Current Medicinal Chemistry, 2009, 16, ????-???? 1
0929-8673/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.
Expanding the Chemical Biologist’s Tool Kit: Chemical Labelling Strate-
gies and Its Applications
Souvik Chattopadhaya*,1, Farhana B. Abu Bakar1 and Shao Q. Yao*,1,2,3
1Department of Biological Sciences; 2Department of Chemistry; 3NUS MedChem Program of the Office of Life Sciences,
3 Science Drive 3, National University of Singapore, Singapore-117543
Abstract: Methods that allow visualisation of proteins in living systems, in real time have been key to our understanding
of the molecular underpinnings of life. Although the use of genetically encoded fusions to fluorescent proteins have
greatly advanced such studies, the large size of these tags and their ability to perturb protein activity has been major limi-
tations. Attempts to circumvent these issues have seen the genesis of complementary strategies to chemically label/modify
proteins. Thus, chemical labelling approaches seek to “decorate” biomolecules in live cells through the site-specific intro-
duction of a small, non-native chemical tag (or reporter group). The introduced tag is minimally invasive such that the ac-
tivity and/or function of the target molecule in not perturbed/compromised by its inclusion. In most cases, this modifica-
tion is brought about by fusing target biomolecules to protein domains/ peptide tags or via the incorporation of reactive
“handles” by either exploiting the cell’s biosynthetic machinery or during protein synthesis. Selective tagging of the bio-
molecule then proceeds via a bioorthogonal chemical reaction following exogenous addition of probe(s). Depending on
the nature of the probe, the method can be applied to either visualise/track the dynamics of target molecule(s) in their na-
tive cellular milieu or for affinity enrichment for further downstream applications. The versatility of these approaches has
been demonstrated by their ability to tag not just proteins but also intractable biomolecules like lipids and glycans. In this
review, we summarise the various strategies available to “chemically” tag proteins and provide a comparative analysis
their advantages and disadvantages. We also highlight the many creative applications of such methodologies and discuss
their future prospects.
Until recently, genetic fusions to fluorescent proteins
(FPs) had been the mainstay for non-destructive optical im-
aging of proteins in live cells. The availability of green fluo-
rescent protein (GFP) and its spectral variants, their stability
together with the exquisite specificity of genetic targeting
made FPs ideal candidates to study the spatiotemporal distri-
bution, trafficking, binding interactions, stability, fate and
function of either individual or multiple protein species in
intact cells . The information garnered from these studies
has allowed complex intracellular processes to be correlated
with protein function. But several caveats exist with the use
of fluorescent proteins- due to their large size (~220 amino
acids), FPs have a tendency to interfere with the folding,
activity and localisation of target proteins. Red fluorescent
proteins (RFPs), in particular, are prone to aggregation lead-
ing to cellular cytotoxicity . Modification of FPs so as to
modulate their spectral and biochemical characteristics are
limited by finite possibilities as such changes are time con-
suming and usually associated with significant trade-offs.
For example, conversion of the tetrameric red fluorescent
protein to its monomeric form resulted in an 80% loss in
fluorescence intensity . Besides optical readouts, the use
of FPs for applications such as probes for cellular cues like
changes in pH, ion concentrations have been hampered by
their slow response times and limited dynamic range .
Lastly, FPs require long time scales to mature and when
*Address correspondence to these authors at the Departments of Chemistry
and Biological Sciences, 3 Science Drive 3, National University of Singa-
pore, Singapore 117543; Tel: (+65) 6516 2925; Fax: (+65) 6779 1691;
Department of Biological Sciences, 14 Science Drive 4, National University
of Singapore, Singapore 117557; Tel: (+65) 6516 1684;
compared to small organic dyes, they are not as bright and
In recent years, attempts to obviate (at least partially)
some of the shortcomings associated with the use of FPs
have seen the introduction of chemical labelling strategies
that seek to combine the specificity of genetic encoding to-
gether with the simplicity, versatility and diversity of small
molecule probes [5-8]. The unifying essence of all these ap-
proaches lies in the incorporation of a “reporter” handle
(chemical moiety/ protein domain/ peptide sequence) into the
target molecule followed by its subsequent modification by
means of exogenously added probe(s). The underlying
modular nature of many of these methods has made it possi-
ble to expand the realm of applications beyond optical imag-
ing applications to encompass more challenging issues such
as study of protein-protein interactions , dissociation and
reassembly of organelles  to the regulation of protein
activity , stability  and monitoring of changes in in-
tracellular pH, ion concentrations in response to environ-
mental stimuli . Since the timing for labelling is under
experimental control, cellular events such as protein turnover
 can be monitored with temporal resolution. The impor-
tance of chemical labelling approaches is underscored by the
fact that these methods have been applied to the study of not
only proteins but also to glycans-biomolecules that were
previously “intractable” to genetic tagging, and in diverse
systems ranging from live cells to whole organisms.
In this review, we begin with considerations that are a
prerequisite for site-specific modification of biomolecules.
We then provide an overview of the various methods avail-
able with emphasising on the chemistries involved in tag-
ging, highlight some of the creative applications and provide
a comparative analysis of the advantages and disadvantages
of these approaches. Last, we outline future prospects of this
2 Current Medicinal Chemistry, 2009 Vol. 16, No. 34
Chattopadhaya et al.
2. CONSIDERATIONS FOR SITE-SPECIFIC MODIFI-
CATION OF BIOMOLECULES
The basic premise of site-specific tagging begins with the
consideration that the tag to be introduced should not cause
perturbations to the folding and activity of the biomolecule
under investigation. These tags usually encode the specificity
element. For enzyme-mediated ligation reactions, specificity
is conferred by the ability of the enzyme to recognise the
“amino acid code” (i.e., the specific peptide sequence or pro-
tein domain fused to the target molecule). In case of chemi-
cal modifications, this information is contained within native
sequence following incorporation of reactive functionalities
via posttranslational modification or by metabolic installa-
tion. Irrespective of the route exploited, size of the unnatural
motif should be small so as to facilitate its introduction by
either the translational apparatus or the biosynthetic en-
zymes. The next step involves the modification of the tag by
a small molecule probe in either a covalent or non-covalent
fashion. The reaction should be highly chemoselective,
bioorthogonal and under physiological conditions, it must
proceed with relatively fast kinetics in the absence of auxil-
iary reagents with no (or innocuous) by-products. Addition-
ally, this modification by the probe should occur in only a
regio- or site-specific manner. Given the abundance of nu-
cleophiles, reducing agents and other functionalities within
the cellular environment, these requirements are not trivial.
In addition, the probe should be metabolically inert and cell
permeable (for intracellular labelling) or impermeable (cell
surface labelling). Cellular toxicity, stability of the probe-
protein adducts (especially for non-covalent labelling) and
bioavailability (for use in whole organism labelling), are
other factors that need consideration. Despite these signifi-
cant requirements, chemical biologists have developed many
innovative strategies-both covalent and non-covalent meth-
ods that seek to augment our understanding of cellular
events. In the sections that follow, we introduce these strate-
gies, highlight some of their applications and summarise
their advantages and disadvantages in perspective of tagging
biomolecules within the cellular context.
3. CHEMICAL LABELLING STRATEGIES
3.1. Non-Covalent Chemical Labelling Approaches
Both short peptide and protein domains have been used
for non-covalent labelling. As the name suggests, these
methods exploit the high affinity binding interactions be-
tween the protein/peptide tag and cognate labels. In compari-
son to protein domains, the use of short peptide tags is ad-
vantageous as it presents a much less invasive option but is
usually accompanied by rapid signal deterioration and non-
specific binding. To circumvent these issues, “engineered”
non-native peptide-ligand pairs have been “evolved”; the
new sequences tend to chelate the ligand more strongly
thereby allowing for stringent washes to reduce background
staining. Nevertheless, non-covalent labelling methods are
particularly invaluable in cases whereby reversible binding is
desired. Table 1 summarises the different non-covalent label-
ling methods currently available.
3.1.1. Specific Chelation of Ligands by Short Peptide Se-
126.96.36.199. Tetracysteine Motifs and Biarsenical Derivatives
A major tour de force in the development of chemical la-
belling strategies was the pioneering efforts of Tsien and co-
workers who showed that a unique combination of amino
acid side chains creates a tag that can specifically chelate an
orthogonal ligand . Accordingly, a short hexapeptide tag
(CCXXCC, X=any amino acid but optimally proline and
glycine) harbouring a tetracysteine (TC) motif was designed
and shown to be specifically labelled with biarsenical deriva-
tives of fluorescein (FlAsH) or resorufin (ReAsH) (Fig. 1A).
