Review
Nature Chemical Biology1, 13-21 (2005)
doi: 10.1038/nchembio0605-13
Chemistry in living systems
Jennifer A Prescher1 andCarolyn R Bertozzi1,2,3,4
Dissecting complex cellular processes requires the ability to track biomolecules as they function within their native habitat. Although genetically encoded tags such as GFP are widely used to monitor discrete proteins, they can cause significant perturbations to a protein's structure and have no direct extension to other classes of biomolecules such as glycans, lipids, nucleic acids and secondary metabolites. In recent years, an alternative tool for tagging biomolecules has emerged from the chemical biology community—the bioorthogonal chemical reporter. In a prototypical experiment, a unique chemical motif, often as small as a single functional group, is incorporated into the target biomolecule using the cell's own biosynthetic machinery. The chemical reporter is then covalently modified in a highly selective fashion with an exogenously delivered probe. This review highlights the development of bioorthogonal chemical reporters and reactions and their application in living systems.
Living systems are composed of networks of interacting biopolymers, ions and metabolites. These components drive a complex array of cellular processes, many of which cannot be observed when the biomolecules are examined in their purified, isolated forms. Accordingly, researchers have begun moving beyond the artificial confines of test tubes to study biological processes in the context of living cells and whole organisms. This endeavor requires the ability to track molecules within their native environs. Few biomolecules are naturally endowed with features that permit their direct detection in complex milieus. Thus, several methods have been developed to equip cellular components with reporter tags for visualization and isolation from biological samples.
The most popular tagging strategy for cellular imaging involves the use of the green fluorescent protein (GFP) and its related variants1, 2, 3. The fusion of these fluorescent probes to a target protein enables visualization by fluorescence microscopy and quantification by flow cytometry. Because they are genetically encoded and require no auxiliary cofactors, GFP tags can be used to analyze protein expression and localization in living cells and whole organisms4, 5. Almost every cellular process has been interrogated using fluorescent protein fusions, including glycoprotein transport in the secretory pathway6 and transcription in the nucleus7. Furthermore, a collection of GFP-like tags is now available with emission wavelengths that span virtually the entire visible spectrum8, 9, 10.
Although fluorescent protein fusions are undoubtedly the most powerful general tools for imaging proteins within living systems, they are not without limitations. These relatively large proteins can be a significant structural perturbation and may therefore influence the expression, localization or function of the protein to which they are attached. Also, fluorescent protein fusions can be visualized only by optical methods, without an obvious extension to other imaging modalities. Finally, GFP variants cannot be applied to visualization of glycans, lipids, nucleic acids or the thousands of small organic metabolites amassed within cells (Fig. 1). Non-proteinaceous materials comprise a significant fraction of cellular biomass11, and the ability to image these species would augment our understanding of cellular biochemistry. Glycans, lipids and inorganic ions are also involved in modulating protein activity by post-translational modification12. Therefore, methods to visualize both proteins and their modifiers would contribute to a more holistic understanding of the proteome.
Figure 1:Composition of a typical mammalian cell11.
Although proteins comprise the largest fraction of a cell's dry mass, it is estimated that more than half are modified with glycans, lipids or other metabolites113. Methods for visualizing both proteins and non-proteinaceous biomolecules would enhance our understanding of living systems.
Full figure and legend (54K) Figures, schemes & tables index
Antibody conjugates have been widely used to track biomolecules in living cells and whole organisms13. They can be generated with specificity for virtually any epitope and are therefore, in principle, applicable to imaging a wide range of biomolecules. However, the large size and physical properties of these reagents hinder their access to antigens within cells and outside of the vasculature in living animals14, 15.
In general, small molecules have better access to intracellular and extravascular compartments. Their use as imaging agents requires a means to selectively target the small probe to a desired biomolecule. Nucleophilic functionality occurs in most types of biopolymers, permitting facile derivatization with biotin, fluorophores and numerous other small-molecule reporters. Established bioconjugation protocols have made these operations trivial for purified biopolymers in vitro16. However, the site-specific chemical modification of biomolecules within their native settings remains a formidable challenge.
