A Bioorthogonal Click Chemistry Toolbox for Targeted Synthesis of Branched and Well-Defined Protein-Protein Conjugates.

Bioorthogonal chemistry holds great potential to generate difficult-to-access protein-protein conjugate architectures. Current applications are hampered by challenging protein expression systems, slow conjugation chemistry, use of undesirable catalysts, or often do not result in quantitative product formation. Here we present a highly efficient technology for protein functionalization with commonly used bioorthogonal motifs for Diels-Alder cycloaddition with inverse electron demand (DAinv ). With the aim of precisely generating branched protein chimeras, we systematically assessed the reactivity, stability and side product formation of various bioorthogonal chemistries directly at the protein level. We demonstrate the efficiency and versatility of our conjugation platform using different functional proteins and the therapeutic antibody trastuzumab. This technology enables fast and routine access to tailored and hitherto inaccessible protein chimeras useful for a variety of scientific disciplines. We expect our work to substantially enhance antibody applications such as immunodetection and protein toxin-based targeted cancer therapies.

Live-Cell Localization Microscopy with a Fluorogenic and Self-Blinking Tetrazine Probe.

Recent developments in fluorescence microscopy call for novel small-molecule-based labels with multiple functionalities to satisfy different experimental requirements. A current limitation in the advancement of live-cell single-molecule localization microscopy is the high excitation power required to induce blinking. This is in marked contrast to the minimal phototoxicity required in live-cell experiments. At the same time, quality of super-resolution imaging depends on high label specificity, making removal of excess dye essential. Approaching both hurdles, we present the design and synthesis of a small-molecule label comprising both fluorogenic and self-blinking features. Bioorthogonal click chemistry ensures fast and highly selective attachment onto a variety of biomolecular targets. Along with spectroscopic characterization, we demonstrate that the probe improves quality and conditions for regular and single-molecule localization microscopy on live-cell samples.

Enzymatic and Site-Specific Ligation of Minimal-Size Tetrazines and Triazines to Proteins for Bioconjugation and Live-Cell Imaging.

Diels-Alder reactions with inverse electron demand (DAinv) have emerged as an indispensable tool for bioorthogonal labeling and the manipulation of biomolecules. In this context, reactions between tetrazines and strained dienophiles have received attention because of high reaction rates. Current methods for the DAinv-mediated functionalization of proteins suffer from slow reactivity, impaired stability, isomerization, or elimination of the incorporated strained dienophiles. We report here a versatile platform for the posttranslational, highly selective, and quantitative modification of proteins with stable dienes. New synthetic access to minimal size tetrazine and triazine derivatives enabled us to synthesize tailored diene substrates for the lipoic acid protein ligase A (LplA) from Escherichia coli, which we employ for the rapid, mild, and quantitative bioconjugation of proteins by DAinv. The presented method benefits from the minimal tag size for LplA recognition and can be applied to proteins from any source organism. We demonstrate its broad suitability by site-specific in vitro protein labeling and live cell labeling for fluorescence microscopy. With this work we expand the scope of DAinv bioorthogonal chemistry for site-specific protein labeling, providing additional experimental flexibility for preparing well-defined bioconjugates and addressing biological questions in complex biological environments.

Site-Specific Protein Labeling Utilizing Lipoic Acid Ligase (LplA) and Bioorthogonal Inverse Electron Demand Diels-Alder Reaction.

Here, we describe a two-step protocol for selective protein labeling based on enzyme-mediated peptide labeling utilizing lipoic acid ligase (LplA) and bioorthogonal chemistry. The method can be applied to purified proteins, protein in cell lysates, as well as living cells. In a first step a W37V mutant of the lipoic acid ligase (LplAW37V) from Escherichia coli is utilized to ligate a synthetic chemical handle site-specifically to a lysine residue in a 13 amino acid peptide motif-a short sequence that can be genetically expressed as a fusion with any protein of interest. In a second step, a molecular probe can be attached to the chemical handle in a bioorthogonal Diels-Alder reaction with inverse electron demand (DAinv). This method is a complementary approach to protein labeling using genetic code expansion and circumvents larger protein tags while maintaining label specificity, providing experimental flexibility and straightforwardness.

Ion source-dependent performance of 4-vinylpyridine, iodoacetamide, and N-maleoyl derivatives for the detection of cysteine-containing peptides in complex proteomics.

Cysteine is unique among the proteinogenic amino acids due to its ability to form disulfide bonds. While this property is of vital importance for protein structures and biological processes, it causes difficulties for the mass spectrometric identification of cysteine-containing peptides. A common approach to overcome these problems in bottom-up proteomics is the reduction and covalent modification of sulfhydryl groups prior to enzymatic digestion. In this study, established alkylating agents and N-maleoyl amino acids with variable hydrophobicity were characterized with respect to a variety of relevant parameters and subsequently evaluated in a large-scale analysis using different ion sources. Depending on the compound, the ion source had a profound impact on the relative and absolute identification of cysteine-containing peptides. The best results were obtained by derivatization of the cysteine residues with 4-vinylpyridine and subsequent matrix-assisted laser desorption ionization (MALDI). Modification with 4-vinylpyridine increased the number of cysteine-containing peptides identified with any other compound using LC-MALDI/MS at least by a factor of 2. This experimental observation is mirrored by differences in the gas-phase basicities, which were computed for methyl thiolate derivatives of the compounds using density functional theory. With electrospray ionization (ESI), complementary use of reagents from three different compound classes, e.g., iodoacetamide, 4-vinylpyridine, and N-maleoyl beta-alanine, was beneficial compared to the application of a single reagent.

Two-step protein labeling by using lipoic acid ligase with norbornene substrates and subsequent inverse-electron demand Diels-Alder reaction.

Inverse-electron-demand Diels-Alder cycloaddition (DAinv ) between strained alkenes and tetrazines is a highly bio-orthogonal reaction that has been applied in the specific labeling of biomolecules. In this work we present a two-step labeling protocol for the site-specific labeling of proteins based on attachment of a highly stable norbornene derivative to a specific peptide sequence by using a mutant of the enzyme lipoic acid ligase A (LplA(W37V) ), followed by the covalent attachment of tetrazine-modified fluorophores to the norbornene moiety through the bio-orthogonal DAinv . We investigated 15 different norbornene derivatives for their selective enzymatic attachment to a 13-residue lipoic acid acceptor peptide (LAP) by using a standardized HPLC protocol. Finally, we used this two-step labeling strategy to label proteins in cell lysates in a site-specific manner and performed cell-surface labeling on living cells.

Two-step protein labeling utilizing lipoic acid ligase and Sonogashira cross-coupling.

Labeling proteins in their natural settings with fluorescent proteins or protein tags often leads to problems. Despite the high specificity, these methods influence the natural functions due to the rather large size of the proteins used. Here we present a two-step labeling procedure for the attachment of various fluorescent probes to a small peptide sequence (13 amino acids) using enzyme-mediated peptide labeling in combination with palladium-catalyzed Sonogashira cross-coupling. We identified p-iodophenyl derivatives from a small library that can be covalently attached to a lysine residue within a specific 13-amino-acid peptide sequence by Escherichia coli lipoic acid ligase A (LplA). The derivatization with p-iodophenyl subsequently served as a reactive handle for bioorthogonal transition metal-catalyzed Sonogashira cross-coupling with alkyne-functionalized fluorophores on both the peptide as well as on the protein level. Our two-step labeling strategy combines high selectivity of enzyme-mediated labeling with the chemoselectivity of palladium-catalyzed Sonogashira cross-coupling.