The TC motif adopts a ?-helical conformation with the bi-
arsenical dye fitting into the i, i+1, i+4 and i+5 positions of
Non-Covalent Labelling Approaches
Name of Tag Tag Size (aa) Label Comments
Specific Chelation of Ligands by Short Peptide Domains:
Tetracysteine (CCXXCC) 6-12
Biarsenicals dyes like
Not applicable for oxidising environments; requires
dithiols; slow on-off kinetics
Oligo-His (His6) 6 Ni-NTA
Fluorescence quenching by Ni2+; unstable; cell-surface
Oligo-Asp (Asp4) 12 Zn2+-DpaTyr Cell surface labelling
Labelling Mediated by Protein Domains:
157 Mtx or TMP
Intracellular labelling; degradation of fusion mediated
FKBP12 (F36V) 108 SLF’ derivatives Protein-ligand dissociation maybe a problem
Fluorogen Activating Proteins >110
Malachite Green / Thiazole
Large size; scFVs fold poorly in cytosol and hence
cannot be applied to oxisiding environments
Expanding the Chemical Biologist’s Tool Kit Current Medicinal Chemistry, 2009 Vol. 16, No. 34 3
the helix. The free, membrane-permeable 1,2-ethanedithiol
(EDT)-bound forms of FlAsH and ReAsH are weakly fluo-
rescent in solution but the protein-bound complexes (Kd=2-4
pm) undergo a marked increase in fluorescence by virtue of a
thiol-arsenic exchange reaction. Furthermore, the EDT sub-
stituents prevent non-specific labelling to biomolecules hav-
ing isolated cysteine residues. In direct comparison to the use
of FPs, the small size of the TC tag provides a decisive ad-
vantage in studies involving protein localisation. Studies
with the ?2-adrenergic receptor  and yeast ?-tubulin 
have shown that the distribution of these proteins remains
normal when tagged with TC motif but show an altered dis-
tribution when GFP fusions are used.
The versatility and usefulness of this technique encom-
passes an ever expansive list of applications that includes,
among many others, the secretion of Shigella flexeneri type
III pathogenic effectors IpaB and IpaC , monitoring of
intracellular dynamics of HIV-1 virion , study the role of
conformational changes in GPCR-mediated cell signalling
 as well as GPCR activation  and even as a reporter
for protein conformation or protein-protein binding when
displayed in a bipartite mode . In several instances, com-
binations of FlAsH and ReAsH have been used for pulse
chase analysis of protein turnover [13,22]. ReAsH, in par-
ticular, can generate singlet oxygen species upon irradiation
and has been used for spatiotemporal inactivation of proteins
 as well as a contrast stain for correlative fluorescence
and electron microscopy to track localisation of a Golgi pro-
tein though cell division cycle .
Potential disadvantages of this approach include the cel-
lular toxicity of arsenic derivatives and high non-specific
staining that invariably necessitates complex wash steps. The
addition of smelly dithiol antidotes such as EDT or 2,3-
dimercaptopropanol (BAL) in washes, inclusion of non-
fluorescent dyes to block hydrophobic sites together with the
availability of new tetracysteine motifs that exhibit higher
binding affinities (>20-fold)  and hence can tolerate
stringent washes, has helped alleviate some of these draw-
backs. Nevertheless the method cannot be applied to organ-
elles that present an oxidising environment such as the cell
surface; simultaneous labelling with of two or more proteins
having tetracysteine tag is also unlikely since these probes
are not modular. New synthetic schemes need to be devised
for each biarsenical dye that may delay the availability of
188.8.131.52. Chelation of Divalent Metal Complexes by Oligohis-
tidine and Oligoaspartic Acid Sequences
In an approach conceptually similar to TC, Guigent et al.
exploited the reversible binding of nickel-nitrilotriacetic acid
(Ni-NTA) derivatised chromophores to fusion proteins bear-
ing an oligohistidine (His6 or His10) tag . The authors
used FRET between a fluorescently labelled receptor an-
tagonist (GR-flu) and rhodamine-NTA derivative to show
selective labelling of the serotonin receptor, 5HT3. Unlike
TC approach, this method can be used rapidly label target
proteins under oxidising conditions. However, this approach
allows only a modest selectivity of labelling and suffers from
the relatively low stability between the Ni-NTA conjugates
and oligohistidine sequence (Kd ~ 0.2-10 μM) as well as
from a significant quenching of fluorescence by the par-
amagnetic Ni2+ (? < 0.10) . Ni2+ is also a toxic heavy
metal and a known human carcinogen. While the affinity has
been modestly improved by the use of three NTA moieties
, cellular toxicity can be avoided by replacing Ni2+ with
other transition metals as was shown by Hauser and Tsien
. The authors made use of a small, membrane imperme-
able fluorescent chelator, HisZiFit, in which Zn2+ replaces
Ni2+ for coordination to the His6 motif without compromis-
ing on binding affinity (Kd = 40 nM). Chemically, HisZiFit
comprises of fluorescein derivatised with pair of 2-
pyridylsulfonamido functionalities that can simultaneously
bind two Zn2+ ions as well as multiple imidazole side chains
of the His6 motif. The replacement of Ni2+ with Zn2+ is ad-
vantageous since Zn2+ is diamagnetic and redox-inert. By
being able to label cell-surface exposed His6 tags of the
STIM1 (stromal interaction molecule 1) protein, the authors
were able to show that in response to Ca2+ depletion, the N-
terminal segment of STIM1 indeed becomes surface exposed
as was purported to be. Similar results could not be obtained
when STIM1fusions to FPs, HRP or HA tags were used as
the large size of these tags potentially interfered with the
externalisation process. Besides His6 tags, complexes to Zn
(II)-ions such as the Zn2+-2,2’-dipicoylamine complex based
on L-tyrosine scaffold (Zn2+-DpaTyr) have also been em-
ployed to specifically label membrane proteins harbouring a
genetically encoded oligoaspartic acid sequence (D4 tag)
3.1.2. Non-Covalent Labelling Using Protein Domains
As the name suggests, these approaches make use of non-
native “engineered” protein-ligand interactions for labelling.
The prototypical example has been the use of single chain
antibody (scFV) that binds a fluorescein-conjugated hapten
but this method could not expanded further since scFV are
known to fold poorly in the cytosol . Drawing inspiration
from this, Nolan and colleagues focussed on an approach
that made use of the tight interaction between a specific mu-
tant of FK-binding protein 12, termed FKBP12 (F36V) and
the synthetic ligand for FKBP12 (SLF’) that was derivatised
with fluorescein (FL-SLF’; Fig. 1B). SLF’ lacks the immu-
nosuppressive effects of FK506 (the original FKBP12
ligand), has sub-nanomolar Kd and >1000 fold selectivity for
F36V mutant over wild-type FKBP12. The readily cell-
permeable and non-toxic FL-SLF’ was shown to be useful in
tagging FKBP12 (F36V)-?-galactosidase fusions directly in
living cells. The ligand also permitted the subsequent photo-
inactivation of these fusions in a spatiotemporally defined
Work from the Cornish laboratory has made use the well
studied non-covalent interaction between the 18 kDa mono-
meric enzyme, dihydrofolate reductase (DHFR) and its cog-
nate inhibitor, methotrexate (Mtx; Fig. 1C). To achieve this,
target proteins as expressed as DHFR fusions and then la-
belled with TexasRed conjugates of Mtx. In spite of the non-
covalent nature of the interaction, the high-binding affinity
and favourable kinetics (koff ~10-4s-1) ensures that such com-
plexes have a reasonable half-life (t1/2) for most applications.
Initial attempts focussed on the use of E. coli DHFR
(eDHFR) fusions localised to the plasma membrane or nu-
cleus that were effectively labelled using Mtx-TexasRed
(Mtx-TR) conjugates . However, the labelling reaction
4 Current Medicinal Chemistry, 2009 Vol. 16, No. 34
Chattopadhaya et al.
was slow (~20 h), required high probe concentrations and
necessitated use of DHFR-/- cell lines since Mtx probes can
bind to endogenous DHFR giving rise to high background.
