A Chemoselective Rapid Azo-Coupling Reaction (CRACR) for Unclickable Bioconjugation

Article abstract of DOI:10.1021/jacs.7b05125

Chemoselective modification of complex biomolecules has become a cornerstone of chemical biology. Despite the exciting developments of the past two decades, the demand for new chemoselective reactions with unique abilities, and those compatible with existing chemistries for concurrent multisite-directed labeling, remains high. Here we show that 5-hydroxyindoles exhibit remarkably high reactivity toward aromatic diazonium ions and this reaction can be used to chemoselectively label proteins. We have previously genetically encoded the noncanonical amino acid 5-hydroxytryptophan in both E. coli and eukaryotes, enabling efficient site-specific incorporation of 5-hydroxyindole into virtually any protein. The 5-hydroxytryptophan residue was shown to allow rapid, chemoselective protein modification using the azo-coupling reaction, and the utility of this bioconjugation strategy was further illustrated by generating a functional antibody-fluorophore conjugate. Although the resulting azo-linkage is otherwise stable, we show that it can be efficiently cleaved upon treatment with dithionite. Our work establishes a unique chemoselective "unclickable" bioconjugation strategy to site-specifically modify proteins expressed in both bacteria and eukaryotes.

Full text of DOI:10.1021/jacs.7b05125

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Communication
A Chemoselective Rapid Azo-Coupling Reaction
(CRACR) for Unclickable Bioconjugation
Partha Sarathi Addy, Sarah B Erickson, James S. Italia, and Abhishek Chatterjee
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b05125 • Publication Date (Web): 08 Aug 2017
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A Chemoselective Rapid Azo-Coupling Reaction (CRACR) for Un-
clickable Bioconjugation
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Partha Sarathi Addy, Sarah B. Erickson, James S. Italia, and Abhishek Chatterjee*
Department of Chemistry, Boston College, 2609 Beacon Street, 246B Merkert Chemistry Center, Chestnut Hill,
Massachusetts 02467, United States. Tel: +1-617-552-1778. email: abhishek.chatterjee@bc.edu
biophysical probes to therapeutic agents.1f, 8-9 However, many
ABSTRACT: Chemoselective modification of complex bio-
of the available chemistries are limited by their slow kinetics,
the need for toxic catalysts, or a lack of compatibility with
other chemistries to allow the simultaneous attachment of
multiple distinct entities at different sites.10 Consequently,
there is continued interest in additional genetically encoded
chemical functionalities that can be rapidly and chemoselec-
tively labeled using catalyst-free conjugation reactions, and
that are also compatible with other available chemistries to
allow concurrent labeling at multiple sites.
molecules has become a cornerstone of chemical biology.
Despite the exciting developments of the last two decades,
the demand for new chemoselective reactions with unique
abilities, and those compatible with existing chemistries for
concurrent multisite-directed labeling, remains high. Here
we show that 5-hydroxyindoles exhibit remarkably high reac-
tivity towards aromatic diazonium ions and this reaction can
be used to chemoselectively label proteins. We have previ-
ously genetically encoded the non-canonical amino acid 5-
hydroxytryptophan in both E. coli and eukaryotes, enabling
efficient site-specific incorporation of 5-hydroxyindole into
virtually any protein. The 5-hydroxytryptophan residue was
shown to allow rapid, chemoselective protein modification
using the azo-coupling reaction, and the utility of this bio-
conjugation strategy was further illustrated by generating a
functional antibody-fluorophore conjugate. Although the
resulting azo-linkage is otherwise stable, we show that it can
be efficiently cleaved upon treatment with dithionite. Our
work establishes a unique chemoselective ‘unclickable’ bio-
conjugation strategy to site-specifically modify proteins ex-
pressed in both bacteria and eukaryotes.
The azo-coupling reaction between an aromatic diazoni-
um ion and aromatic amino-acid residues, such as tyrosine
(Tyr, 1; Figure 1A), has been previously used to chemically
modify proteins.11 However, the inability to achieve site-
specific labeling, owing to the abundance of surface exposed
tyrosine residues commonly found on proteins, significantly
limits the utility of this conjugation strategy. Even though
reactive diazoniums, e.g., 4-nitrobenzenediazonium (4NDz,
3), can label tyrosine residues at physiological pH, less elec-
trophilic counterparts are only able to react at a significantly
elevated pH.11a We anticipated that the identification of aro-
matic groups that exhibit enhanced reactivity towards aro-
matic diazonium ions could form the basis of developing a
new chemoselective reaction for bioconjugation.
