Site‐Specific Dual Labeling of Proteins on Cysteine Residues with Chlorotetrazines

Abstract

Dual-labeled biomolecules constitute a new generation of bioconjugates with promising applications in therapy and diagnosis. Unfortunately, the development of these new families of biologics is hampered by the technical difficulties associated with their construction. In particular, the site specificity of the conjugation is critical as the number and position of payloads can have a dramatic impact on the pharmacokinetics of the bioconjugate. Herein, we introduce dichlorotetrazine as a trivalent platform for the selective double modification of proteins on cysteine residues. This strategy is applied to the dual labeling of albumin with a macrocyclic chelator for nuclear imaging and a fluorescent probe for fluorescence imaging.

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

“…In order to estimate the rate constant of this transformation, Boc-Cys(Tet)-OMe was reacted with TCO-PEG3biotin as a model system under pseudo-rst order conditions; a rate constant of 22.7 M À1 s À1 was observed for the iEDDA reaction (see ESI †). 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.…”

“…5 More recently, s-tetrazines emerged as a powerful tool in bio-orthogonal chemistry, due to specic reactivity with alkynes and alkenes in the inverse-electron demand Diels-Alder reaction (iEDDA). [6][7][8] However, classical methods for the synthesis of tetrazine-conjugates mainly rely on amide bond formation between a carboxylic acid derived tetrazine precursor and an amine. 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).…”

“…13,14 Recent advances towards simple tetrazine unit incorporation into proteins involve site-selective dual-labelling of cysteine residues with 3,6-disubstituted s-tetrazines. 8 Nevertheless, the subsequent reaction with a strained alkyne proved to be demanding, as heating and prolonged reaction times were necessary, which demonstrated the generally slower kinetics when 3,6-disubstituted s-tetrazines are employed, compared with 3-s-tetrazines. 6,8,15 A complementary method to the chemical introduction of the tetrazine moiety into proteins was recently demonstrated by the group of Lang and co-workers, who genetically encoded lysine-based Me-tetrazine amino acids.…”

“…8 Nevertheless, the subsequent reaction with a strained alkyne proved to be demanding, as heating and prolonged reaction times were necessary, which demonstrated the generally slower kinetics when 3,6-disubstituted s-tetrazines are employed, compared with 3-s-tetrazines. 6,8,15 A complementary method to the chemical introduction of the tetrazine moiety into proteins was recently demonstrated by the group of Lang and co-workers, who genetically encoded lysine-based Me-tetrazine amino acids. 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.…”

Cysteine-specific protein multi-functionalization and disulfide bridging using 3-bromo-5-methylene pyrrolones

“…Recently, the need to prepare homogeneous antibody-drug conjugates has inspired a wave of interest in exploring cysteine-specific bioconjugation reagents with improved reactivity, specificity, stability, and biocompatibility 6,7 . To this end, maleimide derivatives [8][9][10] , dehydroalanine 11,12 , perfluoroaromatic reagents [13][14][15][16] , organometallic palladium reagents 17,18 , and many others [19][20][21][22][23] have been developed, and each of these reagents shows unique merits. However, only a few of them permit multifunctionalization of a single Cys residue 22,24,25 , which would be advantageous in certain situations.…”