Cardoso/Hackenberger/Leonhardt

Our long-term goal is to develop and validate novel methods to visualize and manipulate biochemical processes within living cells with fluorescently labeled proteins functionalized for cellular uptake and photoinducible activation. We will utilize artificial cell penetrating peptides and optimize them for intracellular release and bioavailability of cargo proteins. For intracellular binding of target structures we will generate specific nanobodies against the nuclear proteins PCNA and Dnmt1. Different strategies for site-selective modification of proteins including intein expression, incorporation of artificial amino acids by amber suppression and bioorthogonal reactions will be employed to introduce functional modules like fluorescent labels, caged amino acids and artificial cell penetrating groups.

This functionalization will initially be tested with GFP to evaluate the conjugation of cyclic peptides and will be optimized for enhanced cellular uptake. We will monitor the uptake of modified nanobodies by live cell microscopy and will activate them with short light pulses. Physiological effects on target structures including the disruption of protein interactions will be quantified by automated image analysis. This proposed collaboration, combines expertise in nanobody engineering, bioorthogonal chemistry, cellular uptake mechanisms, microscopy and cell biology. Consequently, this joint effort should lead to the development of entirely new research tools to detect, study and manipulate antigens in living cells.

A.F.L. Schneider, L. Benz, M. Lehmann, C.P.R. Hackenberger
Angew. Chem. Int. Ed. 2021, 60(40), 22075-22080
Cell-Permeable Nanobodies Allow Dual-Color Super-Resolution
Link to the article
Angew. Chem.2021, 133(40), 22246-22252
Zellpermeable Nanobodys ermöglichen Zwei-Farben-Superauflösungsmikroskopie in lebenden, nicht transfizierten Zellen
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A.F.L. Schneider, M. Kithil, M.C. Cardoso, M. Lehmann, C.P.R. Hackenberger
Nature Chemistry 2021, 13, 530-539
Cellular uptake of large biomolecules enabled by cell-surface-reactive cell-penetrating peptide additives
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J. Schwach, K. Kolobynina, K. Brandstetter, M. Gerlach, P. Ochtrop, J. Helma, C.P.R. Hackenberger. H. Harz, M.C. Cardoso, H. Leonhardt, A. Stengl
ChemBioChem 2021, 22(7), 1205-1209
Site‐Specific Antibody Fragment Conjugates for Reversible Staining in Fluorescence Microscopy
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M.-A. Kasper, M. Gerlach, A.F.L. Schneider, C. Groneberg, P. Ochtrop, S. Boldt, D. Schumacher, J. Helma, H. Leonhardt, M. Christmann, C.P.R. Hackenberger
ChemBioChem 2020, 21, 113-119
N-Hydroxysuccinimide-Modified Ethynylphosphonamidates Enable the Synthesis of Configurationally Defined Protein Conjugates
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M.-A. Kasper, A. Stengl, P. Ochtrop, M. Gerlach, T. Stoschek, D. Schumacher, J. Helma, M. Penkert, E. Krause, H. Leonhardt, C.P.R. HackenbergerAngew. Chem. Int. Ed. 2019, 58(34), 11631-11636
Ethynylphosphonamidates for the Rapid and Cysteine-Selective Generation of Efficacious Antibody-Drug Conjugates
Link to the article
Angew. Chem. 2019, 131(34), 11757-11762
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M.-A. Kasper, M. Glanz, A. Stengl, M. Penkert, S. Klenk, T. Sauer, D. Schumacher, J. Helma, E. Krause, C. Cardoso, H. Leonhardt, C.P.R. Hackenberger
Angew. Chem. Int. Ed. 2019, 58(34), 11625-11630
Cysteine-Selective Phosphonamidate Electrophiles for Modular Protein Bioconjugations
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Angew. Chem. 2019, 131(34), 11751-11756
Cysteinselektive phosphonamidatbasierte Elektrophile für modulare Biokonjugationen
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M.-A. Kasper, M. Glanz, A. Oder, P. Schmieder, J.P. von Kries, C.P.R. Hackenberger
Chem Sci. 2019, 10, 6322-6329
Vinylphosphonites for Staudinger-induced chemoselective peptide cyclizaton and functionalization
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A. Stengl, M. Gerlach, M.-A. Kasper, C.P.R. Hackenberger, H. Leonhardt, D. Schumacher, J. Helma
OBC 2019, 17, 4964-4969
TuPPL: Tub-tag mediated C-terminal protein-protein-ligation using complementary click-chemistry handles
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A.F.L. Schneider, A.L.D. Wallabregue, L. Franz, C.P.R. Hackenberger
Bioconjugate Chem. 2019, 30(2), 400-404
Targeted Subcellular Protein Delivery Using Cleavable Cyclic Cell-Penetrating Peptides
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M. Effenberger, A. Stengl, K. Schober, M. Gerget, M. Kampick, T.R. Müller, D. Schumacher, J. Helma, H. Leonhardt, D.H. Busch
J Immunol. 2019,202, 2164-2171
FLEXamers: A Double Tag for Universal Generation of Versatile Peptide-MHC Multimers
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M. Gerlach, T. Stoschek, H. Leonhardt, C.P.R. Hackenberger, D. Schumacher, J. Helma
Methods Mol Biol. 2019, 2012, 327-355
Tubulin Tyrosine Ligase-Mediated Modification of Proteins
Link to the article

