Review
Nature Chemical Biology 1, 13-21 (2005)
doi: 10.1038/nchembio0605-13
Chemistry in living systems
Jennifer A Prescher1 and Carolyn 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 GFPlike 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 twostep 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 membranepermeant 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 twocolor 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 histidinerich 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 transsplicing 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.
Figure 4: The Staudinger ligation.
A triarylphosphine and an azide first react to form an aza-ylide intermediate. The
nucleophilic nitrogen atom is trapped in an intramolecular fashion, and the cyclized
intermediate hydrolyzes in water to form a stable amide-linked product. In some cases,
aryl azides (R' = aryl) may react with phosphines to initially form O-alkyl imidates114.
Full figure and legend (11K) Figures, schemes & tables index
The Staudinger ligation has been used to modify glycans on living cells32, enrich
glycoprotein subtypes from various proteomes64, 65 and impart new functionality to
recombinant proteins66. Raines and colleagues and researchers in our laboratory
developed phosphine reagents for a related transformation that produces amide-linked
products without incorporation of the phosphine oxide into the final adducts67, 68.
Although these phosphines have not been used for bioconjugation in living systems, they
have been used to immobilize small molecules69 and proteins70 on glass slides. These and
other applications of the Staudinger ligation have been recently reviewed71.
Copper-catalyzed [3+2] azide-alkyne cycloaddition. In the context of the Staudinger
ligation, the azide serves as an electrophile subject to reaction with soft nucleophiles.
Azides are also 1,3-dipoles that can undergo reactions with dipolarophiles such as
activated alkynes72. These -systems are both extremely rare and inert in biological
systems, further enhancing the bioorthogonality of the azide along this reaction
trajectory. The [3+2] cycloaddition between azides and terminal alkynes to provide stable
triazole adducts was first described by Huisgen more than four decades ago73. The
reaction is thermodynamically favorable by an impressive 30−35 kcal/mol. Without
alkyne activation, however, the process requires elevated temperatures or pressures that
are not compatible with living systems. How can activation be achieved? One possibility
involves the addition of electron-withdrawing groups (such as esters) to the alkyne.
Unfortunately, the resulting , -unsaturated carbonyl compounds can also act as Michael
acceptors for a variety of biological nucleophiles and are therefore not bioorthogonal.
Another possibility involves the use of a catalyst. Sharpless and coworkers and Meldal
and coworkers demonstrated that the rate of cycloaddition between azides and alkynes
can be accelerated 106-fold using catalytic amounts of Cu(I)74, 75. This copper-catalyzed
reaction, termed 'click' chemistry, proceeds readily at physiological temperatures and in
the presence of biological materials to provide 1,4-disubstituted triazoles with nearly
complete regioselectivity (Table 1)33. The copper-mediated reaction has been used to tag
azides installed within virus particles76, nucleic acids77 and proteins from complex tissue
lysates78 with virtually no background labeling. It should be noted that the same reaction
can be carried out using the alkyne as the chemical reporter (Table 1). Like the azide, a
terminal alkyne consists of a mere three atoms.
The primary advantage of the catalyzed azide-alkyne cycloaddition over the Staudinger
ligation is its faster rate. Based on preliminary studies in our laboratory, the coppercatalyzed reaction of azides with alkynes proceeds at least 25 times faster than the
reaction of azides with triarylphosphines in cell lysates. Accordingly, 'click' chemistry
has been used in situations that require detection of very small quantities of azide-labeled
biomolecules78. The primary disadvantage of the copper-catalyzed cycloaddition is the
cellular toxicity of the metal catalyst79. Although more biofriendly metal-ligand
combinations could potentially be discovered, the reaction is not ideal for labeling
biomolecules in living cells.