In recent years, an alternative strategy for tagging biomolecules has emerged that blends the simplicity of genetically encoded tags with the specificity of antibody labeling and the versatility of small-molecule probes. This approach involves the incorporation of unique chemical functionality—a bioorthogonal chemical reporter—into a target biomolecule using the cell's own biosynthetic machinery. Bioorthogonal chemical reporters are non-native, non-perturbing chemical handles that can be modified in living systems through highly selective reactions with exogenously delivered probes. This two-step labeling process can be used to outfit a target biomolecule for detection or isolation, depending on the nature of the probe. Proteins17, 18, 19, 20, glycans21, 22, 23, 24 and lipids25 have all been fashioned with an assortment of chemical reporters in living cells and subsequently ligated with reactive probes. Most recently, the chemical reporter strategy has been applied to monitoring enzyme activities26, 27, 28, 29 and tagging cell surface glycans in whole organisms30. The breadth of these examples underscores the impact of bioorthogonal chemical reporters in expanding the repertoire of biomolecules that can be visualized in living systems.
Here we summarize the development of bioorthogonal chemical reporters and their applications in biology. First, we provide an overview of existing chemical reporters and bioorthogonal reactions. Second, we discuss the applications of these chemistries to monitoring biomolecules and enzyme activities in cellular systems. Third, we highlight the translation of one chemical reporter system from cell-based studies to living animals. Last, we outline future challenges in the field, from the perspective of both chemists and biologists.
Design of chemical reporters and bioorthogonal reactions
The bioorthogonal chemical reporter strategy involves the incorporation of unique functionality into targets of interest, followed by chemical labeling with a small-molecule probe (Fig. 2). Ideally, the chemical reporter (blue circle, Fig. 2) should be integrated into the target scaffold without significant structural perturbation. This is accomplished by appending the reporter to substrates that can be used by the cell's own metabolic machinery. For example, amino acids bearing bioorthogonal functional groups can be accepted by the translational machinery of a cell and incorporated into proteins. Similarly, functionalized monosaccharides can be introduced into cell surface glycans by means of promiscuous enzymes in the biosynthetic pathways of these biopolymers. Regardless of the route exploited, each enzyme involved in the installation process must tolerate the unnatural motif. For this reason, typical biophysical probes, such as fluorescein, cannot be used as direct modifications to metabolic substrates (that is, amino acids, lipids or sugars) as their relatively large size would interfere with enzymatic transformations. A small functional group is more likely to be tolerated by metabolic enzymes. Thus, to date, bioorthogonal chemical reporters have been non-native combinations of endogenous functionality (as discussed below) or small, abiotic functional groups that can slip through existing biosynthetic pathways.
Figure 2:The bioorthogonal chemical reporter strategy.
A chemical reporter (blue circle) linked to a substrate (light green box) is introduced into a target biomolecule through cellular metabolism. In a second step, the reporter is covalently tagged with an exogenously delivered probe (blue arc). Both the chemical reporter and exogenous probe must avoid side reactions with nontarget biomolecules (gray shapes).
Full figure and legend (7K) Figures, schemes & tables index
Once installed in a target biomolecule, the chemical reporter must be reacted with a probe bearing a complementary chemical moiety (blue arc, Fig. 2). The requirements for the covalent reaction between the two components are quite stringent. The reporter and its partner must be mutually reactive in a physiological environment (37 °C, pH 6−8) and, at the same time, remain inert to the surrounding biological milieu. Ideally, the reactants should function similar to an antibody-antigen duo, reacting rapidly with one another, unaided by auxiliary reagents, to form a stable adduct with innocuous (or no) byproducts. Considering the abundance of nucleophiles, reducing agents and other functionality present in cells, the choice of suitable components for the chemical transformation is far from obvious. For instance, amines and isothiocyanates, thiols and maleimides, and other coupling partners typically used for bioconjugation must be avoided to prevent labeling of irrelevant targets. In addition, the chemical reporter and its complementary probe must possess adequate metabolic stability and bioavailability for use in cells or organisms.
Existing bioorthogonal chemical reporters
So far, only a handful of chemical motifs are known to possess the re-quisite qualities of biocompatibility and selective reactivity to function as bioorthogonal chemical reporters in living cells. This elite group comprises peptide sequences that can be ligated with small-molecule imaging probes18, 31, cell surface electrophiles that can be tagged with hydrazide and aminooxy derivatives19, 22, azides that can be selectively modified with phosphines32 or activated alkynes33, 34, and terminal alkynes that can be ligated with azides (Table 1)29. The sections that follow introduce each of these chemical reporters and summarize their advantages and disadvantages in tagging biomolecules in cellular systems.
