Click and bioorthogonal chemistry

What counts as click, and what counts as bioorthogonal

A click reaction is modular, high-yielding, insensitive to oxygen and water, and gives products that are easy to isolate. A bioorthogonal reaction must also ignore amines, thiols, carboxylates and every other group found in a cell, work at 37 °C and pH 7 in water, be non-toxic, and use reaction partners small enough not to disturb the biomolecule they are attached to.

CuAAC

The thermal Huisgen 1,3-dipolar cycloaddition of an azide and a terminal alkyne needs heat and gives a mixture of 1,4- and 1,5-disubstituted 1,2,3-triazoles. In 2002 Meldal and Sharpless independently showed that copper(I) changes the mechanism and makes it fast at room temperature and fully selective for the 1,4-triazole. Copper first forms a σ-acetylide; evidence points to a second copper atom helping to activate the azide, which binds and cyclises to a six-membered copper metallacycle that contracts to a copper triazolide; protonation releases the product. Ruthenium catalysts give the 1,5-isomer instead. CuAAC is excellent for labelling fixed cells, lysates and materials, but Cu(I) produces reactive oxygen species, so in live cells it needs protective ligands such as THPTA and still causes some toxicity.

Strain-promoted azide–alkyne cycloaddition (SPAAC)

Bending an alkyne into an eight-membered ring stores enough strain that a cyclooctyne reacts with an azide without any metal. Bertozzi's group introduced this in 2004. Fluorines next to the alkyne (DIFO) or fused benzene rings (DIBO, DBCO) raise the rate, while BCN is smaller and less lipophilic.

Tetrazine ligation

An electron-poor 1,2,4,5-tetrazine reacts with a strained, electron-rich alkene such as trans-cyclooctene (TCO) by an inverse-electron-demand Diels–Alder reaction, then loses N_2 in a retro-Diels–Alder step, so it is irreversible. The dihydropyridazine product can then oxidise to a pyridazine.

Why rate constants matter

Labelling reagents are used at micromolar concentrations. With the partner at 10 µM, pseudo-first-order kinetics gives t_{1/2} = \ln 2/(k[\mathrm{B}]). A reaction with k = 1 M^{-1} s^{-1} (typical of good SPAAC) has t_{1/2} ≈ 19 hours; a tetrazine–TCO pair at k = 10^4 M^{-1} s^{-1} has t_{1/2} ≈ 7 seconds. Fast kinetics is what makes labelling in whole animals possible.

A typical experiment

Cells fed peracetylated N-azidoacetylmannosamine (Ac_4ManNAz) process it into azido sialic acids that end up on cell-surface glycans. A DBCO-dye conjugate then lights up those glycans.

Current bioorthogonal research is driven by moving reactions from cultured cells into animals and patients.

Pretargeting. A TCO-tagged antibody is injected first and left for a day or more to accumulate on a tumour and clear from the blood. A small radiolabelled tetrazine follows; it finds the antibody within minutes and the excess is excreted quickly. This decouples the slow pharmacokinetics of antibodies from short-lived isotopes such as ^{18}F or ^{64}Cu for PET imaging, and lowers radiation dose to healthy tissue in radioimmunotherapy.

Click-to-release. When a tetrazine reacts with a TCO carrying an allylic carbamate, the tautomerised product eliminates and releases a free amine. Prodrugs built this way, for example doxorubicin caged on TCO and activated by a tetrazine-modified biomaterial injected at the tumour, have reached clinical trials, and the same chemistry decages proteins inside cells.

Fluorogenic probes. Tetrazines quench many attached fluorophores, and the quenching is lost once the ring has reacted, so signal turns on only at the labelled site. This allows no-wash and super-resolution imaging.

Genetic code expansion installs amino acids bearing TCO, BCN or cyclopropene handles at a chosen position in a protein, giving site-specific labelling and homogeneous antibody–drug conjugates.

Open problems. The most reactive tetrazines are also the least stable in serum, and TCO slowly isomerises to the unreactive cis isomer in the presence of thiols and copper-containing proteins; tuning this reactivity–stability balance occupies many groups. Running two or three reactions simultaneously in one cell requires pairs that are mutually orthogonal, not just orthogonal to biology. Newer click families such as SuFEx (sulfur(VI) fluoride exchange) are widening what 'click' means, particularly for covalent probes and polymer chemistry.