In cases wherein DHFR+/+ cell lines were used, media had to
be supplemented with thymidine to mitigate toxic effects of
Mtx. Further work by the same group has helped resolve
some of these issues. By replacing Mtx with trimethoprim
(TMP) conjugates, the authors showed that it was possible to
perform labelling in DHFR+/+ cells since TMP has ~1000-
fold higher selectivity for eDHFR over mammalian DHFR
. The new probes also had fast labelling kinetics and a
nanomolar quantity of the probe was sufficient for labelling.
In a complementary approach, Szent-Gyorgyi et al. de-
veloped single chain catalytic antibodies that can bind (with
nanomolar affinity) and generate fluorescence from other-
wise dark molecules (fluorogens) like thiazole orange (TO)
or malachite green (MG) derivatives . When incorpo-
rated into fusion proteins, these antibodies termed Fluorogen
Activation Proteins (FAPs) served as sensitive reporters for
protein localisation and abundance both at the cell surface
and in subcellular organelles (ER, Golgi). The use of FAPs is
advantageous, as it does away with the requirement to gener-
ate cognate fluorophore tagged ligands. Additionally, by
using a combination of antigenically distinct FAP fusions
and fluorogen derivatives, it is possible to perform multicol-
our imaging so as to dynamically monitor complex cellular
processes. However, since scFV-based FAPs contain internal
disulphide linkages, their usage is primarily limited to non-
reducing environments like the cell surface and secretory
pathways. When MG derivatives are used, phototoxicity
Fig. (1). Various non-covalent labelling schemes. (A) Labelling of proteins displaying a tetracysteine (TC) tag with a biarsenical dye
(FlAsH); (B) Target proteins fused to the FKBP mutant (F36V) can bind the SLF’ ligand with high affinity and specificity; (C) Specific la-
belling of fusions to E. coli dihydrofolate reductase (DHFR) with methotrexate (Mtx) conjugates.
Expanding the Chemical Biologist’s Tool Kit Current Medicinal Chemistry, 2009 Vol. 16, No. 34 5
becomes an issue as MG generates reactive oxygen species
at a rate similar to that of GFP.
3.2. Covalent Chemical Labelling Methods
Although in some cases, the reversible nature and on-off
kinetics for non-covalent labelling methods can be advanta-
geous, most applications require the formation of stable link-
ages between the label and fusion protein (Table 2). In com-
parison to interactions between small molecules and short,
unstructured peptide tags, covalent labelling methods tend to
confer a higher degree of specificity. The bulk of these ap-
proaches depend on enzyme-catalysed reactions and gener-
ally proceed via self labelling or by modification of the tag
fused to target molecules. Covalent labelling has also been
achieved by the introduction of reactive “handles” such as
ketones, aldehydes and azides into target biomolecules, ei-
ther by metabolic incorporation or during protein translation,
followed by the modification of these functionalities by
means of fluorophore bearing small molecule probes that
display orthogonal reactivity.
3.2.1. Covalent Self-Labelling Mediated by Enzyme Fu-
In this method, target proteins are expressed as fusion to
an enzyme that, in the presence of its labelled substrate,
transfers the tag to a residue within the active site and
thereby renders its fusion partner detectable. Recognition
between the enzyme and its substrate ensures that the label-
ling reaction is fast and specific.
184.108.40.206. O6-Alkylguanine Transferase (AGT) Mediated La-
An intracellular labelling system was devised based on
the use of self-alkylation reaction mediated by AGT. In cells,
AGT repairs DNA lesions resulting from O6-alkylation of
guanine by irreversible self-transfer of the alkyl group to an
active site cysteine residue. Following this transfer, AGT is
typically degraded. Human AGT (hAGT) has low substrate
Covalent Labelling Approaches
Tag size (aa) Label Enzyme Comments
Labelling by enzymes involved in post-translational modifications
Acceptor Peptide/ BirA AP (13-15)
Two-step; slow kinetics; cell surface
Acyl Carrier Protein ACP (77) or
CoA-derivatives AcpS Cell-surface labelling only
Peptidyl Carrier Protein PCP (80) or
CoA-derivatives Sfp Cell-surface labelling only
Q-tag PKPQQFM (7)
Slow reaction kinetics; competition from
endogenous TGase; requires high Ca2+ and
Sortagging 5 (LPETG)
Competition between hydrolysis and
Lipoic Acid Tag LAP peptide (22) Alkyl Azide
Cell-surface labelling only
PFTase Cell-based labelling yet to be shown
Aldehyde tag LCTPSR (6)
FGE Cell-based labelling yet to be shown
Enzyme-mediated self labelling
SNAP- or CLIP tag 182
Mutant AGT Large tag size
Cutinase fusions 230
Cutinase Cell-surface labelling
Largest of known tags
Covalent labelling by introduction of bioorthogonal functionalities
Enzymes of Sia,
Restricted by on the promiscuity of the
enzyme to accept unnatural sugar
Amber codon suppression
Requires generation of tRNA and synthetase
pair for each type of amino acid; dominant
negative effect due to premature truncation
6 Current Medicinal Chemistry, 2009 Vol. 16, No. 34
Chattopadhaya et al.
specificity and can readily react with the nucleobase, O6-
benzylguanine (BG) as well as its derivatives. For the pur-
pose of covalent labelling, target proteins were expressed as
fusions to G160W mutant of hAGT (W160hAGT) since it is
known to possess higher activity against BG (Fig. 2A) .
Using either fluorescein (BGFL) or biotin (BGBT) deriva-
tives of BG, it was shown that hAGT-mediated labelling is
fast, proceeds both in vitro and in vivo and in diverse sys-
tems such as E. coli, S. cerevisiae and in mammalian cells-
the only limitation being the probe-induced intracellular deg-
radation of the fusion protein. BG derivatives are chemically
inert and its modular nature permits facile synthesis of dif-
ferent dyes for use in demanding applications such as FRET
measurements and two colour pulse-chase labelling to follow
the changing localisation of a temperature-sensitive protein
upon translation at a higher temperature . Initial efforts
were plagued by background labelling due to binding of BG
derivatives by endogenous AGT. Efforts to decrease non-
specific binding has led to the identification of an inhibitor
against wtAGT together with the generation of a mutant
AGT (MAGT or SNAP tag) that is refractory to this inhibitor,
has reduced affinity for alkylated DNA and exhibits a 50-
fold faster labelling kinetics than wtAGT . The availabil-
ity of different AGT mutants has enabled further expansion
of the realm of applications. For example, by using a combi-
nation of two different mutant AGTs such as MAGT and
LAGT, that accepts the non-natural O6-proparylguanine (PG)
derivative as substrate, it was possible to simultaneously
monitor proteins localised to different organelles following
labelling with BG and PG derivatives that are appended with
spectrally distinct dyes . Further improvements of the tag
itself led to the generation of a yet another mutant termed
CLIP tag that specifically accepts only O2-benzylcytosine
(BC) derivatives . Gautier et al. made use of this or-
thogonal substrate specificities of the SNAP- and CLIP-tags
to demonstrate simultaneous multi-colour pulse chase label-
ling so as to visualise different generations of two distinct
proteins in a single sample. Additionally, since BC deriva-
tives show no cross-reactivity with the mammalian pro-
teome, use of CLIP tag circumvents the issue of high back-
ground observed with the use of BG derivatives. But irre-
spective of the AGT mutant used, an intrinsic downside of
this tag is its large size.
220.127.116.11. Labelling of Cutinase-Fusions with Suicide Inhibi-
Bonasio et al. devised a methodology that is broadly ap-
plicable to cell surface proteins and makes use of fusions to
the fungal serine esterase, cutinase- a 22-kDa globular pro-
tein . The fusion proteins can be targeted using probes
based on the p-nitrophenyl phosphonate (pNPP) scaffold;
pNPP is a mechanism based suicide inhibitor for cutinase
and forms a covalent adduct with the active site serine of
cutinase. Facile modification of sulfhydryl group of pNPP-
SH with maleimide-functionalised fluorophores affords the
cognate fluorescent labels. The authors used this method to
study the dynamic redistribution of the integrin receptor
LFA-1 during cell migration and showed that such fusions
do not perturb the dramatic conformational changes that in-
tegrins have to undergo during such processes.