Strain-promoted cycloaddition. An alternative means of activating alkynes for catalystfree [3+2] cycloaddition with azides involves the use of ring strain34. Constraining the
alkyne within an eight-membered ring creates 18 kcal/mol of strain, much of which is
released in the transition state upon [3+2] cycloaddition with an azide80. As a
consequence, cyclooctynes react with azides at room temperature, without the need for a
catalyst81. This strain-promoted cycloaddition has been used to label biomolecules both in
vitro and on cell surfaces without observable toxic effects34. However, the reaction is
limited by its slow rate. (The second-order rate constant for the reaction of a derivatized
cyclooctyne with benzyl azide in aqueous CD3CN is 0.0012 M-1 s-1, whereas that for the
Staudinger ligation is 0.0025 M-1 s-1; refs. 34,82.) Preliminary results from our laboratory
indicate that the rate of the strain-promoted cycloaddition can be increased by appending
electron-withdrawing groups to the octyne ring (C.R.B., unpublished data).
Introducing ketones, azides and alkynes into biomolecules
Proteins. Ketones, azides and alkynes are not included in the repertoire of side chain
functional groups found in the 20 proteogenic amino acids. To exploit their bioorthogonal
chemistry for protein labeling requires a means for de novo introduction of these
chemical reporters, typically in the form of unnatural amino acids (Fig. 5). This can be
accomplished using a cell's translational machinery in either a residue-specific83 or a sitespecific manner84. As described by Tirrell and coworkers, residue-specific incorporation
of unnatural amino acids into proteins simply involves replacement of a natural residue
with a conservatively modified analog (Fig. 5a). The translational machinery is
sufficiently tolerant of altered substrates that, in the absence of competing natural
substrates, the modified residue is converted to an aminoacyl tRNA that is subsequently
used by the ribosome. By this mechanism, unnatural amino acids bearing bioorthogonal
chemical reporters can be introduced into proteins that are overexpressed in Escherichia
coli. To avoid competition with the endogenous residue, the bacterial strain is rendered
auxotrophic for the natural amino acid. Proteins cannot be overexpressed unless the cells
are supplemented with either that residue or a closely related unnatural analog. For
example, a phenylalanine auxotroph was used to express proteins in which all
phenylalanine residues were replaced with p-azidophenylalanine or pacetylphenylalanine (a keto derivative)85, 86. Similarly, a methionine auxotroph was used
for production of proteins that contained homopropargylglycine or azidohomoalanine at
sites that encode for methionine87, 88. Notably, Link et al. have extended this work to the
labeling of bacterial cell surfaces17, 79. Azido amino acids were installed in outer
membrane protein C (OmpC) of an E. coli methionine auxotroph and the cell surface
azides were then ligated with alkyne probes through both copper(I)-mediated and strainpromoted [3+2] cycloaddition (D.A. Tirrell, personal communication; ref. 79).
Figure 5: Methods for introducing chemical reporters into proteins.
(a) Unnatural amino acids bearing ketones, azides and alkynes can be incorporated into
target proteins in a residue-specific manner using auxotrophic strains of E. coli. (b)
Amino acids with bioorthogonal side chains can be installed into proteins in a sitespecific fashion using nonsense suppression techniques. (c) Chemical reporters can be
introduced into short peptide sequences using the cell's post-translational machinery. In
one example, an analog of biotin ('keto-biotin') is attached to a 15-amino-acid consensus
sequence (blue box) by E. coli biotin ligase (BirA). Similarly, formylglycine-generating
enzyme (FGE) can convert a cysteine residue within a 13-residue consensus sequence
(red box) to formylglycine. Both of the these electrophiles can be labeled with hydrazide
probes.
Full figure and legend (16K) Figures, schemes & tables index
Residue-specific metabolic labeling can produce proteins with multiple copies of a
bioorthogonal functional group, but it has only limited application in cases where a
chemical reporter is desired at a single position within the protein. As pioneered by
Schultz and coworkers, site-specific insertion of a bioorthogonal amino acid has been
achieved using nonsense suppression techniques (Fig. 5b)84. In this approach, a mutually
selective tRNA and aminoacyl-tRNA synthetase are developed so that the unnatural
amino acid can be uniquely activated by the tRNA in vivo. The tRNA's anticodon is
engineered to complement a rare stop codon, which is co-opted to encode the unnatural
amino acid in the corresponding DNA (and intermediate mRNA). Cells transfected with
genes encoding the engineered tRNA, aminoacyl-tRNA synthetase and target protein will
produce the modified protein when supplemented with the unnatural amino acid.