5-hydroxytryptophan (5HTP, 2; Figure 1A) is a stable non-
toxic amino acid which is widely accessible as an over-the-
counter dietary supplement.12 It is also generated in vivo at
low levels as a metabolic precursor to neurotransmitters ser-
otonin and melatonin.13 The 5-hydroxyindole ring of 5HTP is
highly electron rich, as indicated by its propensity to under-
go oxidation under relatively mild conditions.14 To evaluate if
the electron-rich 5-hydroxyindole exhibits enhanced reactivi-
ty towards aromatic diazonium ions, we monitored the ki-
netics of the azo-coupling reaction between 4NDz with
5HTP, and compared it to the corresponding reaction with
tyrosine under ambient conditions, in an aqueous phosphate
buffer at pH 7. The formation of the chromophoric azo-
coupling product in each case was monitored using spectro-
photometry (increased absorption at 450 nm; Figure S1 in the
Supporting Information). 5HTP was found to react with
4NDz at a rate (k2=63000±6500 M-1s-1; Figure S2, S4) approx-
imately 4500 fold faster than tyrosine (k2=14.2±0.1 M-1s-1; Fig-
ure S3, S4). The reaction between 5 μM each of 5HTP and
4NDz in aqueous phosphate buffer (pH 7) was found to
reach near-completion in less than a minute, while an identi-
cal reaction of 4NDz with tyrosine did not show noticeable
The ability to chemoselectively label complex biomole-
cules has emerged as a powerful strategy to both study and
engineer their structure and function.1 To achieve this,
uniquely reactive non-natural chemical functionalities are
incorporated into biomolecules through synthetic or biosyn-
thetic routes, followed by their selective functionalization
using a chemoselective reaction.1 A variety of such reactions
have been developed to date, including the condensation
between
an
aldehyde/ketone
and
an
alkoxy-
amine/hydrazine,2 the Staudinger ligation,3 the Cu(I)-
catalyzed4 and strain promoted cycloaddition5 between al-
kynes and azides, the inverse electron demand Diels-Alder
reactions between tetrazines and strained alkenes,6 various 1,
3-dipolar cycloadditions,7 etc. Many of these chemistries
have been harnessed within a growing collection of genet-
ically encoded non-canonical amino acids (ncAAs).1f, 8 Devel-
opment of engineered nonsense-suppressing aminoacyl-
tRNA synthetase (aaRS)/tRNA pairs that selectively charge
such ncAAs has enabled their facile site-specific incorpora-
tion into proteins expressed in living cells, which can be sub-
sequently used to precisely attach a variety of entities, from
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progress in this timeframe (Figure 1C). However, at elevated
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concentrations of Tyr, slow formation of the coupling prod-
uct was observed (Figure 1C). Even though these observa-
tions indicate that 4NDz may be used to selectively label
5HTP in the presence of Tyr, its non-negligible reactivity
towards the latter prompted us to explore less electrophilic
diazonium compounds with attenuated reactivity.
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Figure 2: Characterizing the product of the azo-coupling
reaction with 5HTP. A) Products of the azo-coupling reaction
between 6, a 5HTP analog, and three aryl-diazoniums were
purified and characterized. B) Crystal structure of the azo-
coupling product of 6 with 4MDz.
We have recently engineered the E. coli tryptophanyl-
tRNA synthetase (EcTrpRS)/tRNA pair to efficiently charge
5HTP in response to the TGA nonsense codon in both eukar-
yotes, as well as in an engineered E. coli strain, where the
endogenous EcTrpRS/tRNATrp pair was functionally replaced
with a eukaryotic counterpart.15 Using this platform, we ex-
pressed super-folder green fluorescent protein incorporating
5HTP at a permissive surface exposed site (sfGFP-151-5HTP;
Figure S11). Incubation of sfGFP-151-5HTP with 40 μM 4CDz
resulted in its rapid and complete covalent modification
(Figure 3A, Figure S12) within 30 min, as observed by mass-
spectrometry analysis, while a nearly identical wild-type
sfGFP (Tyr at 151 position) protein remained unmodified up-
on the same treatment (Figure 3B). We further showed that
sfGFP-151-5HTP, but not wild-type sfGFP, can also be selec-
tively modified using 4NDz (Figure S13) and 4MDz (Figure
S14). As expected, the labeling with 4NDz was significantly
faster, reaching near-completion within a minute (Figure
S13C).These observations confirm that the genetically encod-
ed 5HTP residue can be used to site-specifically label pro-
teins using the chemoselective rapid azo-coupling reaction
(CRACR).