J. Helma, H. Leonhardt, C.P.R. Hackenberger, D. Schumacher
Methods Mol Biol.2018, 1728, 67-93
Tub-Tag Labeling; Chemoenzymatic Incorporation of Unnatural Amino Acids
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H.Y. Chiu, J.A. Bates, J. Helma, H. Engelke, H. Harz, T. Bein, H. Leonhardt*
Nucleus 2018, 9, 530-542
Nanoparticle Mediated Delivery and Small Molecule Triggered Activation of Proteins in the Nucleus
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D. Schumacher, J. Helma, A.F.L. Schneider, H. Leonhardt, C.P.R. Hackenberger
Angewandte Chem. 2018, 130, 2336-2357
Nanobodys: Strategien zur chemischen Funktionalisierung und intrazelluläre Anwendungen.
Angewandte Chem. Int. Ed.2018, 57(9), 2314-2333.
Chemical functionalization strategies and intracellular applications of nanobodies.
Link to the article

A.F.L. Schneider, C.P.R. Hackenberger
Curr. Opin. in Biotechnology 2017, 48, 61-68
Flourescent labeling in living cells.
Link to the article

H.D. Herce, D. Schumacher, A.F.L. Schneider, A.K. Ludwig, F.A. Mann, M. Filies, M.-A. Kasper, S. Reinke, E. Krause, H. Leonhardt, M.C. Cardoso, C.P.R. Hackenberger
Nature Chemistry 2017, 9, 762-771
Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells.
Link to the article
Link to supplementary movie

D. Schumacher, O. Lemke, J. Helma, L. Gerszonowicz, V. Waller, T. Stoschek, P.M. Durkin, N. Budisa, H. Leonhardt, B.G. Keler, C.P.R. Hackenberger
Chem. Sci. 2017, 8, 3471-3478
Broad substrate tolerance of tubulin tyrosine ligase enables one-step site-specific enzymatic protein labeling
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A. Stengl, D. Hörl, H. Leonhardt, J. Helma
SLAS Discovery 2017, 22(3), 309-315
A Simple and Sensitive High-Content Assay for the Characterization of Antiproliferative Therapeutic Antibodies
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R. Röder, J. Helma, T. Preiß, J.O. Rädler, H. Leonhardt, E. Wagner
Pharm. Res. 2017, 34, 161-174
Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells
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J. Bertran-Vicente, M. Penkert, O. Nieto-Garcia, J.-M. Jeckelmann, P. Schmieder, E. Krause, C.P.R. Hackenberger
Nature Comm. 2016, 7, Article number: 12703
Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides.
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S. Schwagerus, O. Reimann, C. Despres, C. Smet-Nocca, C.P.R. Hackenberger
J. Pept. Sci. 2016,22(5), 327-333
Semi-synthesis of a tag-free O-GlcNAcylated tau protein by sequential chemoselective ligation
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H.-Y. Chiu, W. Deng, H. Engelke, J. Helma, H. Leonhardt, T. Bein
Sci. Rep. 2016, 6, 25019
Intracellular chromobody delivery by mesoporous silica nanoparticles for antigen targeting and visualization in real time.
Link to the article

D. Schumacher, C.P.R. Hackenberger, H. Leonhardt, J. Helma
J. Clin. Immunol. 2016, 36, 100-107 (Review)
Current Status: Site-Specific Antobody Drug Conjugates.
Link to the article