18.104.22.168. HaloTag Mediated Labelling
Originally devised by researchers at Promega, HaloTag
protein (HTP) is a ~33 kDa, monomeric engineered haloal-
kane dehalogenase from Rhodococcus rhodochrous that
cleaves carbon-halogen bonds in aliphatic halogenated com-
pounds (Fig. 2B). Nucleophilic attack by the choloroalkane
to Asp106 in the enzyme results in the formation of an ester
bond between the ligand and protein; the ester is then hydro-
Fig. (2). Covalent self-labelling mediated by enzymes fused to target proteins. (A) AGT (SNAP tag) accepts benzylguanine (BG) derivatives
resulting in covalent attachment of label to the active site cysteine residue; (B) Labelling of dehalogenase (HaloTag) fusions with aliphatic
Expanding the Chemical Biologist’s Tool Kit Current Medicinal Chemistry, 2009 Vol. 16, No. 34 7
lysed in a second step to yield alcohol as the final product.
Mutated HTP used as fusion for labelling contains a critical
H272F mutation that prevents hydrolysis of ester bond re-
sulting in HTP retaining the label. Since neither E. coli nor
eukaryotic cells have endogenous dehalogenases, HTP
should permit labelling in different systems when used with
appropriate choloroalkane derivatives. HaloTag ligands can
be easily labelled with small organic dyes such as coumarin
and fluorescein  as well as quantum dots (QD) . The
usefulness of HTP for covalent labelling has been demon-
strated by specific conjugation of QDs to HTP-luciferase
fusions in vitro  while in vivo, the method has been used
to study cellular redistribution of HaloTag fusions to p65 or
I?B . Nevertheless HTP still remains as the largest
among all the currently known tags and caution needs to be
exercised in its use as it may interfere with certain biological
3.2.2. Covalent Labelling by Enzymes Involved in Post-
Cells possess rich machinery for myriad post-
translational (PT) modifications that can be harnessed for the
site-specific covalent labelling of target proteins. Some of
the prosthetic groups like biotin, lipoic acid and 4’-
phosphopantheine are appended only to a few protein species
in a cell and hence the enzymes that catalyse these reactions
highly specific . Posttranslational priming with these
groups is of fundamental importance as several of the target
molecules are enzymes having central roles in primary me-
tabolism. Typically, these enzymes exist in their non-
functional apo- forms; priming with prosthetic groups pro-
vides key functional groups to enable acyl- or carboxyl-
transfer chemistry thereby resulting in a gain of function
. Labelling mediated by enzymes involved in posttrans-
lational modification is context independent and usually
short amino acid sequence(s) suffices as the specificity de-
22.214.171.124. Biotin Ligase
Protein biotinylation mediated by biotin ligase (BirA) is
an important post-translational modification but in nature,
BirA accepts only biotin and its derivatives as substrates.
Ting and colleagues, for the first time, demonstrated that E.
coli BirA accepts a ketone isostere of biotin (in which meth-
ylene groups replace the ureido nitrogens) and transfers it
effectively to the lysine-side chain within the 15 amino acid
acceptor peptide (AP) sequence . This paved the way for
a two-step labelling strategy in which incorporation of ke-
tone into the AP is followed by bio-orthogonal ligation with
hydrazide or hydroxylamine bearing probes (Fig. 3A). The
absence of the keto-functionalities from natural protein, lip-
ids and carbohydrates ensures that modification by the probe
can proceed selectively under physiological conditions. Ad-
ditional advantages include the small size of the tag and ease
of synthesis of hydrazide derivatives. However, the second
step of the labelling procedure is problematic; the second
order rate constant for formation of hydrazone at pH 7 is
below 1 s-1M-1 that is three orders of magnitude below that
seen with AGT fusions. This necessitates the use of millimo-
lar concentrations of the probes to achieve significant label-
ling within short time scales. Besides, the method is applica-
ble to labelling of only cell surface proteins. Notwithstanding
these drawbacks, the approach was shown to be useful for
time-lapse imaging of single AMPA receptors in neurons
following labelling with streptavidin-QD605 as well as for
discerning rapid events such as trafficking of the AMPA
receptors-GluR1 and GluR2 in neurons using a two-colour
pulse chase labelling method . To further expand the
scope of biotin ligase labelling to multi-colour imaging,
phage display has been used to identify new biotin ligase-AP
pairs. Accordingly, 15-mer peptide libraries were screened
against yeast biotin ligase (yBL) to identify a cognate sub-
strate, the yeast acceptor peptide (yAP) . The utility of
the orthogonal yBL-yAP and BirA-AP pairs for two-colour
imaging was shown by the simultaneous labelling of yAP
and AP fusions in the same cell with spectrally distinct QDs.
Transglutaminases (TGases), ubiquitous enzymes in mul-
ticellular organisms catalyse amide bond formation between
glutamine and lysine side chains and are functionally active
during apoptosis, wound healing and migration events .
Using guinea pig liver transglutaminase (gpTGase) that ex-
hibits a wide tolerance for structure of the amine-containing
substrate, Ting and colleagues labelled cell surface proteins
that had been genetically appended with the “Q-tag”
(PKPQQFM; Fig. 3B). In the presence of biotin- or Al-
exa568 derivatives of cadaverine, only the Q tag was specifi-
cally recognized and modified by TGase. . Besides visu-
alisation of target proteins, the authors were able to interro-
gate the homodimerisation of p50 transcription factor by
using a benzophenone spermine photoaffinity probe. Even
though TGase mediated labelling is a single step, it requires
high concentration of both Ca2+ and the probe. Therefore, it
is unlikely that this approach will be extended to tag intracel-
lular proteins given the low basal levels of Ca2+ and competi-
tion from endogenous TGase substrates.
126.96.36.199. Phosphopantetheine Transferase (PPTase)
This method makes use PPTases to modify only cell sur-
face targets that have been fused to a carrier protein (CP).
Acyl carrier proteins (ACP) and peptidyl carrier proteins
(PCP) are used as fusion tags for this purpose, which in gen-
eral range in size from 77-100 amino acids. Modification of
tag is brought about by the action of a PPTase, either AcpS
(for ACP only)  or Sfp (for both PCP and ACP)  that
transfers the phosphopantetheinyl unit (Ppant) from CoA-
derivatives to a conserved serine residue within the carrier
protein (Fig. 3C). PPTases lack discrimination for different
substitutions at the terminal thiol of CoA, thereby permitting
easy transfer of fluorophores or other probes to ACP- and
PCP-tagged proteins. Vivero-Pol et al. used three different
CoA derivatives in sequence to track the dynamic localisa-
tion of the cell wall protein Sag1p during cell division of the
budding yeast . It has also been possible to harness the
substrate specificity of the PPTases- AcpS and Sfp to label
of two different CP-fusions in tandem . In particular, the
studies on GPCRs have greatly benefitted from the use of
ACP strategy as labelling is exclusively restricted to the cell
surface and therefore provides a high signal-to-noise ratio.
Using ACP fused to the amino terminal of the GPCR, neu-
rokinin-1 (NK1R) and limiting amounts of CoA-Cy5 to ob-
tain a low density of labelled receptors, Prummer et al was
able to study mobility of NK1R down to single receptor sen-
8 Current Medicinal Chemistry, 2009 Vol. 16, No. 34
Chattopadhaya et al.
sitivity . By choosing optimal fluorophores set and using
them at a precise molar ratio, Meyer et al. used quantitative
FRET analysis to gain insights into the monomeric organisa-
tion of the NK1R in live cells together with the functional
significance of its sequestration into membrane microdo-
mains [54,55]. Prior to this work, the very existence of mi-
crodomains remained elusive as data provided biochemical
analysis, FRAP or plasmon-resonance spectroscopy were
inconclusive. Besides GPCRs, the trafficking of the transfer-
rin receptor (TfR1) has also been studied. . To achieve
this, Yin et al. made use of the PPTase, Sfp and CoA-
Alexa488 conjugates to label TfR1-PCP fusions at the car-
Given that the 8-10 kDa size of the CP tags can poten-
tially interfere with the function of target proteins, recent
improvements to carrier protein-based labelling has emer-
gence of short peptide tags that can effectively substitute
protein domains without compromising of labelling efficacy,
specificity and speed. The first such peptide substrate for Sfp
was the “ybbR” tag identified by Yin et al . More re-
cently, screening of phage-display peptide libraries has
yielded a pair of 12-amino acid peptides that are differen-
tially labelled by AcpS and Sfp . The tags, A1 and S6
were used to label two different receptors on the surface of
the same cell with distinct CoA-dye conjugates  or by a
combination of CoA-fluorophore and CoA-QD . Since
the PCP strategy can be used in both oxidising and reducing
cellular environments, efforts have also been expended to
synthesise phosphopantetheine analogues that are cell per-
meable and can be efficiently converted to the corresponding
CoA-derivatives. This not only simplifies the synthesis of
CoA derivatives but also permits tagging of intracellular
proteins. So far, the method was shown to work in E. coli
overexpressing the target protein and Sfp . It remains to
be seen if the same approach can be extended to label pro-
teins in eukaryotic cells.
