Stable Pyrrole‐Linked Bioconjugates through Tetrazine‐Triggered Azanorbornadiene Fragmentation

An azanorbornadiene bromovinyl sulfone reagent for cysteine‐selective bioconjugation has been developed. Subsequent reaction with dipyridyl tetrazine leads to bond cleavage and formation of a pyrrole‐linked conjugate. The latter involves ligation of the tetrazine to the azanorbornadiene‐tagged protein through inverse electron demand Diels–Alder cycloaddition with subsequent double retro‐Diels–Alder reactions to form a stable pyrrole linkage. The sequence of site‐selective bioconjugation followed by bioorthogonal bond cleavage was efficiently employed for the labelling of three different proteins. This method benefits from easy preparation of these reagents, selectivity for cysteine, and stability after reaction with a commercial tetrazine, which has potential for the routine preparation of protein conjugates for chemical biology studies.     

Squaramate‐Modified Nucleotides and DNA for Specific Cross‐Linking with Lysine‐Containing Peptides and Proteins

Squaramate‐linked 2′‐deoxycytidine 5′‐O‐triphosphate was synthesized and found to be good substrate for KOD XL DNA polymerase in primer extension or PCR synthesis of modified DNA. The resulting squaramate‐linked DNA reacts with primary amines to form a stable diamide linkage. This reaction was used for bioconjugations of DNA with Cy5 and Lys‐containing peptides. Squaramate‐linked DNA formed covalent cross‐links with histone proteins. This reactive nucleotide has potential for other bioconjugations of nucleic acids with amines, peptides or proteins without need of any external reagent.     

Stable,Reactive, and Orthogonal Tetrazines: Dispersion Forces Promote the Cycloaddition with Isonitriles

The isocyano group is a structurally compact bioorthogonal functional group that reacts with tetrazines under physiological conditions. Now it is shown that bulky tetrazine substituents accelerate this cycloaddition. Computational studies suggest that dispersion forces between the isocyano group and the tetrazine substituents in the transition state contribute to the atypical structure–activity relationship. Stable asymmetric tetrazines that react with isonitriles at rate constants as high as 57 L mol^?1 s^?1 were accessible by combining bulky and electron‐withdrawing substituents. Sterically encumbered tetrazines react selectively with isonitriles in the presence of strained alkenes/alkynes, which allows for the orthogonal labeling of three proteins. The established principles will open new opportunities for developing tetrazine reactants with improved characteristics for diverse labeling and release applications with isonitriles.     

Proximity‐Based Sortase‐Mediated Ligation

Protein bioconjugation has been a crucial tool for studying biological processes and developing therapeutics. Sortase A (SrtA), a bacterial transpeptidase, has become widely used for its ability to site‐specifically label proteins with diverse functional moieties, but a significant limitation is its poor reaction kinetics. In this work, we address this by developing proximity‐based sortase‐mediated ligation (PBSL), which improves the ligation efficiency to over 95 % by linking the target protein to SrtA using the SpyTag–SpyCatcher peptide–protein pair. By expressing the target protein with SpyTag C‐terminal to the SrtA recognition motif, it can be covalently captured by an immobilized SpyCatcher–SrtA fusion protein during purification. Following the ligation reaction, SpyTag is cleaved off, rendering PBSL traceless, and only the labeled protein is released, simplifying target protein purification and labeling to a single step.     

A Hexasaccharide Containing Rare 2‐O‐Sulfate‐Glucuronic Acid Residues Selectively Activates Heparin Cofactor II

Glycosaminoglycan (GAG) sequences that selectively target heparin cofactor II (HCII), a key serpin present in human plasma, remain unknown. Using a computational strategy on a library of 46 656 heparan sulfate hexasaccharides we identified a rare sequence consisting of consecutive glucuronic acid 2‐O ‐sulfate residues as selectively targeting HCII. This and four other unique hexasaccharides were chemically synthesized. The designed sequence was found to activate HCII ca. 250‐fold, while leaving aside antithrombin, a closely related serpin, essentially unactivated. This group of rare designed hexasaccharides will help understand HCII function. More importantly, our results show for the first time that rigorous use of computational techniques can lead to discovery of unique GAG sequences that can selectively target GAG‐binding protein(s), which may lead to chemical biology or drug discovery tools.