Table 1: Chemical reporters and bioorthogonal reactions used in living systems.
Full tableFigures, schemes & tables index
Bioorthogonal peptide sequences. No single proteogenic amino acid side chain can function as a unique chemical moiety for target-specific tagging. However, Tsien and coworkers have demonstrated that unique combinations of side chains can create new functionality that satisfies the criteria of a bioorthogonal chemical reporter. They designed a short peptide sequence containing a tetracysteine motif (CCXXCC, where XX are virtually any two amino acids, but optimally proline and glycine) that reacts selectively with biarsenicals18, 31. The hexapeptide chemical reporter can be fused to target proteins at the genetic level and covalently labeled in living cells with membrane-permeant biarsenical dyes, such as the fluorescein derivative FlAsH and the resorufin derivative ReAsH (Table 1). The ethanedithiol substituents of these reagents prevent the labeling of biomolecules bearing isolated cysteine residues. Furthermore, the biarsenical probes are only weakly fluorescent when free in solution and undergo a marked increase in fluorescence when bound to the target sequence. Target singularity is ensured by the rarity of the hexapeptide motif among endogenous proteins.
The tetracysteine reporter group has been used to image a variety of proteins, including some whose distribution was known to be perturbed by GFP labeling35, 36. In several studies, combinations of FlAsH and ReAsH were used to observe the real-time assembly, trafficking and degradation of the protein targets (Fig. 3)37, 38. ReAsH also doubles as a photosensitizer, generating singlet oxygen to selectively inactivate proteins to which it is fused39 or to produce contrast stains for electron microscopy37.
Figure 3:Bioorthogonal chemical reporters and cellular imaging.
HeLa cells expressing tetracysteine-fused connexin were treated with FlAsH (green), incubated in medium for 4 hours, then treated with ReAsH (red) and imaged. This two-color pulse-chase labeling experiment demonstrated that newly synthesized connexin is incorporated at the outer edges of existing gap junctions (indicated by white arrows)37. Figure reproduced from ref. 37 by permission of the American Association for the Advancement of Science.
Full figure and legend (25K) Figures, schemes & tables index
The tetracysteine-biarsenical method has also inspired the deve-lopment of several complementary approaches for attaching small molecules to proteins40, 41, 42, 43. Many of these strategies involve enzymatic reactions or ligand-receptor binding. For example, proteins can be labeled by fusion to an enzyme (for example, human O6-alkylguanine transferase44) or receptor (for example, FKBP12(F36V)45, dihydrofolate reductase46, 47) that is capable of binding functionalized probes. Additionally, proteins can be labeled by fusion to peptide sequences that bind small-molecule reagents. These include histidine-rich peptides recognized by functionalized Ni-NTA probes48, peptide aptamers engineered to bind the fluorophore Texas Red49 and acidic peptides that can bind luminescent lanthanides50. Muir and coworkers have also reported the use of trans-splicing inteins for tagging proteins in living cells51. Further optimization of all these labeling methodologies may permit their more widespread application in biological systems.
In summary, the tetracysteine-biarsenical system affords a powerful alternative to GFP tagging for protein visualization. The hexapeptide tag is a minimal structural perturbation relative to fluorescent proteins. Still, as a modification to metabolic substrates such as amino acids and monosaccharides, the hexapeptide tag is unlikely to be tolerated by biosynthetic enzymes. Thus, metabolic labeling of biopolymers other than proteins requires an alternative—and even smaller—chemical reporter. As described below, carefully chosen, simple functional groups can fulfill this purpose.
Ketones and aldehydes. Comprising only a handful of atoms, ketones and aldehydes are bioorthogonal chemical reporters that can tag not only proteins, but also glycans and other secondary metabolites (Table 1). These mild electrophiles are attractive choices for modifying biomolecules as they are readily introduced into diverse scaffolds, absent from endogenous biopolymers and essentially inert to the reactive moieties normally found in proteins, lipids and other macromolecules. Although these carbonyl compounds can form reversible Schiff bases with primary amines such as lysine side chains, the equilibrium in water favors the carbonyl. By contrast, the stabilized Schiff bases with hydrazide and aminooxy groups (hydrazones and oximes, respectively) are favored in water and are quite stable under physiological conditions52.