188.8.131.52. Lipoic Acid Ligase
In E. coli, lipoic acid ligase (LplA) catalyses the ATP-
dependent ligation of lipoic acid to an enzyme involved in
oxidative metabolism. Previous studies had indicated that
LplA could be promiscuous in terms of its substrate require-
ments thereby indicating substantial plasticity within the
active site of the enzyme . Ting and colleagues capital-
ised on this to devise a two-step LplA mediated, covalent
labelling strategy  but before they could achieve this, the
authors had to address two key issues- (i) identify an alkyl
azide substrate that can be recognised and effectively incor-
porated by wtLplA instead of lipoic acid and (ii) create a
small tag to replace LplA’s natural substrate and this led to
the identification of a 22-amino acid tag designated as LplA
acceptor peptide (LAP) that could be appended to either ter-
minal of target protein. Alkyl azides were selected since the
azide functionality is abiotic, resistant to oxidation and do
not cross-react with amines or other nucleophiles abundant
in biological systems. Though stable, azides have high in-
trinsic energy content that makes them particularly amenable
undergo bioorthogonal reactions such as Staudinger ligation
with phosphines and [3+2] cycloaddition reactions with acti-
Though a single step ligation with a fluorophore would
have offered a simpler labelling alternative, a two-step label-
ling process was opted as constraints placed by the size of
the lipoate binding pocked of LplA necessitated the use of a
small “functional handle” and incorporation of the azide tag
provided greater versatility in terms of subsequent modifica-
tion with diverse probes. LplA ligates the alkyl azide to a ?-
NH2 of lysine side chain within the LAP sequence followed
by its derivatisation with a cyclo-octyne conjugate suitably
modified with dyes, QDs or biotin to afford a triazole adduct
(Fig. 3D). Using this method, the authors were specifically
able to label LAP-low density lipoprotein receptor (LDLR)
fusions expressed on the cell surface in a manner that did not
alter receptor activity/trafficking . Though labelling me-
diated by LplA and biotin ligase is conceptually similar, or-
thogonal and involves two steps, LplA-approach is advanta-
geous as it has faster labelling kinetics.
184.108.40.206. “Sortagging”- Sortase (SrtA) Mediated Labelling
“Sortagging” has focussed on the use of SrtA from the
Staphylococcus aureus. SrtA recognises the LPXTG (X is
any amino acid) motif in structurally and functionally di-
verse substrates, cleaves between threonine and glycine and
subsequently links, via a peptide bond, the free -COOH
group of threonine with the N-terminal of a oligoglycine
nucleophile that is provided in vivo by the cell wall precursor
. This transpeptidation activity results in tethering of
surface proteins to the cell wall peptidoglycan, a process
important for bacterial infection. Sortagging as a versatile
method for protein labelling was demonstrated by work from
two individual groups. While Popp et al. probed labelling in
intact HEK293T cells transfected with CD40L , Tanaka
et al. used osteoclast differentiation factor (ODF) as target
protein . Both CD40L and ODF are Type II membrane
proteins and had the LPETG tag appended to the C-terminal
extracellular domain. In each case, specific labelling was
essentially accomplished in a single step, required only re-
combinant SrtA and an oligoglycine probe and proceeded in
the presence of serum-containing media without addition of
any cofactors (Fig. 3E). Common with other enzyme-
mediated labelling approaches, SrtA exhibits high sequence
specificity and activity with an estimated kcat/KM > 105 M-1s-1
. Small size of the tag, modular nature of the oligogly-
cine probes and their facile synthesis by standard solid-phase
peptide chemistry, are added advantages. A major drawback
of this approach is that both the transpeptidation and hy-
drolysis reactions proceed competitively with the kcat for
ligation being only 12-fold higher than cleavage . In ad-
dition, at physiological pH, only a small fraction of the en-
zyme is catalytically active . Therefore, SrtA mediated
ligation requires excess probe and enzyme to drive the
transpeptidation reaction. Given these requirements, it is
unlikely that sortagging can be extended to label intracellular
proteins. Evolving SrtA mutant(s) that exhibit only transpep-
tidase activity will greatly help expand the utility of this
Yamamoto and Nagamune have extended the utility of
“sortagging” to label proteins at the N-terminal . To
achieve this, the authors used a two-step labelling process-a
LPETG5 (9 amino acid) tag was introduced at the N-terminal
of the protein of interest. Preincubation of cells with SrtA
and triglycine allowed cleavage of the threonine-glycine
Expanding the Chemical Biologist’s Tool Kit Current Medicinal Chemistry, 2009 Vol. 16, No. 34 9
Fig. (3). Covalent labelling of peptide/protein domains mediated by enzymes involved in posttranslational modification. (A) Labelling of
acceptor peptide (AP) with biotin ligase; (B) Transglutaminase mediated labelling of Q-tag in presence of cadaverine derivatives; (C) PPTase
(AcpS or Sfp) recognises and transfers a labelled phosphopantatheine from CoA derivative to a conserved serine residue within the CP do-
main; (D) Lipoic acid ligase transfers alkyl azides to the ?-NH2 of the lysine residue within the LAP tag; in a second step, the azide reacts
with cyclo-octyne conjugates to label target proteins; (E) “Sortagging” in presence of oligoglycine probes; (F) FGE mediated conversion of
cysteine (within the aldehyde tag) to formylglycine (FGly) followed by modification with aminooxy probes.
bond, release of the LPET motif and presentation of the G5
tag at the N-terminal of target proteins. In the second step,
upon incubation with SrtA and oligoglycine bearing probes,
transpeptidation proceeds between the probe and G5 tag at
the amino terminal of proteins leading to a site-specific la-
belling of the proteins. The LPETG5 motif was selected, as
sortase is known to prefer a pentaglycine motif as substrate
instead of short oligoglycines as was indicated Km measure-
ments in which Km values decreased with an increase in
length of oligoglycine chain .
10 Current Medicinal Chemistry, 2009 Vol. 16, No. 34
Chattopadhaya et al.
220.127.116.11. Protein Farnesyltranferase (PFTase) and Formylgly-
cine Generating Enzymes (FGE)
It is possible to enzymatically introduce protein bio-
orthogonal functionalities into proteins using either PFTase
or FGE. PFTase appends modified farnesyl moieties onto
proteins that bear the four amino acid farnesylation motif
(CVIA) at the C-terminal. Duckworth et al. used the PFTase-
mediated prenylation reaction to introduce an alkyne handle
into the C-terminal of EGFP protein that had the CVIA se-
quence appended to it; subsequently the alkyne handle was
modified via the cycloaddition reaction with azide-TexasRed
Activation of sulfatases requires the post-translational
modification of the first cysteine (in eukaryotes and pro-
karyotes) or serine (in prokaryotes) within the conserved
pentapeptide motif, (C/S)X(P/A)XR, to formylglycine
(FGly) and this is mediated by FGEs . Work by Bertozzi
and colleagues showed that a >90% conversion of the cys-
teine to FGly can be achieved in E. coli when FGE from
Mycobacterium tuberculosis and target protein that had been
appended with the “aldehyde tag”-LCTPSR, are coexpressed
. Condensation of the aldehyde moiety of FGly with
aminooxy- or hydrazide functionalised probes enabled the
chemoselective labelling of proteins (Fig. 3F). It remains to
be seen if both PFTase and FGE can be applied to label pro-
teins in vivo.