Figure 1: 5HTP 2 exhibit significantly higher reactivity to-
wards aryl diazonium ions relative to Tyr 1. A) Azo-coupling
reaction of aryl-diazoniums with Tyr and 5HTP. B) Struc-
tures of aryl-diazonium ions used. C) Observed rate of azo-
coupling reaction of 4NDz 3 with 5HTP and Tyr at indicated
concentrations in 100 mM phosphate buffer (pH 7, room
temperature). D) Measurement of the rate of the azo-
coupling reaction between 4CDz and 5HTP under pseudo-
first order conditions. E) Second-order rate constants of the
azo-coupling reactions between the indicated partners (M-1s-
1). Each rate represents an average of three independent ex-
periments, and error represents standard deviation.
We evaluated 4-carboxybenzenediazonium (4CDz, 4) and
4-methoxybenzenediazonium (4MDz, 5), which reacted with
5HTP with rate constants of 193±37 M-1s-1 (Figure 1D) and
2.3±0.1 M-1s-1 (Figure S5). In contrast, prolonged incubation of
4CDz with tyrosine at pH 7 resulted in significant levels of
diazonium hydrolysis but no azo-coupling (Figure S6-S8);
although, some coupling was observed at pH 9, corroborat-
ing previous reports.11f Owing to its selective yet robust reac-
tivity towards 5HTP at pH 7, we focused on the 4CDz scaf-
fold for further studies. The reaction between 5HTP (0.1 mM)
and 4CDz (10 μM) in 100 mM phosphate buffer (pH 7) was
found to reach near-completion within minutes, while 4CDz
failed to react with tyrosine and other canonical amino acids
under similar conditions (Figure S9-S10). To characterize the
product of the azo-coupling reaction with 5-hydroxyindole,
three diazonium compounds were reacted with methyl 2-(5-
hydroxy-1H-indol-3-yl)acetate (6; Figure 2A), a 5HTP analog,
and the resulting products were isolated and characterized
by NMR (1H and 13C) and mass-spectrometry. The product
with 4MDz was also characterized by X-ray crystallography
(Figure 2B) to further confirm that the azo coupling occurs
through the 4-position of the 5-hydroxyindole ring.
We generated the fluorescent diazonium compound 7
(Figure 3C) from 6-aminofluorescein, which retains the 4CDz
scaffold. Incubation with 7 resulted in selective and complete
labeling of sfGFP-151-5HTP, but not wild-type sfGFP, as mon-
itored by SDS-PAGE followed by fluorescence imaging (Fig-
ure 3D), and mass-spectrometry (Figure S15). Recently, the
aryl-trizabutadiene group has been established to be a facile
precursor to the aryl-diazonium species, the release of which
can be triggered by light or low pH.16 We synthesized a con-
jugate (8, Figure 3C) between biotin and a triazabutadiene,
which upon brief irradiation releases biotin-4CDz (Figure
S16A). We showed selective biotinylation of sfGFP-151-5HTP,
but not wild-type sfGFP, upon treatment with biotin-4CDz
by a western-blot analysis using a streptavidin-HRP probe
(Figure 3E), and MS analysis (Figure S16).
The ability to site-specifically label antibodies has been a
valuable technology to generate therapeutically important
antibody-drug conjugates, as well as covalent conjugates
with biophysical probes that are important as diagnostics as
well as research tools.9, 17 To evaluate if CRACR can be used
to generate such antibody-conjugates in a site-specific man-
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Journal of the American Chemical Society
ner, we expressed the previously described Fab fragment18 of
clicked” upon a dithionite treatment. Indeed, treatment of
the anti-Her2 antibody Herceptin19 in our engineered E. coli
strain, site-specifically incorporating 5HTP at position 169.
The wild-type Fab protein was also expressed as a control
(lysine at position 169). SDS-PAGE and ESI-MS analysis was
used to confirm successful expression of both proteins (Fig-
ure 4A-B). 80 μM of the fluorescent diazonium compound 7
was incubated with both the wild-type and the 5HTP-mutant
anti-Her2 Fab for 15 min on ice. Subsequent ESI-MS analysis
and SDS-PAGE, followed by fluorescence imaging, confirmed
complete labeling of the 5HTP-mutant and no labeling of the
wild-type antibody (Figure 4A-B). The resulting fluorescently
labeled antibody was dialyzed to remove the unreacted
fluorophore, and was shown by fluorescence-activated cell
sorting (FACS) analysis to successfully bind to the SK-BR-3
breast cancer cells, which overexpresses the Her2 receptor
(Figure 4C).
