine – TCO ligation and verify this with diverse physical chemical characterization techniques. This approach allows for the immobilization of bioactive molecules as well as complex functional proteins at the surface of our biomaterials, en route to meet nature’s complexity.
Materials and Methods: As supramolecular base material polycaprolactone telechelically modified with UPy moieties was used (PCLdiUPy). UPy-modified tetrazine (UPy-Tz) moieties that can be incorporated into our materials were synthesized, commenced with the synthesis of a UPy-carboxylic acid functionalized synthon, where the last step comprised of reaction of the Tz with the UPy-synthon. Both a TCO-modified model compound as well as a TCO-modified protein were synthesized and subsequently reacted at the surface-water interface, enabling surface functionalization. Surfaces were characterized using different techniques, including X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), water contact angle measurements, surface matrix assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), 3D time-of-flight secondary ion mass spectrometry (3D TOF-SIMS) and fluorescence spectroscopy.
Results and Discussion: XPS measurements have shown that UPy-Tz is preferentially present at the surface of the supramolecular films. Moreover, upon click reaction with a TCO-model compound equipped with an iodine atom, a significant increase in iodine signal is observed in films consisting of PCLdiUPy with UPy-Tz as compared to PCLdiUPy films. Fluorescence spectroscopy showed that upon incorporating increasing amounts of UPy-Tz in the material, higher fluorescence signals originating from TCO-modified fluorescent protein were observed. Surface MALDI-TOF-MS experiments revealed presence of the UPy-Tz – TCO-modified model compound click product, a direct proof of the success of this novel strategy. 3D TOF-SIMS experiments allowed to reconstruct a density map of the different compounds incorporated in our materials and provided useful insights in their spatial distribution.
Conclusion: We report on a novel, elegant and successful strategy to functionalize supramolecular material surfaces via an orthogonal click reaction between a UPy-Tz moiety, modularly incorporated into our supramolecular polymer, and a TCO-modified model compound. The strategy presented here allows us to adapt this chemistry to obtain multi-functional materials, where we envision ultimate application as highly sophisticated biomaterials in the field of cardiovascular tissue engineering.
E. W. Meijer
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