3.2.3. Covalent Labelling Via Introduction of Bioorthogo-
Bioorthogonal chemical handles are non-native, non-
perturbing entities that can be modified in living systems
through highly selective reactions with exogenously deliv-
ered probes in a two-step reaction. Besides azides (section
18.104.22.168), ketones and aldehydes have also been employed as
bioorthogonal chemical reporters as these functionalities can
be used to tag both proteins and glycans. Ketones and alde-
hydes react with hydrazide- or aminooxy-probes to form
Schiff’s bases (hydrazones and oximes, respectively) that are
quite stable under physiological conditions. However, in
complex physiological settings they are not truly bioor-
thogonal since cells have an abundance of these functionali-
ties in the form of free sugar, cofactors (like pyridoxal phos-
phate) etc. The pH optimum for these reactions is 5-6, which
is difficult to attain in vivo. Not surprisingly, the use of ke-
tones and aldehydes have been restricted to applications such
as cell surface labelling as these environments lack carbonyl
22.214.171.124. Metabolic Incorporation of Bioorthogonal Reactive
Glycans mediate a plethora of cellular functions ranging
from cell surface recognition to transcriptional control.
Changes in glycosylation pattern have been frequently asso-
ciated with malignant transformations. Therefore, methods
that allow monitoring of glycans in terms of their distribu-
tion, interaction and structure would provide a more detailed
understanding of their roles under both normal and patho-
logical states. Since FPs and other genetically encoded tags
cannot be applied to the study of glycans, metabolic labelling
has been a particularly useful strategy for such studies. This
methodology exploits endogenous biosynthetic pathways to
incorporate chemical handles that can be subsequently elabo-
rated with small molecule probes in a second step (Fig. 4).
Central to its success is the ability of the enzymes in the
biosynthetic pathways to tolerate the unnatural sugar. In so
far, the approach has been successfully used to study
glycoconjugates that contain the monosaccharide sialic acid
acetylglucosamine (GlcNAc) and fucose.
Among the various metabolic pathways, the sialic acid
biosynthetic pathway has been extensively used because of
its permissiveness to unnatural N-acyl substituents. Both
keto- and azide functionalities have been introduced into cell
surface sialoglycoconjugates using the respective precursor
sugars- N-levulinoylmannosamine (ManLev)  and N-
azidoacetylmannosamine (ManNAz) . Of particular use
has been the azido sialic acid residues (SiaNAz) resulting
from the metabolic incorporation of ManNAz as the azide
functionality could be modified both by phosphine-bearing
probes via Staudinger ligation  or a strain-promoted
[3+2] cycloaddition reaction with alkynes . Besides
mannosamines, free sialic acid analogs themselves can be
used to deliver both ketones and azides to cell surface sialo-
glycans . Since these analogs enter the pathway down-
stream of the more restrictive enzymes, they can be used to
deliver bulkier reporters. Besides the sialoside biosynthetic
pathway, GalNAc and GlcNAc salvage pathways have been
used to introduce keto-  and azido  sugars into
mucin-type O-linked glycoproteins using the respective N-
glucosamine (GlcNAz) precursors. On a similar note, Wong
and colleagues have made use of the permissiveness of fu-
cose salvage pathway to incorporate azido-fucose analogs
into fucosylated glycans. Subsequent click modification of
the azido functionalities using naphthalimide probes permit-
ted imaging of fucosylated glycoproteins both on the cell
surface and in the Golgi .
Since keto- and azide functionalities display orthogonal
reactivity, it is possible to simultaneously visualise the dis-
tribution of different glycan subtypes in the same cell. To
achieve this, Chang et al. fed Jurkat cells with peracetylated
ManLev (Ac4ManLv) and
(Ac4GalNAz), co-incubated the cells with phosphine-dye
conjugate and biotin hydrazide, followed by FITC-avidin
staining. The authors were able to confirm the dual labelling
with both dyes by flow cytometry analysis . In terms of
imaging applications, Cu(I)-catalysed cycloaddition reac-
tions are preferred since “click” reactions proceed 25-fold
faster than Staudinger’s ligation. To circumvent toxicity is-
sues of using Cu(I), Bertozzi and colleagues replaced the
canonical Cu(I) catalyst with constrained difluorinated cy-
clo-octyne (DIFO) derivatives that greatly enhanced the re-
action rate [74,80]. With DIFO-fluorophore conjugates the
authors were able to visualise dynamic events such as glycan
trafficking in cells as well as observe spatiotemporal patterns
of glycan expression, trafficking and tissue distribution dur-
ing early developmental stages in live zebrafish embryos
126.96.36.199. Incorporation of Reactive Handles During Protein
Schultz and colleagues have pioneered the approach of
exploiting the cell’s translational machinery to incorporate
Expanding the Chemical Biologist’s Tool Kit Current Medicinal Chemistry, 2009 Vol. 16, No. 34 11
Fig. (4). Metabolic incorporation of azido handles using tetracetylated N-azidoacetylmannosamine (Ac4ManNAz) via the sialic acid biosyn-
thetic pathway. In a second step, the azide handle is modified by phosphine- or alkyne bearing probes via the Staudinger ligation or [3+2]
cycloaddition reaction, respectively.
unnatural amino acids site-specifically into proteins and have
shown that it is broadly applicable for protein modification
in E. coli, yeast and mammalian cells . The method in-
volves nonsense codon suppression mutagenesis by use of an
orthogonal tRNA/ aminoacyl-tRNA synthetase pair. The
tRNA synthetase activates and grafts the unnatural amino
acid only onto its cognate tRNA while the tRNA’s anticodon
loop is engineered to complement the rare stop codon. When
supplemented with the unnatural amino acid, cells encoding
the genes for the tRNA-synthetase pair and target protein
incorporate the non-natural functionality with high fidelity to
afford the site-specifically modified protein (Fig. 5). Appli-
cations for protein labeling usually involve the use of non-
proteinogenic amino acids that introduce reactive handles
like keto-, azido- or alkyne functionalities allowing for fur-
ther derivatisation via hydrazone formation, Staudinger liga-
tion or [3+2] cycloaddition reactions. In one such example,
m-acetyl-phenylalanine was specifically incorporated into
the LamB membrane protein and subsequent labelling with
membrane impermeant hydrazide dyes allowed visualisation
of the protein in different colours . Besides a two-step
modification, unnatural amino acids having fluorescent
groups like dansyl, amino- or hydroxycoumarin and even an
environmentally sensitive coumarin analogue have been di-
rectly incorporated into proteins . Excellent specificity,
minimal perturbation to protein structure and versatility in
terms of small molecule label that can be incorporated are
the major advantages of this approach. Nevertheless the
method still necessitates generation of a tRNA/synthetase
pair for each unnatural amino acid; other limiting issues in-
cludes the natural prevalence of amber codons in eukaryotes
and low suppression efficiencies resulting premature protein
truncation which in some cases, may produce a dominant
In a complementary approach, Tirrell and coworkers ex-
ploited the promiscuity of the methionyl-tRNA synthetase to
introduce alkynyl sites into proteins via the co-translational
incorporation of homopropargylglycine (Hpg) or ethynyl-
phenylalanine (Eth)  (Fig. 5). This permitted visualisa-
tion of newly synthesised proteins following modification of
the alkyne handle with 3-azido-7-hydroxycoumarin via the
[3+2] cycloaddition reaction. Initially, the coumarin dye is
quenched but subsequent to the addition reaction, it under-
goes a marked increase in fluorescence. Though the method
has been applied to visualize proteins in both E. coli  and
Cu-free [3+2] cycloaddition
12 Current Medicinal Chemistry, 2009 Vol. 16, No. 34
Chattopadhaya et al.
Fig. (5). Incorporation of reactive handles during protein translation. Non-canonical amino acids can be incorporated in a residue specific
manner (for instance substitution of methionine with unnatural amino acids) or an orthogonal tRNA and aminoacyl tRNA synthetase pair can
be used to incorporate the functionality in response to the amber stop codon; in the later case, proteins are modified in a site-specific manner.
mammalian cells , it does not allow study of any particu-
lar target protein. Susceptibility to labelling is not dictated by
the identity of the protein but rather by the extent to which it
is translated during “pulse-chase” with the alkynyl amino
acid. Since the extent of labelling is governed by the preva-
lence of methionine residues in the amino acid sequence of
the proteins, it invariably leads to a heterogeneously labelled
protein pool. While incorporation of unnatural functionalities
at positions other than methionine is not possible, the use of
alkyl amino acids restricts its applicability to fixed cells as
the cycloaddition reaction requires overnight incubations
with toxic Cu(I) catalyst.