E. Hoffmann, K. Streichert, N. Nischan, C. Seitz, T. Brunner, S. Schwagerus, C.P.R. Hackenberger, M. Rubini
Molecular BioSystems2016, 12, 1750-1755
Stabilization of bacterially expressed erytropoietin by single site-specific introduction of short branched PEG chains at naturally occurring glycosylaton sites.
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K. Klamecka, P.M. Severin, L.F. Milles, H.E. Gaub, H. Leonhardt
Phys. Biol. 2015, 12, 056009
Energy profile of nanobody-GFP complex under force
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M. Rajan, O. Mortusewicz, U. Rothbauer, F.D. Hastert, K. Schmidthals, A. Rapp, H. Leonhardt*, M.C. Cardoso*
FEBS Open Bio2015, 5, 779-788
Generation of an alpaca-derived nanobody recognizing γ-H2AX
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J. Helma, M.C. Cardoso, S. Muyldermans, H. Leonhardt
J Cell Biol2015, 209, 633-644
Nanobodies and recombinant binders in cell biology
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D. Schumacher, J. Helma, F.A. Mann, G. Pichler, F. Natale, E. Krause, M.C. Cardoso, C.P.R. Hackenberger, H. Leonhardt
Angew. Chem. Int. Ed. 2015, 54(46), 13787-13791
Versatile and efficient site-specific protein functionalization by tubulin tyrosine ligase
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M. Mühlberg, M.G. Hösl, C. Kühne, J. Dernedde, N. Budisa, C.P.R. Hackenberger
Beilstein JOC 2015, 11, 784-791
Orthogonal Dual-Modification of Proteins for the Engineering of Multivalent Protein Scaffolds
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C. Witte, V. Martos, H.M. Rose, R. Reinke, S. Klippel, L. Schröder, C.P.R. Hackenberger
Angew. Chem. Int. Ed. 2015, 54(9), 2806-2810 (VIP-Paper)
Live-cell MRI of xenon Hyper-CEST biosensors targeted in metabolically-labeled cell-surface glycans
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N. Nischan, H.D. Herce, F. Natale, N. Bohlke, N. Budisa, M.C. Cardoso, C.P.R. Hackenberger
Angew. Chem. Int. Ed. 2015, 54(6), 1950-1953
Covalent Attachment of Cyclic TAT Peptides to GFP Results in Protein Delivery into Live Cells with Immediate Bioavailability
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O. Reimann, C. Smet-Nocca, C.P.R. Hackenberger
Angew. Chem. Int. Ed. 2015, 54, 306-310
Traceless Purification and Desulfurization of Tau Protein Ligation Products
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M.R.J. Vallée, P. Majkut, D. Krause, M. Gerrits, C.P.R. Hackenberger
Chem. Eur. J. 2015, 21, 970-974
Chemoselective Bioconjugation of Triazole Phosphonites in Aqueous Media
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D. Aschenbrenner, D.A. Pippig, K. Klamecka, K. Limmer, H. Leonhardt, H.E. Gaub
PLoS One 2014, e115049
Parallel force assay for protein-protein interactions.
Link to the article

H.D. Herce, A.E. Garcia, M.C. Cardoso
J. Am. Chem. Soc. 2014, 136, 17459-17467
Fundamental molecular mechanism for the cellular uptake of guanidinium-rich molecules
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R. Wieneke, N. Labòria, M. Rajan, A. Kollmannsperger, F. Natale, M.C. Cardoso, R. Tampé
J. Am. Chem. Soc. 2014, 136, 13975-13978
Live-cell Targeting of His-tagged Proteins by Multivalent N-Nitrilotriacetic Acid Carrier Complexe
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D. Schumacher, C.P.R. Hackenberger
Curr. Opin. Chem. Biol. 2014, 22, 62-69
More than add-on: chemoselective reactions for the synthesis of functional peptides and proteins
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J. Bertran-Vicente, R.A. Serwa, M. Schümann, P. Schmieder, E. Krause, C.P.R. Hackenberger
J. Am Chem. Soc. 2014, 136(39), 13622-13628
Site-Specifically Phosphorylated Lysine Peptides
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P. Majkut, I. Claußnitzer, H. Merk, C. Freund, C.P.R. Hackenberger, M. Gerrits
PLoS ONE 2013, 8(12): e82352
Completion of Proteomic Data Sets by K_d Measurement Using Cell-Free Synthesis
of Site-Specifically Labeled Proteins
Link zum Artikel

H.D. Herce, W. Deng, J. Helma, H. Leonhardt, M.C. Cardoso
Nat. Commun 2013, 4, 2660
Visualization and targeted disruption of protein interactions in living cells.
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N. Nischan, A. Chakrabarti, R.A. Serwa, P.H.M. Bovee-Geurts, R. Brock, C.P.R. Hackenberger
Angew. Chem.2013, 125, 45, 12138-12142
Stabilisierung von Peptiden für intrazelluläre Anwendungen mit Phosphoramidat-verzweigten Polyethylenglycol-Ketten
Angew. Chem. Int. Ed.2013, 52, 45, 11920-11924
Stabilization of Peptides for Intracellular Applications by Phosphoramidate-Linked PEG Chains
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M.R.J. Vallée, L.M. Artner, J. Dernedde, C.P.R. Hackenberger
Angew. Chem. 2013, 125, 9682-9686
Angew. Chem. Int. Ed.2013, 52, 9504-9508
Alkyne phophonites for sequential azide-azide couplings
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