The term bioorthogonal chemistry refers to any chemical reaction that can occur inside of living systems without interfering with native biochemical processes. The term was coined by Carolyn R. Bertozzi in 2003. Since its introduction, the concept of the bioorthogonal reaction has enabled the study of biomolecules such as glycans, proteins, and lipids in real time in living systems without cellular toxicity. A number of chemical ligation strategies have been developed that fulfill the requirements of bioorthogonality, including the 1,3-dipolar cycloaddition between azides and cyclooctynes (also termed copper-free click chemistry), between nitrones and cyclooctynes, oxime/hydrazone formation from aldehydes and ketones, the tetrazine ligation, the isocyanide-based click reaction, and most recently, the quadricyclane ligation.
The use of bioorthogonal chemistry typically proceeds in two steps. First, a cellular substrate is modified with a bioorthogonal functional group (chemical reporter) and introduced to the cell; substrates include metabolites, enzyme inhibitors, etc. The chemical reporter must not alter the structure of the substrate dramatically to avoid affecting its bioactivity. Secondly, a probe containing the complementary functional group is introduced to react and label the substrate.
Although effective bioorthogonal reactions such as copper-free click chemistry have been developed, development of new reactions continues to generate orthogonal methods for labeling to allow multiple methods of labeling to be used in the same biosystems. Carolyn R. Bertozzi was awarded the Nobel Prize in Chemistry in 2022 for her development of click chemistry and bioorthogonal chemistry.
Etymology The word bioorthogonal comes from Greek bio- "living" and orthogōnios "right-angled". Thus literally a reaction that goes perpendicular to a living system, thus not disturbing it.
Requirements for bioorthogonality To be considered bioorthogonal, a reaction must fulfill a number of requirements: Selectivity: The reaction must be selective between endogenous functional groups to avoid side reactions with biological compounds Biological inertness: Reactive partners and resulting linkage should not possess any mode of reactivity capable of disrupting the native chemical functionality of the organism under study. Chemical inertness: The covalent link should be strong and inert to biological reactions. Kinetics: The reaction must be rapid so that covalent ligation is achieved prior to probe metabolism and clearance. The reaction must be fast, on the time scale of cellular processes (minutes) to prevent competition in reactions which may diminish the small signals of less abundant species. Rapid reactions also offer a fast response, necessary in order to accurately track dynamic processes. Reaction biocompatibility: Reactions have to be non-toxic and must function in biological conditions taking into account pH, aqueous environments, and temperature. Pharmacokinetics are a growing concern as bioorthogonal chemistry expands to live animal models. Accessible engineering: The chemical reporter must be capable of incorporation into biomolecules via some form of metabolic or protein engineering. Optimally, one of the functional groups is also very small so that it does not disturb native behavior.
Staudinger ligation The Staudinger ligation is a reaction developed by the Bertozzi group in 2000 that is based on the classic Staudinger reaction of azides with triarylphosphines. It launched the field of bioorthogonal chemistry as the first reaction with completely abiotic functional groups although it is no longer as widely used. The Staudinger ligation has been used in both live cells and live mice.
Bioorthogonality The azide can act as a soft electrophile that prefers soft nucleophiles such as phosphines. This is in contrast to most biological nucleophiles which are typically hard nucleophiles. The reaction proceeds selectively under water-tolerant conditions to produce a stable product.
Phosphines are completely absent from living systems and do not reduce disulfide bonds despite mild reduction potential. Azides had been shown to be biocompatible in FDA-approved drugs such as azidothymidine and through other uses as cross linkers. Additionally, their small size allows them to be easily incorporated into biomolecules through cellular metabolic pathways.
Mechanism
Classic Staudinger reaction
The nucleophilic phosphine attacks the azide at the electrophilic terminal nitrogen. Through a four-membered transition state, N2 is lost to form an aza-ylide. The unstable ylide is hydrolyzed to form phosphine oxide and a primary amine. However, this reaction is not immediately bioorthogonal because hydrolysis breaks the covalent bond in the aza-ylide.
Staudinger ligation
The reaction was modified to include an ester group ortho to the phosphorus atom on one of the aryl rings to direct the aza-ylide through a new path of reactivity in order to outcompete immediate hydrolysis by positioning the ester to increase local concentration. The initial nucleophilic attack on the azide is the rate-limiting step. The ylide reacts with the electrophilic ester trap through intramolecular cyclization to form a five-membered ring. This ring undergoes hydrolysis to form a stable amide bond.
Limitations The phosphine reagents slowly undergo air oxidation in living systems. Additionally, it is likely that they are metabolized in vitro by cytochrome P450 enzymes.
The kinetics of the reactions are slow with second order rate constants around 0.0020 M−1•s−1. Attempts to increase nucleophilic attack rates by adding electron-donating groups to the phosphines improved kinetics, but also increased the rate of air oxidation.
The poor kinetics require that high concentrations of the phosphine be used which leads to problems with high background signal in imaging applications. Attempts have been made to combat the problem of high background through the development of a fluorogenic phosphine reagents based on fluorescein and luciferin, but the intrinsic kinetics remain a limitation.