3.2.4. Covalent Labelling via Expressed Protein Ligation
EPL is a semisynthetic variant of the widely popular na-
tive chemical ligation (NCL) in which two peptides, one
having a carboxy terminal thioester moiety and other having
Protein folding and
RESIDUE SPECIFIC INCORPORATION
SITE SPECIFIC INCORPORATION
Expanding the Chemical Biologist’s Tool Kit Current Medicinal Chemistry, 2009 Vol. 16, No. 34 13
a cysteine residue at the N-terminal, are joined via a peptide
bond . In EPL, one or both of the reactants is a recombi-
nant (expressed) protein . For years EPL has been a
mainstay for protein engineering approaches and has been
widely used in a variety of applications ranging from intro-
duction of fluorophores, stable isotopes to segmental isotope
labelling and most importantly, in the generation of proteins
having posttranslational modifications such as phosphoryla-
tion, lipidation and glycosylation . Giriat and Muir
showed that protein semi-synthesis can proceed in a cellular
context  but it was work by Yao and colleagues that ex-
tended EPL based methodologies into the realm of imaging
applications . Since the pre-requirement for proteins to
undergo NCL is the availability of a free amino terminal
cysteine residue (?-Cys), Yeo et al. resorted to using intein-
mediated protein splicing to generate such protein. Inteins
are naturally occurring self-splicing proteins that can sponta-
neously excise themselves so as to join the two flanking re-
gions (exteins) by a peptide bond . For labelling to oc-
cur, target proteins were expressed as intein fusions such that
spontaneous cleavage leads to the in situ generation of ?-
Cys-proteins that could be specifically labelled with
thioester-bearing probes. Using a suite of cell permeable
thioester probes bearing dyes, biotin and crosslinkers the
specific labelling of target molecules in bacterial  and
mammalian cells . However, this approach had notable
drawbacks-the size of the intein used was large; uncon-
trolled, spontaneous splicing invariably gave rise to a differ-
entially labelled protein pool and necessitated the use of long
labelling times with high concentrations of the probe. The
extent of splicing was also dependent on the nature of its
To alleviate these concerns, the group made use of a
highly specific protease to generate the amino terminal cys-
teine residue . Tobacco Etch Virus (TEV) protease was
used as this cysteine protease is highly specific, has exquisite
amino acid requirements at the P1, P3 and P6 positions while
being tolerant to P1’ substitutions. Accordingly, the glycine
residue at P1’ position of the canonical sequence, ENLYFQG
was changed to cysteine. Proteins appended with the modi-
fied TEV recognition and cleavage sequence were efficiently
processed (>90% cleavage) in bacterial cells to generate N-
terminal cysteine proteins that could be labelled in a site-
specific manner with membrane permeant thioester probes
(Fig. 6A). The method was successfully used to label an im-
portant bacterial cell division protein, FtsZ, in manner that
did not alter the localisation and function of the protein.
Since cleavage and labelling proceeds simultaneously, la-
belled proteins are obtained in a single step. The thioester
probes can be easily fashioned with different fluorophores
and affinity handles like biotin. In comparison approaches
using protein/peptide domains, this method is essentially
“tag” free as only a single, innocuous amino acid is intro-
duced. However the method can only be applied to label
proteins at the amino terminal and cannot be extended to
include those proteins having an organellar targeting/sorting
sequence at this position. Besides TEV, the 3C protease
(3Cpro) from Foot-and-Mouth Disease Virus (FMDV) has
also been used to generate ?-Cys proteins that can be modi-
fied by NCL .
3.2.5. Activity Based Probes as Reporters of Enzyme Activ-
As opposed to overexpression systems, a more funda-
mental understanding of multifarious roles mediated by pro-
teins stems from the study of endogenous proteins within the
context of their native cellular milieu. Enzymes, by virtue of
their catalytic activity, are particularly amenable to studies
using reporter substrates since enzyme-mediated turnover
comprises of an inherent signal amplification step (and hence
a high signal-to-noise ratio) . But a persistent problem of
these methods has been the difficulty in ascribing the ob-
served signal with specific target identified using biochemi-
cal methods since enzyme-substrate pairs are not covalently
linked. This in turn confounds analysis of imaging data.
Activity based probes (ABPs)  originally developed
to profile mechanistically distinct enzymes classes are well
positioned to address this limitation. Blum et al. demon-
strated that ABPs serve as valuable tools to not only con-
tinuously monitor activity but also to covalently tag the en-
zymes in vivo (Fig. 6B). The group made use of a fluores-
cently quenched dipeptidyl ABP (GB117) based on the acy-
loxymethyl ketone (AOMK) warhead to specifically detect
cysteine proteases . The probes were capped with
BODIPY and QSY7 as the donor-quencher pair. The fluoro-
phore pair was chosen so as to maintain cellular permeability
of probe with the QSY7 moiety being appended to the acy-
loxy-leaving group. Enzymatic cleavage of the peptide scaf-
fold led to dequenching of the fluorescence due to a loss of
QS7 fluorophore together with the concomitant tagging of
the enzyme thereby rendering the enzyme-probe adduct visi-
ble. The extent of probe modification served as an indirect
readout of enzyme activity since the binding of the probe is
solely governed by activity. This strategy allowed the
authors to dynamically image cathepsin activity in lysosomes
in cultured monolayers of the NIH3T3 fibroblast cell line.
Further extending on this work, Blum et al. performed non-
invasive imaging of cathepsin activity in mice bearing tu-
mour xenografts . In this case, quenched probes with
near infrared emission tags (qNIRF-ABP) were intrave-
nously injected into mice to produce spatially resolvable
signals that correlated with levels of active cathepsin in those
tissues. Limitations of ABPs as imaging tools comprise of
the lack of signal amplification since labelling does not in-
volve enzymatic turnover. This makes it difficult to gauge
the extent of enzyme activity required for a sufficient signal
to noise ratio. Also, the warhead permits only en masse im-
aging of all members of a given enzyme class making it im-
possible to study the contribution of individual members
towards a diseased condition. In its current format, ABPs can
only be used to image enzymes.
4. APPLICATIONS OF CHEMICAL LABELLING AP-
PROACHES: BEYOND IMAGING
Applications of chemical labelling strategies extend be-
yond the realm of spatiotemporal imaging of protein dynam-
ics. Creative use of these methods has led to development
FRET-bases sensors to monitor protease activity . To-
wards this end, two orthogonal AGT domains-MAGT and
LAGT, were linked by the caspase-3 sequence, DEVD. Fol-
lowing labelling with FRET compatible BG and PG deriva-
14 Current Medicinal Chemistry, 2009 Vol. 16, No. 34
Chattopadhaya et al.
Fig. (6). (A) Covalent labelling via expressed protein ligation (EPL). TEV mediated generation of N-terminal cysteine proteins can be modi-
fied site-specifically by thioester-bearing probes via the native chemical ligation (NCL) reaction. (B) Cell permeable, quenched activity based
probes have been used to visualise protease activity. The acyloxymethyl ketone (AOMK) warhead was used to specifically target cysteine
proteases while the QSY7 served to quench the fluorescence from the donor, BODIPY. Cleavage of dipeptide sequence resulted in unmasking
of the donor fluorescence.
tives, enzyme activity was monitored by a decrease in FRET
efficiency. Tour et al. developed a cell permeable biarsenical
Ca2+ sensor, Calcium Green FlAsH that undergoes a 10-fold
increase in fluorescence upon Ca2+ binding and exhibits mil-
lisecond response to changes in ion flux . When targeted
to ?1C L-type calcium channel tagged with the TC motif, the
authors were able to identify hotspots, that is, regions that
have highest calcium influx in response to membrane depo-
larisation. Similarly, advances in the synthesis of CoA-
derivatives for use with the carrier protein labelling strategy
has seen the availability of the new derivative CoA-CypHer
that is non-fluorescent at neutral pH but becomes highly
fluorescent at acidic pH. While labelling is restricted to sur-
face exposed receptors, only the internalised receptors in
acidic vesicles are visible. Monitoring the trafficking of the
OR170-40 odorant receptor labelled with this dye revealed
that this receptor is rapidly internalised and recycled even in
absence of the ligand, a behaviour unique to ORs and indica-
tive of the mechanism behind rapid recovery of odour per-
ception . Dissecting such nuances in behaviour would
not be possible with the use of FP fusions. In vitro applica-
tions of the CP approach have focused on the generation of
protein microarrays. PCP-fusion proteins could be site-
specifically biotinylated by Sfp and CoA-biotin directly in
bacterial cell extracts; these proteins could then be specifi-
cally captured on streptavidin slides to generate a protein
microarray . In another study, phages having the PCP
domain fused to the pIII coat protein were used to monova-
lently display a library of small molecules . CoA de-
rivatives of the small molecules were ligated to the PCP-pIII
protein by Sfp while a 20-nucleotide sequence of the
phagemid served to encode the identity of the small mole-
cule. Following panning and selection, a DNA decoding ar-
ray was used to identify the small molecule target(s) thereby
showing that this strategy is amenable for high throughput
screening of chemical libraries.