The unnatural amino acid mutagenesis method has been used to introduce chemical
reporter groups into proteins in both E. coli20, 89, 90, 91and yeast92, 93 (Fig. 5b). For example,
m-acetylphenylalanine was site-specifically incorporated into LamB, an outer-membrane
protein of E. coli, and subsequently labeled with membrane-impermeant hydrazide
dyes20. Similarly, azido and alkynyl amino acids related to tyrosine were installed in
proteins within both E. coli and yeast90, 93. After cell lysis, the derivatized proteins were
tagged by copper-catalyzed [3+2] cycloaddition.
The above methods use the cell's translational machinery to incorporate bioorthogonal
functionality into proteins. Cells also possess a rich machinery for post-translational
modification that might be exploited for similar purposes. This notion was recently
explored by Ting and coworkers using E. coli biotin ligase (BirA), an enzyme capable of
attaching a biotin prosthetic group to a 15-residue consensus sequence19. BirA can
recognize this sequence irrespective of its surrounding context and can also tolerate
subtle modification to the biotin structure. These features were combined in a general
tagging strategy wherein the consensus sequence served as a gene-encoded tag for
enzymatic ligation of a keto-biotin analog (Fig. 5c). The ketone could then be modified
with fluorescent hydrazide probes. Further engineering of BirA might enable enzymatic
transfer of biotin analogs bearing other chemical reporters. The method is technically
straightforward and potentially generalizable across a broad range of proteins and cell
types. Other strategies for enzymatic labeling of a target peptide sequence have also been
reported recently, and these might be considered alternative avenues for the delivery of
chemical reporters to proteins94, 95.
The direct enzymatic conversion of an amino acid side chain to a chemical reporter
would be an appealing means for site-specific protein labeling. An opportunity to achieve
this was recently presented by the discovery of the formylglycine-generating enzyme
(FGE)96. Responsible for converting sulfatases from an inactive to an active state, this
enzyme converts a critical cysteine residue to formylglycine (bearing an aldehyde at the
C- position) within a conserved 13-residue consensus sequence. Like BirA, FGE will
modify its target sequence irrespective of the surrounding context. Thus, the FGE
consensus sequence can be imported into heterologous proteins and function as a general
'aldehyde tag' for subsequent chemical labeling with aminooxy or hydrazide reagents
(Fig. 5c and C.R.B., unpublished data).
Glycans and glycoconjugates. A powerful feature of chemical reporters is their
applicability to labeling not just proteins but many classes of biopolymers. Indeed,
chemical reporters may turn out to be as broadly applicable for labeling of glycans, lipids
and other metabolites as GFP has been for proteins. In the field of glycobiology, ketones
and azides have already proven to be useful markers for visualizing glycans within their
native environment. These biopolymers are known to mediate cell surface recognition
events97 and intracellular trafficking98 and, in recent years, have also been implicated in
transcriptional regulation99, 100. Glycans can participate in direct interactions with
receptors, or they can exert their biological activities in an indirect fashion by modulating
the functions of the proteins or lipids to which they are attached. The ability to monitor
glycans both independently of, and in conjunction with, the scaffold to which they are
attached could provide fundamental insights into their roles in cell biology. At the
cellular level, changes in glycosy-lation are known to correlate with malignant
transformation101 and the development of a chronic inflammatory state102. Visualization
of these changes at the level of glycan structures would add a new dimension to our
understanding of the underlying pathology.
Chemical reporters can be embedded within glycans using endo-genous biosynthetic
pathways (a process we have previously termed metabolic oligosaccharide engineering),
then elaborated with small-molecule probes for detection or isolation103. This two-step
tagging scheme has been used to study glycoconjugates containing the monosaccharides
sialic acid (Sia), N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc).