Copper-free click chemistry
Copper-free click chemistry is a bioorthogonal reaction first developed by Carolyn Bertozzi as an activated variant of an azide alkyne Huisgen cycloaddition, based on the work by Karl Barry Sharpless et al. Unlike CuAAC, Cu-free click chemistry has been modified to be bioorthogonal by eliminating a cytotoxic copper catalyst, allowing reaction to proceed quickly and without live cell toxicity. Instead of copper, the reaction is a strain-promoted alkyne-azide cycloaddition (SPAAC). It was developed as a faster alternative to the Staudinger ligation, with the first generations reacting over sixty times faster. The bioorthogonality of the reaction has allowed the Cu-free click reaction to be applied within cultured cells, live zebrafish, and mice.
Copper toxicity The classic copper-catalyzed azide-alkyne cycloaddition has been an extremely fast and effective click reaction for bioconjugation, but it is not suitable for use in live cells due to the toxicity of Cu(I) ions. Toxicity is due to oxidative damage from reactive oxygen species formed by the copper catalysts. Copper complexes have also been found to induce changes in cellular metabolism and are taken up by cells.
There has been some development of ligands to prevent biomolecule damage and facilitate removal in in vitro applications. However, it has been found that different ligand environments of complexes can still affect metabolism and uptake, introducing an unwelcome perturbation in cellular function.
Bioorthogonality The azide group is particularly bioorthogonal because it is extremely small (favorable for cell permeability and avoids perturbations), metabolically stable, and does not naturally exist in cells and thus has no competing biological side reactions. Although azides are not the most reactive 1,3-dipole available for reaction, they are preferred for their relative lack of side reactions and stability in typical synthetic conditions. The alkyne is not as small, but it still has the stability and orthogonality necessary for in vivo labeling. Cyclooctynes are traditionally the most common cycloalkyne for labeling studies, as they are the smallest stable alkyne ring.
Mechanism
The reaction proceeds as a standard 1,3-dipolar cycloaddition, a type of asynchronous, concerted pericyclic shift. The ambivalent nature of the 1,3-dipole should make the identification of an electrophilic or nucleophilic center on the azide impossible such that the direction of the cyclic electron flow is meaningless. [p] However, computation has shown that the electron distribution amongst nitrogens causes the innermost nitrogen atom to bear the greatest negative charge.
Regioselectivity Although the reaction produces a regioisomeric mixture of triazoles, the lack of regioselectivity in the reaction is not a major concern for most current applications. More regiospecific and less bioorthogonal requirements are best served by copper-catalyzed Huisgen cycloaddition, especially given the synthetic difficulty (compared to the addition of a terminal alkyne) of synthesizing a strained cyclooctyne.
Development of cyclooctynes Cyclooctyne Second order rate constant (M−1s−1) OCT 0.0024 ALO 0.0013 MOFO 0.0043 DIFO 0.076 DIBO 0.057 BARAC 0.96 DIBAC (ADIBO) 0.31 DIMAC 0.0030
OCT was the first cyclooctyne developed for Cu-free click chemistry. While linear alkynes are unreactive at physiological temperatures, OCT was able readily react with azides in biological conditions while showing no toxicity. However, it was poorly water-soluble, and the kinetics were barely improved over the Staudinger ligation. ALO (aryl-less octyne) was developed to improve water solubility, but it still had poor kinetics.
Monofluorinated (MOFO) and difluorinated (DIFO) cyclooctynes were created to increase the rate through the addition of electron-withdrawing fluorine substituents at the propargylic position. Fluorine is a good electron-withdrawing group in terms of synthetic accessibility and biological inertness. In particular, it cannot form an electrophilic Michael acceptor that may side-react with biological nucleophiles. DIBO (dibenzocyclooctyne) was developed as a fusion to two aryl rings, resulting in very high strain and a decrease in distortion energies. It was proposed that biaryl substitution increases ring strain and provides conjugation with the alkyne to improve reactivity. Although calculations have predicted that mono-aryl substitution would provide an optimal balance between steric clash (with azide molecule) and strain, monoarylated products have been shown to be unstable.
BARAC (biarylazacyclooctynone) followed with the addition of an amide bond which adds an sp2-like center to increase rate by distortion. Amide resonance contributes additional strain without creating additional unsaturation which would lead to an unstable molecule. Additionally, the addition of a heteroatom into the cyclooctyne ring improves both solubility and pharmacokinetics of the molecule. BARAC has sufficient rate (and sensitivity) to the extent that washing away excess probe is unnecessary to reduce background. This makes it extremely useful in situations where washing is impossible as in real-time imaging or whole animal imaging. Although BARAC is extremely useful, its low stability requires that it must be stored at 0 °C, protected from light and oxygen.
Further adjustments variations on BARAC to produce DIBAC/ADIBO were performed to add distal ring strain and reduce sterics around the alkyne to further increase reactivity. Keto-DIBO, in which the hydroxyl group has been converted to a ketone, has a three-fold increase in rate due to a change in ring conformation. Attempts to make a difluorobenzocyclooctyne (DIFBO) were unsuccessful due to the instability.