Site-Selective Cysteine-Cyclooctyne Conjugation

Abstract

We report a site-selective cysteine-cyclooctyne conjugation reaction between a seven-residue peptide tag (DBCO-tag, Leu-Cys-Tyr-Pro-Trp-Val-Tyr) at the N or C terminus of a peptide or protein and various aza-dibenzocyclooctyne (DBCO) reagents. Compared to a cysteine peptide control, the DBCO-tag increases the rate of the thiol-yne reaction 220-fold, thereby enabling selective conjugation of DBCO-tag to DBCO-linked fluorescent probes, affinity tags, and cytotoxic drug molecules. Fusion of DBCO-tag with the protein of interest enables regioselective cysteine modification on proteins that contain multiple endogenous cysteines; these examples include green fluorescent protein and the antibody trastuzumab. This study demonstrates that short peptide tags can aid in accelerating bond-forming reactions that are often slow to non-existent in water.

Keywords: bioconjugation; bioorthogonal chemistry; cysteine-cyclooctyne reaction; dibenzocyclooctyne; protein modification.

Figure 1.

Protein modification with dibenzocyclooctyne (DBCO) reagents. (a) Proteins with azides can react with DBCO reagents via strain-promoted azide-alkyne cycloaddition (SPAAC) reactions; thiol-modified side products can be generated with cysteine-containing proteins. (b) Previous thiol-yne conjugations using DBCO reagents are not regioselective and generate mixtures of products with proteins containing multiple cysteines. (c) The DBCO-tag enables site-selective thiol-yne reaction that modifies the DBCO-tag cysteine in the presence of other competing cysteine residues on the same protein.

Figure 2.

DBCO-tag enabled site-selective mEGFP labelling. (a) Site-selective conjugation between DBCO-tag-mEGFP and DBCO-(PEG)₄-biotin (1). A small amount of G₃-mEGFP was present in the starting material and reaction mixture due to incomplete sortagging reaction. Reaction conditions: 50 μM DBCO-tag-mEGFP, 1 mM DBCO-(PEG)₄-biotin (1), 0.2 M phosphate, 1 mM DTT, pH 8.0, 6 % DMSO, 37°C, 16 hours. (b) N-ethylmaleimide protein labelling resulted in heterogeneous products. Reaction conditions: 50 μM DBCO-tag-mEGFP, 0.2 M phosphate, 1 mM DTT, pH 8.0, 37°C, 15 minutes, then add 5 mM N-ethylmaleimide to the reduced protein and reacted at 37°C for 1 hour.

Figure 3.

DBCO-tag enabled site-selective antibody labelling without impairing the protein binding function. (a) Site-selective antibody modification using DBCO-tag. The DBCO-tag was placed at the C-terminus of antibody heavy chain. A small amount of C-terminal truncation (minus Lys-Gly) was present in the starting material, and was labeled as ‘-KG’. Reaction conditions: 100 μM DBCO-tag-trastuzumab, 2 mM DBCO-(PEG)₄-biotin, 0.2 M phosphate, 10 mM DTT, pH 8.0, 10% DMSO, 37°C, 4 hours. (b) Site-selectively biotinylated trastuzumab retains its binding affinity to recombinant HER2 in Octet BioLayer Interferometry assay. 20 μM site-selectively biotinylated DBCO-tag-trastuzumab was immobilized on the streptavidin biosensors and sampled with serially diluted concentrations of recombinant HER2 (concentrations shown next to each sensorgram); see Figure S24 for fitting and data analysis.