The term bioorthogonal chemistry refers to any chemical reaction

Chemical reaction

A chemical reaction is a process that leads to the transformation of one set of chemical substances to another. Chemical reactions can be either spontaneous, requiring no input of energy, or non-spontaneous, typically following the input of some type of energy, such as heat, light or electricity...

 that can occur inside of living systems

Living systems

Living systems are open self-organizing living things that interact with their environment. These systems are maintained by flows of information, energy and matter....

 without interfering with native biochemical processes. The term was coined by Carolyn R. Bertozzi

Carolyn R. Bertozzi

Carolyn Ruth Bertozzi is an American chemist. She is the T.Z. and Irmgard Chu Distinguished Professor of Chemistry and Professor of Molecular and Cell Biology at the University of California, Berkeley; Professor of Molecular and Cellular Pharmacology at the University of California, San...

 with the development of the Staudinger ligation

Staudinger reaction

The Staudinger reaction or Staudinger reduction is a chemical reaction in which the combination of an azide with a phosphine or phosphite produces an iminophosphorane intermediate. Combined with the hydrolysis of the aza-ylide to produce a phosphine oxide and an amine, this reaction is a mild...

 in 2000. Since its introduction, the concept of the bioorthogonal reaction has enabled the study of biomolecules such as glycans

Glycans

The term glycan refers to a polysaccharide or oligosaccharide. Glycans usually consist solely of O-glycosidic linkages of monosaccharides. For example, cellulose is a glycan composed of beta-1,4-linked D-glucose, and chitin is a glycan composed of beta-1,4-linked N-acetyl-D-glucosamine...

, proteins, and lipids in real time in living systems without cellular toxicity. In addition, a number of chemical ligation

Ligation

Ligation may refer to:* In molecular biology, the covalent linking of two ends of DNA molecules using DNA ligase* In medicine, the making of a ligature * Chemical ligation, the production of peptides from amino acids...

 strategies have been developed that fulfill the requirements of bioorthogonality, including the 1,3-dipolar cycloaddition

Cycloaddition

A cycloaddition is a pericyclic chemical reaction, in which "two or more unsaturated molecules combine with the formation of a cyclic adduct in which there is a net reduction of the bond multiplicity." The resulting reaction is a cyclization reaction.Cycloadditions are usually described by the...

 between azides and cyclooctynes (also termed copper-free click chemistry

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 Sharpless et. al. Unlike the CuAAC, Cu-free click chemistry has been modified to be bioorthogonal by eliminating a...

), between nitrones and cyclooctynes, oxime

Oxime

An oxime is a chemical compound belonging to the imines, with the general formula R1R2C=NOH, where R1 is an organic side chain and R2 may be hydrogen, forming an aldoxime, or another organic group, forming a ketoxime. O-substituted oximes form a closely related family of compounds...

/hydrazone

Hydrazone

Hydrazones are a class of organic compounds with the structure R1R2C=NNH2. They are related to ketones and aldehydes by the replacement of the oxygen with the NNH2 functional group...

 formation from aldehydes and ketones, the tetrazine

Tetrazine

Tetrazine is an unstable compound that consists of a six-membered aromatic ring containing four nitrogen atoms with the molecular formula C2H2N4. The name tetrazine is used in the nomenclature of derivatives of this compound...

 ligation, the isonitrile-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. Secondly, a probe containing the complementary functional group is introduced to react and label the substrate.

Requirements of bioorthogonality

To be considered bioorthogonal, a reaction must fulfill a number of requirements. First, the reaction must be selective and the reactive partners should not possess any mode of reactivity capable of disrupting the native chemical functionality of the organism under study. Second, 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 (minute), to prevent competition in reactions, which may diminish the small signals of less abundant species. Rapid reactions also offer the a fast response, necessary in order to accurately track dynamic processes. The functional groups and resulting covalent linkage must be inert to biological reactions to avoid perturbing the system. Additionally, reactivity must be achievable within the limitations of biocompatibility with living cells. Solubility in water and pharmacokinetics are a growing concern as bioorthogonal chemistry expands to live animal models.In addition, one of the reactive partners (the bioorthogonal chemical reporter

Bioorthogonal chemical reporter

In chemical biology, bioorthogonal chemical reporter is a non-native chemical functionality that is introduced into the naturally occurring biomolecules of a living system, generally through metabolic or protein engineering. These functional groups are subsequently utilized for tagging and...

) must be capable of incorporation into biomolecules via some form of metabolic or protein engineering.