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

Abstract

Diels−Alder reactions with inverse electron demand (DA inv ) 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 DA inv -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 DA inv . 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 DA inv bioorthogonal chemistry for site-specific protein labeling, providing additional experimental flexibility for preparing well-defined bioconjugates and addressing biological questions in complex biological environments.

3-Bromotetrazine: labelling of macromolecules via monosubstituted bifunctional s-tetrazines

“…In comparison, the kinetics of the iEDDA reaction of a diene functionalised with two thioethers required 14.5 h and heating to 37 C. 8 Ultimately, fast reaction rates are indispensable for bio-orthogonal reactions in order to ensure full conversion of the target to the labelled compound, especially at physiologically relevant mM-concentrations of the protein. 11 Thus, this fast protocol clearly demonstrates the utility of Br-Tet (2) for bio-orthogonal labelling and shows that with this approach the most likely smallest uorophore available can easily be attached onto a protein under biologically relevant conditions without the use of any additives such as base. 21 More importantly, the very short reaction times required for the labelling procedure, as well as the subsequent iEDDA reaction, which can be carried out in a one-pot fashion, greatly enhance the utility of this approach.…”

“…9 A major drawback of this method is the presence of a hydrophobic linker, which has a negative effect on the physiochemical properties of the moiety attached to the tetrazine (Scheme 1). 10,11 In this context, the target engagement, metabolism and pharmacological prole of the payloads linked to the tetrazine can be altered drastically. 10,12 Whereas there are several reports of the introduction of tetrazine moieties into proteins in response to an amber codon, there are few general methods available for the direct, chemical synthesis of small-/macromolecules functionalised with a minimal tetrazine.…”

“…14 While this approach guarantees high degrees of labelling and high selectivity, in general, the heterologous expression of functionalised proteins and purication thereof can pose a challenge. 11,14,16 Based on these observations, the development of novel, small tetrazine chemical probes for the fast and easy labelling of macromolecules, peptides and proteins in Scheme 1 Labelling of small-/macromolecules with tetrazines. a selective fashion is of great interest.…”

Divergent Synthesis of Monosubstituted and Unsymmetrical 3,6‐Disubstituted Tetrazines from Carboxylic Ester Precursors

“…[10] Fort his approach, linkers are required that can add bulk and hydrophilicity to the conjugate. [11] Thed irect preparation of tetrazines from carboxylic precursors could greatly expand access to functionalized derivatives.D escribed here is an ew strategy for the one-pot synthesis of 3-thiomethyltetrazines from carboxylic esters of commercially available 3-methyl-3oxetanemethanol. Thec ompounds serve as versatile intermediates for the divergent synthesis of ab road range of functionalized tetrazines through Pd-catalyzed cross-coupling and in the first example of catalytic thioether reduction to form monosubstituted tetrazines ( Figure 1).…”

Iridium(iii) complexes of 1,2,4-triazines as potential bioorthogonal reagents: metal coordination facilitates luminogenic reaction with strained cyclooctynes

“…Recently, 1,2,4-triazines have been reported as potential bioorthogonal reagents offering excellent stability. [4][5][6][7][8][9][10][11] Unfortunately, 1,2,4-triazines react much slower in IEDDA reactions. Although it has been shown that the kinetics can be improved by the introduction of electronwithdrawing groups, 9,10 the rate constant is two-three orders of magnitude lower than for tetrazines.…”