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Synthesis and characterization of chemically fueled supramolecular materials driven by carbodiimide-based fuels

Nature Protocols volume 16, pages 3901–3932 (2021)Cite this article

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Abstract

Many supramolecular materials in biological systems are driven to a nonequilibrium state by the irreversible consumption of high-energy molecules such as ATP or GTP. As a result, they exhibit unique dynamic properties such as a tunable lifetime, adaptivity or the ability to self-heal. In contrast, synthetic counterparts that exist in or close to equilibrium are controlled by thermodynamic parameters and therefore lack these dynamic properties. To mimic biological materials more closely, synthetic self-assembling systems have been developed that are driven out of equilibrium by chemical reactions. This protocol describes the synthesis and characterization of such an assembly, which is driven by carbodiimide fuels. Depending on the amount of chemical fuel added to the material, its lifetime can be tuned. In the first step, the protocol details the synthesis and purification of the peptide-based precursors for the fuel-driven assemblies by solid-phase peptide synthesis. Then, we explain how to analyze the kinetic response of the precursors to a carbodiimide-based chemical fuel by HPLC and kinetic models. Finally, we detail how to study the emerging assembly’s macro- and microscopic properties by time-lapse photography, UV-visible spectroscopy, shear rheology, confocal laser scanning microscopy and electron microscopy. The procedure is described using the example of a colloid-forming precursor Fmoc-E-OH and a fiber-forming precursor Fmoc-AAD-OH to emphasize the differences in characterization depending on the type of assembly. The characterization of a precursor’s transient assembly can be done within 5 d. The synthesis and purification of a peptide precursor requires 2 d of work.

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Fig. 1: In chemically fueled self-assembly, molecules are activated and deactivated for assembly by a chemical reaction cycle.
Fig. 2: Kinetic analysis of the reaction cycle.
Fig. 3: Material properties of 10 mM precursor 1 in response to fuel.
Fig. 4: Material properties of precursor 2 in response to fuel.
Fig. 5: Overview of the experimental design.
Fig. 6: Reaction scheme of the proposed solid-phase peptide synthesis of 2.
Fig. 7: Reactions involved in the chemical reaction cycle.
Fig. 8: Time-lapse photography setup.
Fig. 9: HPLC chromatograms of precursor Fmoc-AAD-OH (2) after purification detected at 254 nm and the applied pump gradient.
Fig. 10: Nile red fluorescence intensity at 635 nm against concentration of the precursor.
Fig. 11: Calibration curves.
Fig. 12: Time-lapse series of photographs shows the activation of 10 mM Fmoc-E-OH (1) with several amounts of EDC.
Fig. 13: Preparation of a microscope glass slide with a grease chamber for microscope imaging.

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Data availability

The authors declare that all the data supporting the findings of this study are available within the article, the source data files and the Supplementary Information files. Source data are provided with this paper.

Software availability

The MATLAB code used in this protocol together with an exemplary dataset is provided at https://github.com/BoekhovenLab/Nature_protocols. Source data are provided with this paper.

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Acknowledgements

This research was conducted within the Max Planck School Matter to Life supported by the German Federal Ministry of Education and Research (BMBF) in collaboration with the Max Planck Society. J.B., F.S., A.M.B., B.W. and O.L. acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – SFB-863 – Project ID 111166240 (Project B11) and funding by the European Research Council (ERC starting grant 852187). J.R.F. acknowledges the Deutsche Forschungsgemeinschaft for project 411722921. Cryo-TEM measurements were performed using infrastructure contributed by the Dietz Lab and the TUM EM Core Facility. We acknowledge the technical support provided by F. Kohler.

Author information

Authors and Affiliations

  1. Department of Chemistry, Technical University of Munich, Garching, Germany

    Fabian Schnitter, Alexander M. Bergmann, Jennifer Rodon Fores & Job Boekhoven

  2. Department of Mechanical Engineering and Munich School of Bioengineering, Technical University of Munich, Garching, Germany

    Benjamin Winkeljann & Oliver Lieleg

  3. Institute for Advanced Study, Technical University of Munich, Garching, Germany

    Job Boekhoven

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  1. Fabian Schnitter
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Contributions

F.S. and A.M.B. contributed equally to this protocol. They designed and performed the experiments and wrote the manuscript. B.W. and J.R.F. carried out the experiments. J.B. designed experiments, outlined and wrote the manuscript, and supervised the project. O.L. designed the experiments.

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Correspondence to Job Boekhoven.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Dibyendu Das and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Tena-Solsona, M. et al. Nat. Commun. 8, 15895 (2017): https://doi.org/10.1038/ncomms15895

Schnitter, F. & Boekhoven, J. ChemSystemsChem 3, e2000037 (2021): https://doi.org/10.1002/syst.202000037

Donau, C. et al. Nat. Commun. 11, 5157 (2020): https://doi.org/10.1038/s41467-020-18815-9

Dai, K. et al. J. Am. Chem. Soc. 142, 33 (2020): https://doi.org/10.1021/jacs.0c04203

Kriebisch, B. A. K. et al. J. Am. Chem. Soc. 142, 49 (2020): https://doi.org/10.1021/jacs.0c10486

Extended data

Extended Data Fig. 1 Cryo-TEM microscope micrograph.

The control micrograph of a 10 mM precursor 2 stock solution excludes preassembly of the inactivated precursor in the absence of EDC fuel.

Extended Data Fig. 2 EDC hydrolysis and Fmoc deprotection kinetics.

a,b, The addition of the quenching solution freezes the reaction cycle of Fmoc-E-OH (1) (a) and Fmoc-AAD-OH (2) (b). In the timescale of the HPLC analysis, the degradation of EDC and Fmoc deprotection does not falsify the concentration determination.

Source data

Extended Data Fig. 3 The absorbance of light as a measure for turbidity.

a, Usage of different sample containers affects the measurement quality as sedimentation in a 96-well plate wrongly extends the system’s lifetime. b, Regarding colloids formed by 1, a cuvette is a better choice as sample container.

Source data

Supplementary information

Reporting Summary

Supplementary Video 1

Macroscopic property of reaction cycle shown by evolution of turbidity

Supplementary Video 2

Macroscopic property of reaction cycle shown by evolution of a hydrogel

Source data

Source Data Fig. 2

HPLC signal areas.

Source Data Fig. 3

UV-VIS absorbance.

Source Data Fig. 4

Rheology storage and loss modulus.

Source Data Fig. 10

Nile Red fluorescence intensity.

Source Data Fig. 11

HPLC calibration.

Source Data Extended Data Fig. 2

EDC hydrolysis and Fmoc deprotection kinetics.

Source Data Extended Data Fig. 3

Effect of sample container on UV-VIS measurements.

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Schnitter, F., Bergmann, A.M., Winkeljann, B. et al. Synthesis and characterization of chemically fueled supramolecular materials driven by carbodiimide-based fuels. Nat Protoc 16, 3901–3932 (2021). https://doi.org/10.1038/s41596-021-00563-9

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