Milan Vrábel

Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry

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Rastislav Dzijak

Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry

Thomas Carell

Ludwig-Maximilians-Universität München

Michal Hocek

Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry

Martin Dračínský

Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry

Radek Pohl

Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry

Juraj Galeta

Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry

Michal Rahm

Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry

VŠ

Veronika Šlachtová

Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry

Michael J. Gattner

AbbVie (Germany)

Hannes Mikula

TU Wien

Cooperative Institutions

Czech Academy of Sciences, Institute of Organic Chemistry and Biochemistry

Czech Academy of Sciences

Ludwig-Maximilians-Universität München

Center for Integrated Protein Science Munich

Charles University

TU Wien

Center for NanoScience

Nanosystems Initiative Munich

University of Chemistry and Technology, Prague

Czech Academy of Sciences, Institute of Biophysics

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Genetic Code Expansion, Protein Expression, and Protein Functionalization in Bacillus subtilis

ACS Synthetic Biology (2020)

Christopher M. Scheidler Milan Vrábel Sabine Schneider

The site-specific chemical modification of proteins through incorporation of noncanonical amino acids enables diverse applications, such as imaging, probing, and expanding protein functions, as well as to precisely engineer therapeutics. Here we report a general strategy that allows the incorporation of noncanonical amino acids into target proteins using the amber suppression method and their efficient secretion in the biotechnological relevant expression host Bacillus subtilis. This facilitates efficient purification of target proteins directly from the supernatant, followed by their functionalization using click chemistry. We used this strategy to site-specifically introduce norbornene lysine into a single chain antibody and functionalize it with fluorophores for the detection of human target proteins.

Genetic Code

Surface Modification

Protein Engineering

Synthetic Biology

10.1021/acssynbio.9b00458

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Citations (16)

Structural Basis for the Site-Specific Incorporation of Lysine Derivatives into Proteins

PLoS ONE (2014)

Veronika Flügel Milan Vrábel Sabine Schneider

Posttranslational modifications (PTMs) of proteins determine their structure-function relationships, interaction partners, as well as their fate in the cell and are crucial for many cellular key processes. For instance chromatin structure and hence gene expression is epigenetically regulated by acetylation or methylation of lysine residues in histones, a phenomenon known as the 'histone code'. Recently it was shown that these lysine residues can furthermore be malonylated, succinylated, butyrylated, propionylated and crotonylated, resulting in significant alteration of gene expression patterns. However the functional implications of these PTMs, which only differ marginally in their chemical structure, is not yet understood. Therefore generation of proteins containing these modified amino acids site specifically is an important tool. In the last decade methods for the translational incorporation of non-natural amino acids using orthogonal aminoacyl-tRNA synthetase (aaRS):tRNAaaCUA pairs were developed. A number of studies show that aaRS can be evolved to use non-natural amino acids and expand the genetic code. Nevertheless the wild type pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei readily accepts a number of lysine derivatives as substrates. This enzyme can further be engineered by mutagenesis to utilize a range of non-natural amino acids. Here we present structural data on the wild type enzyme in complex with adenylated ε-N-alkynyl-, ε-N-butyryl-, ε-N-crotonyl- and ε-N-propionyl-lysine providing insights into the plasticity of the PylRS active site. This shows that given certain key features in the non-natural amino acid to be incorporated, directed evolution of this enzyme is not necessary for substrate tolerance.

Genetic Code

Amino Acyl-tRNA Synthetases

10.1371/journal.pone.0096198

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Citations (20)

Sulfonated Hydroxyaryl‐Tetrazines with Increased pKa for Accelerated Bioorthogonal Click‐to‐Release Reactions in Cells

Angewandte Chemie International Edition (2024)

Michal Rahm Patrick Keppel Rastislav Dzijak Martin Dračínský Veronika Šlachtová

Bioorthogonal reactions that enable switching molecular functions by breaking chemical bonds have gained prominence, with the tetrazine‐mediated cleavage of trans‐cyclooctene caged compounds (click‐to‐release) being particularly noteworthy for its high versatility, biocompatibility, and fast reaction rates. Despite several recent advances, the development of highly reactive tetrazines enabling quantitative elimination from trans‐cyclooctene linkers remains challenging. In this study, we present the synthesis and application of sulfo‐tetrazines, a class of derivatives featuring phenolic hydroxyl groups with increased acidity constants (pKa). This unique property leads to accelerated elimination and complete release of the caged molecules within minutes. Moreover, the inclusion of sulfonate groups provides a valuable synthetic handle, enabling further derivatization into sulfonamides, modified with diverse substituents. Significantly, we demonstrate the utility of sulfo‐tetrazines in efficiently activating fluorogenic compounds and prodrugs in living cells, offering exciting prospects for their application in bioorthogonal chemistry.