the purified model azo molecule 9 (Figure 5A) with 1.5 mM
dithionite led to its rapid reductive cleavage, as observed by
HPLC-coupled MS analysis (Figure 5A, Figure S17). To
demonstrate that the azo-linkage generated on a protein
through a 5HTP residue can also be cleaved this way, we
treated the aforementioned conjugate (Figure 3E) between
sfGFP-151-5HTP and biotin-4CDz 8 with dithionite and con-
firmed the removal of the biotin by a western-blot analysis
(Figure 5B). However, it should be noted that the 4-amino-5-
hydroxytryptophan residue generated upon this reduction is
readily oxidized under ambient conditions, which precluded
the MS-analysis of this cleavage reaction on the protein. The
remarkable speed and selectivity of the 5HTP-directed
CRACR, and the ability to subsequently cleave the resulting
azo-linkage, underscores unique advantages of this conjuga-
tion strategy.
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Figure 4: A functional Herceptin-Fab-fluorophore conjugate
created using 5HTP-directed CRACR. A) ESI-MS analysis of
the wild-type Fab (expected mass 47752 Da), Fab-169-5HTP
(expected mass 47826 Da), and the conjugate between Fab-
169-5HTP and 7 (expected mass 48186 Da). B) SDS-PAGE
followed by Coomassie staining (top) and fluorescence imag-
ing (bottom) reveals selective fluorescence-labeling of Fab-
169-5HTP, but not wild-type Fab by 7. C) The conjugate be-
tween 7 and Fab-169-5HTP conjugate can bind the Her2-
overexpressing SK-BR-3 cells, as shown by FACS analysis.
Percentage of cells fluorescing within the R4 gate is shown in
each case.
Figure 3: Chemoselective protein labeling using 5HTP-
directed azo-coupling reaction. A) Incubating 10 μM sfGFP-
151-5HTP with 40 μM 4CDz at room temperature for 30
minutes results in complete protein labeling (expected mass
27784 Da). B) The wild-type protein does not undergo modi-
fication upon the same treatment. C) Structures of the fluo-
rescent diazonium compound 7, and the biotin-
triazabutadiene conjugate 8, synthesized for protein labeling
through CRACR. D) Selective fluorescence-labeling of sfGFP-
151-5HTP by 7, shown by fluorescence imaging following
SDS-PAGE. E) Selective biotinylation of sfGFP-151-5HTP us-
ing photolyzed 8, revealed by western blot (using a streptav-
idin-HRP probe) following SDS-PAGE.
In summary, we have established a general strategy to la-
bel recombinant proteins using 5HTP-directed CRACR. The
ability to 1) site-specifically incorporate 5HTP, a stable and
cheap ncAA, into any protein of interest expressed in both E.
coli and eukaryotic cells, 2) chemoselectively functionalize
this non-natural residue with readily generated diazonium
ions through a rapid (>190 M-1s-1 for the 4CDz scaffold) cata-
lyst-free reaction, 3) systematically tune the coupling chemis-
try by rational substitution on the aryl-diazonium, and 4)
Even though the azo-linkage is stable under physiological-
ly relevant conditions, it is known to undergo facile cleavage
20
upon treatment with dithionite,11a,
a strong but protein-
compatible reducing agent. Given that the linkages generat-
ed by most available bioconjugation reactions are challeng-
ing to selectively undo under mild conditions, we aimed to
explore if the conjugate made through CRACR can be “un-
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ly different from the canonical amino acid tryptophan. It is
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with existing bioorthogonal conjugation reactions (e.g., az-
ide-alkyne click reaction)21 – a possibility that we are actively
exploring – which will enable site-specific labeling of pro-
teins with multiple different entities.
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Figure 5: The azo-linkage created through 5HTP-directed
CRACR can be cleaved using dithionite. A) Dithionite-
mediated cleavage of the model azo-compound 9. B) HPLC-
MS analysis of this reaction shows the production of the ex-
pected products (also see Figure S17). C) Dithionite-mediated
cleavage of the conjugate between photolyzed 8 and sfGFP-
151-5HTP monitored by SDS-PAGE followed by coomassie
staining and western blot (Streptavidin-HRP). Treating
sfGFP-151-5HTP (left lane) with photolyzed 8 leads to its bio-
tinylation (middle lane). Subsequent treatment of this puri-
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group (right lane).
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ASSOCIATED CONTENT
Supporting Information
Experimental methods, additional figures, NMR and MS-
characterization of small molecules. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*abhishek.chatterjee@bc.edu
ORCID Abhishek Chatterjee: orcid.org/0000-0002-6231-5302
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
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