The sialic acid biosynthetic pathway is permissive of unnatural N-acyl substituents, and
this site has been identified as suitable for the addition of chemical reporters103, 104. Mahal
et al. reported that mammalian cells metabolize the precursor sugar Nlevulinoylmannosamine (ManLev), an unnatural keto analog of N-acetylmannosamine
(ManNAc), to the corresponding keto sialic acid (SiaLev) on cell surface glycans22. The
unnatural residues can be tagged with a variety of probes, including fluorophores and
MRI contrast reagents105. Similarly, azides have been incorporated into cell surface
glycans by metabolism of the unnatural azido sugar N-azidoacetylmannosamine
(ManNAz) (Fig. 6)32. The resulting azido sialic acid (SiaNAz) residues can be labeled
with various probes through Staudinger ligation with phosphines32 or [3+2] cycloaddition
with alkynes34. In addition, free sialic acid analogs themselves can be used to deliver both
ketones and azides to cell surface sialoglycoconjugates23. These intermediates enter the
metabolic pathway downstream of the corresponding mannosamines. They bypass the
most restrictive enzymes in the pathway and can therefore be adorned with larger
chemical reporters (such as aryl azides).
Figure 6: Azides can be incorporated into glycoconjugates using glycan biosynthetic pathways.
Azido analogs of ManNAc (ManNAz) and sialic acid (SiaNAz) are metabolized by cells
and converted to cell surface azido sialosides. Similarly, an azido analog of GalNAc
(GalNAz) can be metabolically introduced at the core position of mucin-type O-linked
glycoproteins. An azido analog of GlcNAc (GlcNAz) can be incorporated into cytosolic
and nuclear glycoproteins.
Full figure and legend (25K) Figures, schemes & tables index
In addition to the sialoside biosynthetic pathway, both the GalNAc and GlcNAc salvage
pathways are tolerant of unnatural sugars bearing bioorthogonal functionality. By this
route, keto106 and azido64 GalNAc analogs (such as N-azidoacetylgalactosamine or
GalNAz (Fig. 6) can be incorporated into mucin-type O-linked glycoproteins. Similarly,
cells incubated with N-azidoacetylglucosamine (GlcNAz, Fig. 6) will append the
unnatural residue to cytosolic and nuclear proteins at sites normally occupied by OGlcNAc65. The azide-labeled glycoproteins can be tagged in a secondary reaction with
fluorescent probes or affinity tags, enabling them to be visualized and enriched from
complex cell and tissue lysates. In the future, such experiments might be used to
inventory changes in glycoprotein profiles in normal versus diseased cells.
Lipids and other biomolecules. Lipids are a class of biomolecules that serve critical
roles in cellular function and can also modulate the activities of other biomolecules, such
as proteins and glycans. Like glycans, they cannot be readily labeled using genetic
methods. Chemical reporters incorporated into lipids by metabolism of their biosynthetic
precursors could provide the means to inventory and image these biomolecules within
their native environments. The first example of this was demonstrated in a proteomic
analysis of farnesylated proteins25. Protein farnesylation is a post-translational
modification that can relocalize cytosolic proteins to membranes, modulate protein
conformational changes and potentially mediate protein-protein interactions107. The
complete repertoire of farnesylated proteins is not known and is difficult to predict by
analysis of primary sequence alone. Zhao and coworkers applied the bioorthogonal
chemical reporter strategy to label farnesylated proteins in cells for subsequent
enrichment and proteomic analysis25. They incubated cells with azido analogs of either
farnesol or farnesyl pyrophosphate and then tagged the modified proteins by reaction of
cell lysates with a biotin-derivatized phosphine. Purification by avidin capture followed
by mass spectrometric analysis revealed several farnesylated proteins involved in a
variety of processes, including nucleosome assembly and peroxisome biogenesis. A
similar approach might be applied to profiling proteins modified with other lipid groups
such as geranylgeranyl, palmitoyl or myristoyl moieties.
