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Bio-additive-based screening: toward evaluation of the biocompatibility of chemical reactions

Nature Protocols volume 14, pages 2599–2626 (2019)Cite this article

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Abstract

A requirement for biochemical labeling strategies is a pronounced biocompatibility of the underlying reaction methodology. This protocol enables a systematic evaluation of the biocompatibility of (new) reaction methodologies that are potentially attractive for biochemical applications. The cellular environment for in vitro and in vivo applications is mimicked by the one-by-one addition of diverse bio-additives to the reaction. The influence of the bio-additives on the product yield, termed bio-robustness, is quantified by gas chromatography (GC) or NMR techniques, whereas qualitative analysis of the level of biomolecule preservation by ultra-HPLC–mass spectrometry (UHPLC–MS) or gel electrophoresis enables monitoring of the effects of the reaction conditions on the biomolecule stability, e.g., bio-additive modification or degradation. The 22 chosen bio-additives and the required controls can be completely evaluated within 5–7 working days, depending on reaction time, instrument and the general equipment availability of the lab. We illustrate this protocol by assessing the reaction biocompatibility of a copper-catalyzed N-arylation of sulfonamides. The hereby obtained results are compared to those for a reaction that is characterized by high reaction biocompatibility: the energy-transfer-enabled disulfide–ene reaction.

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Fig. 1: Conceptual sketch of bio-additive-based reaction biocompatibility screening.
Fig. 2: Workflow of bio-additive-based reaction biocompatibility screening.
Fig. 3
Fig. 4
Fig. 5
Fig. 6: UHPLC–MS analysis of preservation of ATP as bio-additive (entry 14) in the copper-catalyzed N-arylation and comparison with untreated ATP as control.
Fig. 7: Analysis of ssDNA.
Fig. 8: Visualization of bio-robustness and biomolecule preservation.

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Data availability

The data supporting the findings of this study are available within the paper and its supplementary information, or from the corresponding authors upon reasonable request.

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Acknowledgements

We thank S. Hüwel, W. Dörner and S. Wulff for experimental and technical assistance (all WWU Münster). This work was supported by the Deutsche Forschungsgemeinschaft (Leibniz Award to F.G. and RE2796/6-1 to A.R.) and by the Fonds der Chemischen Industrie (doctoral fellowship to L.A. and Dozentenpreis to A.R.). M.T. thanks SusChemSys 2.0 for general support.

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Author notes

  1. These authors contributed equally: Lea Anhäuser, Michael Teders.

Authors and Affiliations

  1. Institut für Biochemie, Westfälische Wilhelms-Universität Münster, Münster, Germany

    Lea Anhäuser & Andrea Rentmeister

  2. Organisch-Chemisches Institut, Westfälische Wilhelms-Universität Münster, Münster, Germany

    Michael Teders & Frank Glorius

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  1. Lea Anhäuser
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  2. Michael Teders
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Contributions

L.A., M.T., A.R. and F.G. designed the concept and the protocol. L.A. and M.T. performed all experimental work. All authors co-wrote the paper.

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Correspondence to Andrea Rentmeister or Frank Glorius.

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Peer review information: Nature Protocols thanks Gonçalo Bernardes and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

Teders, M. et al. Nat. Chem. 10, 981–988 (2018): https://www.nature.com/articles/s41557-018-0102-z

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Supplementary Information

Supplementary Figures 1–23, Supplementary Table 1, Supplementary Note 1 and Supplementary Procedure 1

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Anhäuser, L., Teders, M., Rentmeister, A. et al. Bio-additive-based screening: toward evaluation of the biocompatibility of chemical reactions. Nat Protoc 14, 2599–2626 (2019). https://doi.org/10.1038/s41596-019-0190-2

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