The introduction of aryl azide photocrosslinkers via the
W37V mutant of LplA permitted the study of protein-protein
interaction (PPI) between FKBP and FRB . The heterodi-
mer was observed only in presence of rapamycin and UV
exposure. Instead of a covalent trapping of the interacting
partners, use of BirA allows detection of PPI by proximity-
induced biotinylation. In this case, FRB-BirA fusions were
used to detect bona fide interactions with FKBP having the
AP sequence. In presence of biotin and rapamycin, FKBP-
FRB interaction could be detected by the appearance of bi-
otinylated FKBP on streptavidin blots . PPI in live cells
has also been detected using AGT fusions in conjunction
with small molecules that had two BG units connected by a
flexible linker  but this method has the disadvantage it
leads higher ‘background” due to the formation of homodi-
mers in addition to heterodimerisation. BG-Mtx derivatives
have been used as “chemical inducers of dimerisation” (CID)
to control transcription in yeast. In an essentially three-
hybrid system, BG-Mtx was used to recruit the DNA binding
domain (LexA-AGT) and transcriptional activation domain
(B42-DHFR) to constitute a functional transcription factor
and this was assessed by viability assays using the histidine
auxotroph strain, L40 .
In a more profound application of chemical labelling
methodology that can have important implications in tissue
Expanding the Chemical Biologist’s Tool Kit Current Medicinal Chemistry, 2009 Vol. 16, No. 34 15
engineering and regenerative medicine, Yarema and col-
leagues demonstrate that pluripotent human embryoid body-
derived (hEBD) stem cells can be coaxed to differentiate into
neuronal cells . A simple installation of thiol residues
onto cell surface exposed sialic acid residues via metabolic
incorporation of Ac5ManTGc (a ManNAc analogue) was
able to induce the lineage specific differentiation. This opens
up yet another exciting avenue for influencing stem cell
fates-one mediated by ”engineering” of glycosylation path-
5. CHEMICAL LABELLING AND DRUG DISCOV-
Current trends in drug discovery process have seen a shift
from single target assays to high throughput (HT) and high
content (HC) image-based analysis. This has been made pos-
sible, in part, by the availability of automated imaging and
analysis systems that permit quantitative analysis of cellular
events and visualisation of relevant cellular phenotypes
. Screening approaches are now “target based” which
assumes that disruption of a single gene or molecular
mechanism is the key event in disease ontogeny. Together
with the availability of alternative validation tools such as
small interfering RNA (siRNA), target discovery process has
greatly matured and fluorescent microscopy based methods
are well poised to meet the demands of assessing the targets
within a cellular context. For example, it has been possible to
observe dynamic changes in gene expression by placing GFP
under the control of hypoxia-response elements. Following
treatment, any damage resulting from hypoxia or oxidative
could be conveniently monitored via the activation of the
transcription of the hypoxia-inducible factor 1?. Microscopy
platforms also permit the functional analysis of phenotypic
changes that are difficult to assess such as neurite growth
and cell migrations. These approaches are versatile as they
provide flexibility in terms of resolution-at low magnifica-
tion, phenotypic changes across entire cell populations can
be quantified while at high resolution it is possible to discern
subcellular changes. However, most published image based
screens have been, at least in the pharmaceutical industry, at
the secondary screening stage/ lead optimisation stage.
Given that chemical labelling approaches are still in its
infancy, it is would be interesting to speculate as to how the
emergence of these methods may impact HC screening. One
area that is likely to benefit from the introduction of such
approaches is the understanding of GPCR oligomerisation. A
recent analysis suggested that approximately 45% of all
known pharmaceutical drugs are directed at transmembrane
receptors with GPCRs being the predominant target .
Within cells, GPCR do not exist and function as non-
interacting species but rather as homo- or heterodimers
and/or oligomers . Chemical labelling approaches per-
mit exclusive surface labelling of receptors and therefore it is
possible to exclude signal contributions by internal-
ised/subcellular receptors in assays of receptor oligomerisa-
tion using FRET, TR-FRET . Such analysis would pro-
vide a better understanding of GPCRs and may offer novel
concepts for drug design. Next to GPCRs, proteases are im-
portant drug targets given that aberrant proteolysis leads to a
plethora of diseases like inflammation, arteriosclerosis, neu-
rodegeneration and infection . Blum et al. showed that
by using quenched activity based probes, it is possible to
monitor proteases like cathepsins in both in cultured cells-
 as well as in vivo . It is possible that similar ap-
proaches will be adopted to study other protease class since
ABPs are known to exist for different enzyme classes. Be-
sides imaging, these probes will also serve as important tools
to validate the efficacy and pharmacodynamic properties of
drugs against target protease(s) and thereby accelerate the
drug discovery process.
A persistent problem facing the field of glycobiology has
been the difficulty is dissecting the precise role of an indi-
vidual glycan in tumour. This is despite the fact that mount-
ing clinical evidence point to the fact that changes in glyco-
sylation patterns accompany tumourigenesis. The direct im-
aging of cellular glycans via the oligosaccharide engineering
approach may help address this issue. By chemically tagging
glycans with affinity probes, it may also be possible to enrich
and identify disease-associated glycans . In a recent
report, Laughlin et al. followed the developmental fate of
glycans in live zebrafish embryos . This further raises
the possibility that it will be possible to perform noninvasive
imaging of glycans not only for HCS cell based assays but
also in mouse models of cancer and inflammation.
These are exciting times for cell biology as developments
in microscopy techniques and instrumentation are pushing
the limits of resolution barriers. It is now possible to image
structures in cells at molecular resolution given the availabil-
ity of methods like PALM, STORM and STED. To meet the
demands of these technological innovations and bridge the
requirements of specificity and minimal perturbation to the
experimental system, it is important that parallel advance-
ments are made in the development of new fluorophores and
labelling strategies. In all likelihood, chemical labelling
strategies will continue to complement the use of FPs to seek
answers to fundamental cell biology questions as no single
method is “perfect”. Future development in chemical label-
ling strategies is likely to see innovations in design of probes
to include photoactivable and/or environmentally sensitive
fluorophores. So far, much of effort has been expended in
developing the different strategies and applying them in
model systems. It is only appropriate that these methods find
greater acceptance and undergo more rigorous evaluation.
Jansen et al. to exploited the fine temporal control of SNAP-
tag mediated labelling to investigate the role of the centro-
mere protein A (CENP-A), a histone H3 variant, in cell divi-
sion . Studies like this sets the precedent and heralds
the possibility of groundbreaking work in the near future. To
further harness the strengths of chemical labelling, it is likely
that multicolour labelling using two or more orthogonal la-
belling approaches will be applied to one sample.
Currently, a range of small molecule based fluorescent
sensors is available to detect metal ions, second messenger
molecules (NO) etc [113,114]. Specific targeting/ localisa-
tion of these sensors to subcellular organelles using chemical
labelling approaches would permit a more detailed under-
standing of the cellular microenvironment. Ratiometric or
FRET-bases sensors for important cofactors like NADH,
16 Current Medicinal Chemistry, 2009 Vol. 16, No. 34
Chattopadhaya et al.
NAD+, cellular ATP, is another exciting area of application.
Development of FP-based sensors for these metabolites has
been challenging given the conformational constraints in-
volved with the use of such bulky molecules. Besides pro-
teins, glycans it will be worthwhile to explore the possibili-
ties of using bioorthogonal labelling methods to tag lipids
and mRNA as well as understand role of other posttransla-
tional modifications such as actetylation and methylation.
Developments in novel bioorthogonal reactions will also
greatly improve the scope and range of chemical labelling
approaches. One thing is for sure; the future of chemical
labelling is bright, as it will continue being an important tool
for both chemical biologists and biologists alike.
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