In principle, any biomolecule can be studied using the bioorthogonal chemical reporter
strategy, as long as its biosynthetic pathway is tolerant of modified precursors. Azidemodified nucleotides have been incorporated into nucleic acids both in vitro and in living
cells to study protein-DNA and DNA-DNA interactions60, 108. Thus, a simple
extrapolation indicates that chemical reporters could be incorporated into nucleic acids
within living cells and covalently labeled with chemical probes. We anticipate that the
bioorthogonal chemical reporter strategy will also find utility in the profiling of other
biomolecules (such as cofactors) and post-translational modifications (such as acetylation
or methylation) in living systems. Indeed, syntheses of azide-bearing flavonoids109 and Sadenosylmethionine derivatives have recently been reported110, 111.
Chemical reporters as readouts of enzyme function
In addition to their use in monitoring biomolecule expression and localization, chemical
reporters can provide a readout of enzyme function. In this case, the target protein is
labeled with the chemical reporter by virtue of its catalytic activity on a modified
substrate, rather than through the cell's metabolic machinery. Termed activity-based
protein profiling by Cravatt and coworkers, this approach has been used to identify active
glutathione S-transferases26, glycosidases28 and proteasome molecules27. In each case, a
mechanism-based covalent inhibitor of the target protein class was designed to
incorporate the azide group. Catalytically active proteins were covalently labeled with the
inhibitor and then selectively tagged with phosphine- or alkyne-modified probes,
permitting analysis by western blotting or enrichment for mass spectrometric analysis.
This approach to activity-based labeling can be applied, in principle, to any enzyme class
for which a selective covalent modifier is available. In a broader profiling experiment,
overall levels of enzymes with active-site nucleophiles were compared in various breast
cancer cell lines29. In this case, an electrophilic substrate bearing an alkyne reporter was
found to give cleaner labeling than the corresponding azido analog.
Bioorthogonal chemical reporters in living organisms
One of the most dramatic applications of GFP-protein fusions has been noninvasive
imaging of protein expression and localization in living organisms ranging from
Caenorhabditis elegans to mice4, 112. Chemical reporters might provide similar
opportunities for other classes of biomolecules. Already, both proteins and glycans have
been labeled with azides in laboratory mice, using covalent enzyme inhibitors26 and azido
sugars30, respectively. For noninvasive imaging applications, the secondary tagging
reaction with a phosphine or alkyne probe must also be accomplished in the living
organism.
The demands on bioorthogonal reactions in this context are far more stringent than those
for cellular systems. Aside from having extraordinary chemical selectivity, the reagents
must not be prone to rapid metabolic breakdown or excretion, and they must not
accumulate in cells or organs nonspecifically on the timescale of the reaction. Very few
covalent chemistries have been relocated from the round-bottom flask to a living
organism. We recently investigated whether cells labeled with azido sugars in laboratory
mice were capable of further chemical modification by Staudinger ligation30. Mice were
injected with either ManNAz or GalNAz for several days and then administered a
phosphine probe. After several hours, the anticipated product of the Staudinger ligation
was observed on splenocyte cell surfaces and serum glycoproteins (C.R.B., unpublished
data, ref. 30). In the future, this chemical reporter−bioorthogonal reaction system might
enable noninvasive imaging of glycan expression.
Summary and future outlook
The bioorthogonal chemical reporter strategy offers a means to visualize multiple classes
of biomolecules in living systems. Substrates linked to chemical reporters can be
metabolized by cells and incorporated into proteins, glycans, lipids and other cellular
species. After covalent reaction with complementary probes, these classes of
biomolecules can be visualized in living cells and, in some cases, living organisms. Both
discrete biopolymers (such as tetracysteine-fused proteins) and entire subsets of
biomolecules (such as metabolically labeled glycans or lipids) can be tracked in their
native habitats using this technology.