Bioorthogonal Chemistry

Tetrazine

Biocompatibility

Bioconjugation

Functional group

Cyclooctene

10.1002/anie.202411713

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Citations (1)

CCDC 1406413: Experimental Crystal Structure Determination

The Cambridge Structural Database (2016)

Juraj Galeta Michal Šála Martin Dračínský Milan Vrábel Zdeněk Havlas

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

10.5517/ccdc.csd.cc1j6h5d

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Single-Step Formation of Pyrimido[4,5-d]pyridazines by a Pyrimidine-Tetrazine Tandem Reaction

Organic Letters (2016)

Juraj Galeta Michal Šála Martin Dračínský Milan Vrábel Zdeněk Havlas

A straightforward synthesis of pyrimido[4,5-d]pyridazines from pyrimidines and tetrazines under basic conditions is reported. Deprotonated, substituted 5-halopyrimidines readily react with variously substituted tetrazines in a highly regioselective manner via a complex reaction pathway, which was supported by DFT calculations. This mechanism leads to the empirically observed regioisomers without going through the conceivable hetaryne intermediate. These results on 5-halopyrimidines led to development of the methodology for preparation of opposite regioisomers based on 6-halopyrimidines.

Structural isomer

Tetrazine

Pyridazine

Tandem

10.1021/acs.orglett.6b01601

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Citations (14)

Bioorthogonal Fluorescence Turn‐On Labeling Based on Bicyclononyne−Tetrazine Cycloaddition Reactions that Form Pyridazine Products

ChemPlusChem (2019)

Sebastian J. Siegl Juraj Galeta Rastislav Dzijak Martin Dračínský Milan Vrábel

Fluorogenic bioorthogonal reactions enable visualization of biomolecules with excellent signal-to-noise ratio. A bicyclononyne-tetrazine ligation that produces fluorescent pyridazine products has been developed. In stark contrast to previous approaches, the formation of the dye is an inherent result of the chemical reaction and no additional fluorophores are needed in the reagents. The crucial structural elements that determine dye formation are electron-donating groups present in the starting tetrazine unit. The newly formed pyridazine fluorophores show interesting photophysical properties the fluorescence intensity increase in the reaction can reach an excellent 900-fold. Model imaging experiments demonstrate the application potential of this new fluorogenic bioorthogonal reaction.

Bioorthogonal Chemistry

Tetrazine

Pyridazine

Biomolecule

10.1002/cplu.201900176

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Citations (32)

An optimized protocol for the synthesis of peptides containing trans-cyclooctene and bicyclononyne dienophiles as useful multifunctional bioorthogonal probes

Zenodo (CERN European Organization for Nuclear Research) (2021)

Agustina La‐Venia Rastislav Dzijak Robert Rampmaier Milan Vrábel

Bioorthogonal Chemistry

Cyclooctene

10.5281/zenodo.5079314

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Citations (1)

Uncovering mechanisms of thiazolidinediones on osteogenesis and adipogenesis using spatial fluxomics

Metabolism (2025)

Kristýna Brejchová Michal Rahm Andrea Beňová Veronika Domanska Paul Reyes-Gutierez

Highlights•MSDC-0602K differentially affects BM-MSCs and AT-MSCs.•Bioorthogonal click chemistry allowed measurement of pyruvate pools.•Subcellular metabolic flux analysis revealed rewiring of pyruvate pathways.•Metabolic flux analysis of TG synthesis showed distinct adipogenic strategies.AbstractObjectiveInsulin-sensitizing drugs, despite their broad use against type 2 diabetes, can adversely affect bone health, and the mechanisms underlying these side effects remain largely unclear. Here, we investigated the different metabolic effects of a series of thiazolidinediones, including rosiglitazone, pioglitazone, and the second-generation compound MSDC-0602 K, on human mesenchymal stem cells (MSCs).MethodsWe developed 13C subcellular metabolomic tracer analysis measuring separate mitochondrial and cytosolic metabolite pools, lipidomic network-based isotopologue models, and bioorthogonal click chemistry, to demonstrate that MSDC-0602 K differentially affected bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived MSCs (AT-MSCs). In BM-MSCs, MSDC-0602 K promoted osteoblastic differentiation and suppressed adipogenesis. This effect was clearly distinct from that of the earlier drugs and that on AT-MSCs.ResultsFluxomic data reveal unexpected differences between this drug's effect on MSCs and provide mechanistic insight into the pharmacologic inhibition of mitochondrial pyruvate carrier 1 (MPC). Our study demonstrates that MSDC-0602 K retains the capacity to inhibit MPC, akin to rosiglitazone but unlike pioglitazone, enabling the utilization of alternative metabolic pathways. Notably, MSDC-0602 K exhibits a limited lipogenic potential compared to both rosiglitazone and pioglitazone, each of which employs a distinct lipogenic strategy.ConclusionsThese findings indicate that the new-generation drugs do not compromise bone structure, offering a safer alternative for treating insulin resistance. Moreover, these results highlight the ability of cell compartment-specific metabolite labeling by click reactions and tracer metabolomics analysis of complex lipids to discover molecular mechanisms within the intersection of carbohydrate and lipid metabolism.Graphical abstract

Pioglitazone

Rosiglitazone

Metabolic flux analysis

Troglitazone

10.1016/j.metabol.2025.156157

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Citations (0)

Bioorthogonal Chemistry—Introduction and Overview

Topics in current chemistry collections (2016)

Thomas Carell Milan Vrábel

Bioorthogonal Chemistry

Bioconjugation