Rideout and coworkers recognized the potential use of ketones and aldehydes for chemoselective drug assembly in the presence of living cells53, 54, 55. They reported that decanal and octyl aminoguanidine—both independently harmless to cells—react selectively to form a hydrazone-linked detergent capable of lysing cultured erythrocytes. This same strategy was used to generate inhibitors of protein kinase C from the in situ assembly of aldehyde and hydrazide precursors56. As described in more detail later, this transformation has been used to chemically modify mammalian cell surfaces19, 22, 23, 57, 58. More recently, Sadamoto and coworkers introduced ketones into bacterial cell walls and labeled the reporters with a hydrazide-based fluorophore59.
Although suitable for chemical modifications in the presence of cultured cells, ketone (and aldehyde) condensations are somewhat limited in the context of living organisms. The pH optimum of these reactions is 5−6, values that cannot be achieved in most tissues in vivo. Additionally, ketones and aldehydes are not truly bioorthogonal in more complex physiological settings. Keto and aldehydic metabolites are abundant within cells and in biological fluids in the form of free sugars, pyruvate, oxaloacetate and various cofactors (such as pyridoxal phosphate). Therefore, aldehydes and ketones are best used in environs devoid of carbonyl electrophiles (namely, on cell surfaces or in the extracellular environment) and should be considered 'biorestricted' chemical reporters.
Azides. In contrast to aldehydes and ketones, azides are viable chemical reporters for labeling all classes of biomolecules in any biological locale (Table 1). This versatile functional group is abiotic in animals and absent from nearly all naturally occurring species. (Only one naturally occurring azido metabolite has been reported to date, isolated from unialgal cultures.)60 Azides do not react appreciably with water and are resistant to oxidation. Additionally, azides are mild electrophiles; but unlike aldehydes, they do not react with amines or the other 'hard' nucleophiles that are abundant in biological systems. Rather, they require 'soft' nucleophiles for reaction. Azides are therefore susceptible to reduction by free thiols, including the ubiquitous cellular reductant, glutathione. However, reactions between monothiols and alkyl azides typically require vigorous heating (100 °C for several hours) or auxiliary catalysts60, 61.
Despite its exquisite bioorthogonality, the azide has only recently been used as a chemical reporter in living systems. This may be due to perceptions of the azide as unstable, toxic or both. Azides are prone to decomposition at elevated temperatures, but they are quite stable at physiological temperatures60. Whereas aryl azides are well-known photocrosslinkers, alkyl azides do not photodecompose in the presence of ambient light. Finally, although azide anion (for example, in the form of NaN3) is a widely used cytotoxin, organic azides have no intrinsic toxicity. Indeed, organic azides are components of clinically approved drugs such as AZT60.
Although kinetically stable, azides are predisposed to unique modes of reactivity owing to their large intrinsic energy content. This feature has been exploited for the development of bioorthogonal reactions, including the Staudinger ligation of azides with functionalized phosphines and the [3+2] cycloaddition of azides with activated alkynes. These reactions can be used for the selective labeling of azide-functionalized biomolecules.
Staudinger ligation. In 1919, Hermann Staudinger reported that azides react with triphenylphosphines (soft nucleophiles) under mild conditions to produce aza-ylide intermediates62. These intermediates can be subsequently hydrolyzed in water or trapped by myriad electrophiles to provide a pair of products: an amine and the corresponding phosphine oxide63. The bioorthogonal nature of this transformation suggested potential applications of the azide as a chemical reporter, provided a covalent link could be forged between the two reactants. We modified the classic Staudinger reaction by introduction of an intramolecular trap into the phosphine (Fig. 4)32. Now known as the Staudinger ligation, this transformation ultimately produces a covalent link between one nitrogen atom of the azide and the triarylphosphine scaffold. The Staudinger ligation can be used to covalently attach probes to azide-bearing biomolecules. Like the azide, phosphines do not react appreciably with biological functional groups and are therefore also bioorthogonal. Additionally, the reaction proceeds readily at pH 7 with no apparent toxic effects. Oxidation of the phosphine by air or metabolic enzymes is the only potentially problematic side reaction that may diminish the amount of probe that is available in biological systems.