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Title

Live‐Cell‐Templated Dynamic Combinatorial Chemistry

AuthorsCarbajo López, Daniel; Pérez, Yolanda ; Bujons, Jordi; Alfonso, Ignacio
KeywordsDynamic combinatorial chemistry
Extracellular matrix
Glycosaminoglycans
on-cell NMR spectroscopy
Molecular recognition
Issue Date29-Apr-2020
PublisherWiley-Blackwell
CitationAngewandte Chemie - International Edition (2020)
AbstractDynamic covalent chemistry combines in a single step the screening and synthesis of ligands for biomolecular recognition. In order to do that, a chemical entity is used as template within a dynamic combinatorial library of interconverting species, so that the stronger binders are amplified due to the efficient interaction with the target. Here we employed whole A549 living cells as template in a dynamic mixture of imines, for which amplification reflects the efficient and selective interaction with the corresponding extracellular matrix. The amplified polyamine showed strong interaction with the A549 extracellular matrix in on‐cell NMR experiments, while combination of NMR, SPR, and molecular dynamics simulations in model systems provided insights on the molecular recognition event. Notably, our work pioneers the use of whole living cells in dynamic combinatorial chemistry, which paves the way towards the discovery of new bioactive molecules in a more biorelevant environment. Dynamic combinatorial chemistry (DCC) proposes the use of dynamic combinatorial libraries (DCL) for the generation of species able to exchange through reversible covalent bonds.1 These molecular systems are responsive to external stimuli by modification of the DCL composition,2 with the stabilized members being amplified at the expense of the other components in the mixture.3 Within the chemical biology field, the DCC approach has led to the discovery of new protein ligands,4 nucleic acids binders,5 and even replicators.6 For biological applications, it should be desirable that the conditions used for the DCC screening resemble those in the place of action.7 Inspired by Sander's comparison of DCC with the immune system,8 we envisioned targeting the extracellular matrix (ECM).9 The external surface of the cells is formed by a complex network of glycoproteins and anionic polysaccharides that is fundamental for processes such as cell communication,10 regeneration,11 metastasis,12 and host‐pathogen infection.13 The ECM is the first barrier for a molecule (i.e. a drug) to enter inside the cell; thus, navigating the ECM is fundamental in biomedicine and in chemical biology.14 However, the chemical and structural complexity of the ECM have hindered its detailed molecular characterization and frustrated the rational design of synthetic ligands.15 Paradoxically, the intrinsic complexity of the ECM offers an ideal playground for the realization of the self‐organizing features of DCC (Figure 1 A), which has demonstrated its power for the discovery of strong binders to challenging biomolecules.1, 2-4, 5-8 Considering our recent results in the identification of a strong heparin binder16 and the chemical similarity between heparin and the glycosaminoglycans (GAGs) of the ECM,14 we designed a library (Figure 1 B) combining spermine as a cationic polyamine scaffold17 with a set of aromatic aldehydes, which would mediate binding through CH–aryl interactions with the saccharide units.18 Thus, the dynamic mixture of imines (2XY ) obtained by the reaction between spermine (1 ) and an equimolecular mixture of four aromatic aldehydes (A , B , K , L ) was incubated with living cells and reduced in situ with NaBH3CN to the corresponding polyamines (3XY ). As an initial model, we used the A549 human lung adenocarcinoma cell line since the ECM of these cells is rich in anionic GAGs.19 The supernatant was analyzed by UPLC‐MS allowing the identification and quantification of each member of the library (Figure 1 C). The normalized area of the UPLC‐MS peaks for the reactions performed in the presence (A T) and in the absence (A 0) of cells was compared by the calculation of the corresponding amplification factors (AF=A T/A 0).
Publisher version (URL)https://doi.org/10.1002/anie.202004745
URIhttp://hdl.handle.net/10261/215824
DOIhttp://dx.doi.org/10.1002/anie.202004745
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