Several challenges remain with respect to both metabolic labeling and chemical tagging
in biological systems. In many cases, competition with endogenous substrates, such as
natural amino acids, lipids and sugars, cannot be avoided. In such cases, one should
expect metabolic substitution with the reporter-modified building block to be incomplete.
The fraction of biomolecules labeled with the chemical reporter may be an important
parameter when interpreting results and therefore must be quantified in some
circumstances. Another consideration is the physiological consequence associated with
the addition of a chemical reporter to a biomolecule, particularly in living organisms.
Even subtle perturbations to the structure of a protein, glycan or lipid may affect its
biological activity, localization or stability. A third issue to address is the problem of
slow reaction kinetics, which can undermine the use of bioorthogonal reactions for
biomolecule tagging. The chemical labeling step involves a bimolecular reaction with a
second-order rate constant that is typically far below that of a noncovalent binding event
(such as an antibody-antigen interaction). Rapid reactions are essential for the
observation of biological events that occur on a very short time scale or among
biomolecules of low abundance. This problem might be solved by engineering a fast,
reversible association of the reaction partners that precedes an irreversible covalent
labeling step.
Even at this early point in the development of the technique, it is clear that chemical
reporters and bioorthogonal reactions have a rich future in the field of chemical biology.
Some interesting future directions include imposing temporal and spatial control over
metabolic labeling with the reporter functional group. This might be accomplished using
caged substrates that are released by light- or tissue-specific enzymes. A future challenge
for synthetic chemists will be to craft novel bioorthogonal transformations for use in
living organisms. Just as combinations of fluorescent proteins (such as CFP and YFP)
have proven useful in studying multicomponent processes, an arsenal of bioorthogonal
reactions could find use in monitoring collections of species that function together in
living systems.
Top of page
Acknowledgments
J.A.P. is supported by a Howard Hughes Medical Institute predoctoral fellowship. We
thank N. Agard, J. Baskin, I. Carrico, D. Dube, S. Laughlin and C. McVaugh for critical
reading of the manuscript.
Competing interests
The authors declared no competing interests.
Top of page
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1. Department of Chemistry, University of California, Berkeley, California 94720,
USA.
2. Department of Molecular and Cell Biology, University of California, Berkeley,
California 94720, USA.
3. Howard Hughes Medical Institute, University of California, Berkeley, California
94720, USA.
4. Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley,
California 94720, USA.
5. Email: crb@berkeley.edu
Correspondence to: Carolyn R Bertozzi1,2,3,4 Email: crb@berkeley.edu
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.
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).
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 twocolor 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.
A triarylphosphine and an azide first react to form an aza-ylide intermediate. The
nucleophilic nitrogen atom is trapped in an intramolecular fashion, and the cyclized
intermediate hydrolyzes in water to form a stable amide-linked product. In some cases,
aryl azides (R' = aryl) may react with phosphines to initially form O-alkyl imidates114.
(a) Unnatural amino acids bearing ketones, azides and alkynes can be incorporated into
target proteins in a residue-specific manner using auxotrophic strains of E. coli. (b)
Amino acids with bioorthogonal side chains can be installed into proteins in a sitespecific fashion using nonsense suppression techniques. (c) Chemical reporters can be
introduced into short peptide sequences using the cell's post-translational machinery. In
one example, an analog of biotin ('keto-biotin') is attached to a 15-amino-acid consensus
sequence (blue box) by E. coli biotin ligase (BirA). Similarly, formylglycine-generating
enzyme (FGE) can convert a cysteine residue within a 13-residue consensus sequence
(red box) to formylglycine. Both of the these electrophiles can be labeled with hydrazide
probes.
Azido analogs of ManNAc (ManNAz) and sialic acid (SiaNAz) are metabolized by cells
and converted to cell surface azido sialosides. Similarly, an azido analog of GalNAc
(GalNAz) can be metabolically introduced at the core position of mucin-type O-linked
glycoproteins. An azido analog of GlcNAc (GlcNAz) can be incorporated into cytosolic
and nuclear glycoproteins.