Bringing chemistry to life

Michael Boyce & Carolyn R Bertozzi Bioorthogonal

Chemistry allows a wide variety of biomolecules to be specifically labeled and probed in living cells and whole organisms. Here we discuss the history of bioorthogonal reactions and some of the most interesting and important advances in the field.

Since the galvanic rediscovery of Gregor Mendel’s work over a century ago, the modern biosciences have made astounding advances in our mechanistic understanding of living systems. Genetics, biochemistry, molecular biology and allied fields have provided especially impressive insight into the structures and functions of DNA, RNA and proteins, leading to such recent achievements as the sequencing of the human genome. In modern cell biology, proteins can be visualized using fluorescent protein fusions and knocked down by RNA-mediated silencing. Rapid progress in the life sciences continues as new technologies such as DNA deep sequencing, genome-wide expression profiling and mass spectrometry–based proteomics transform how biology is done. However, not all biological molecules and processes are within the easy reach of genetics or genomics. Glycans, lipids, small metabolites and myriad post-translational modifications are not encoded directly by the genome, making them challenging to study with traditional biological tools alone. Furthermore, many dynamic biological processes occur on short time scales not amenable to genetic or biochemical interrogation. Post-genomic science has set in sharp relief the need for new technologies that take aim at these molecules and processes. The field of bioorthogonal chemistry thus emerged from a perceived technology gap that rendered many biomolecules, initially glycans1,2, invisible to available probing strategies. Though considered a relatively new sector of chemical biology, bioorthogonal chemistry seeks to solve an old problem: finding a needle in a haystack. That is, among all of the molecular diversity inherent to cells and organisms, how can one type of biomolecule be singled out for analysis? In the 20th century, the monoclonal antibody transformed the biosciences as we had known them3. Antibodies are unrivaled in their ability to seek out a single molecular target among millions of distractions and bind with high affinity. But antibodies are not a panacea: they generally cannot enter live cells, restricting their use to the extracellular environment; they have poor tissue penetrance in animals; and they must be laboriously generated de novo for each new antigen. Thus, in addition to its aim to target new classes of biomolecules, bioorthogonal chemistry was a solution to the challenge of replicating the exquisite selectivity of antibody-antigen binding with a single covalent reaction among complementary functional groups. The term bioorthogonal chemistry refers to chemical transformations among abiotic reactants that can proceed in living systems (for example, cells or organisms) without interfering with, or interference from, the surrounding biological milieu. Devising such reactions presents a major and largely unfamiliar challenge to chemists, as most of us were trained that such offending substances as water and air can be excluded from our reactions, competing functional groups protected, catalysts added and temperature modulated. However, to be maximally useful in biological research, bioorthogonal reactions must proceed smoothly in water at physiological pH, temperature and pressure, provide good yield and reasonable kinetics at low reagent concentrations, remain inert to abundant biological nucleophiles, electrophiles and redox-active metabolites, and produce only nontoxic (or no) side products. The notion that single-target selectivity can be attained by covalent reaction in live cells was validated by groundbreaking work by Roger Tsien and co-workers in 1998, using bisarsenical-functionalized fluorescent dyes4,5 (Fig. 1a). They designed these abiotic molecules to react selectively with a tetracysteine motif that is genetically engineered into the protein of interest4,5. Although the term ‘bioorthogonal chemistry’ had not yet been coined, Tsien’s work sparked the imagination of chemists, who felt empowered to attempt covalent reactions in cells with entirely abiotic reactants. Notably, Tsien’s work also modeled what is now becoming a common theme in chemical biology—tool development motivated by specific biological problems. In this case, the challenge at hand was the perturbing effects that a large fluorescent protein fusion can have on an imaging target of interest. By contrast, the tetracysteine motif was a small addition to the target protein, with its bioorthogonality derived from the unique combination of natural amino acids that was virtually absent (we later learned) from mammalian proteomes. Subsequently, other groups have exploited such genetically encoded, orthogonal peptides by using engineered enzymes to covalently modify the tag, producing a useful chemical label6,7 (Fig. 1b). For example, elegant recent work with biotin ligase in a neuronal synapse model system8 demonstrated how © 2011 Nature America, Inc. All rights reserved. NATURE METHODS | VOL.8 NO.8 | AUGUST 2011 | 639 COMMENTARY | SPECIAL FEATURE O S HN H H NH NH2 H2NHN N N H H N N H H N OCH3 PPh2 FIAsH CCXXCC O HO HO HO OH HN O N3 O OH OH OH OH H O N O CO2 – HO O N3 HO OH As OH N3 O OH OH OH N3 S O O OH COOH S As S S O S HN H H NH BirA, biotin ATP QD605- streptavidin Metabolism Metabolism Aqueous buffer, pH 6.8 N N N O CoASH, acyl CoA synthetase N-myristoyltransferase OH Q O O O CO2H O O O O ManNAz Fluorophore-conjugated antibody Staudinger ligation metabolism PPh2 O O O O HO HO OH OH ( )11 O N H H N O ( )11 O N H H N O ( )11 Cu(I) N3 O OH F F OH OH F F O N O OH HO NH O Raman spectroscopy a d ef g b c O CO2 – O O N N N Figure 1 | Bioorthogonal reactions. (a) The biarsenical fluorescent dye FlAsH specifically reacts with target proteins genetically tagged with the tetracysteine motif4. (b) Small, genetically encoded peptide tags (red rectangle) can be specifically modified in or on live cells using engineered enzymes, such as the biotin ligase BirA6,7. Biotin-tagged target proteins can then be visualized by streptavidin-conjugated probes, such as quantum dots (QD605). (c) In an example of carbonyl condensations as a bioorthogonal method, unnatural, ketone-functionalized amino acids can be metabolically incorporated into proteins and then detected by reaction with a hydrazide-functionalized fluorescent probe (yellow star)11. (d) In an example of the Staudinger ligation, an azidosugar analog of N-acetylmannosamine (termed ManNAz) is used to metabolically label sialic acid residues on cell-surface glycoproteins. The azidosugar is detected by reaction with a tagged phosphine probe (orange rectangle) and visualized with an antibody to the tag2,21. (e) In an example of CuAAC chemistry, an alkynyl analog of myristic acid is metabolically processed to an acetyl coenzyme-A metabolite and then attached to protein N termini via N-myristoyltransferase17. The tagged protein can then be labeled via CuAAC using an azide probe (yellow star). (f) In an example of copper-free click chemistry, an azidosugar analog of fucose is metabolically incorporated into cell-surface glycans in live zebrafish embryos and detected by reaction with a fluorescent difluorinated cyclooctyne (yellow star)39,49. (g) DNA labeled with metabolically incorporated 5b-ethynyl-2’-deoxyuridine is visualized via direct Raman spectroscopic detection of the intact alkyne48. Katie Vicari these techniques can shed light on aspects of biology that would be difficult to investigate with conventional techniques alone.

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