Chemoselective bioconjugation based on modular click chemistry with 4-halocoumarins and aryl sulfonates

Article abstract of DOI:10.1039/d1ra03271b

We report chemoselective and modular peptide bioconjugation using stoichiometric amounts of 4-halocoumarin and arylsulfonate agents that undergo metal-free C(sp²)-heteroatom bond formation at micromolar concentrations. The underlyingipso-substi

Full text of DOI:10.1039/d1ra03271b

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Chemoselective bioconjugation based on modular
click chemistry with 4-halocoumarins and aryl
sulfonates†
Cite this: RSC Adv., 2021, 11, 18960
*
F. Yushra Thanzeel
and Christian Wolf
We report chemoselective and modular peptide bioconjugation using stoichiometric amounts of 4-
halocoumarin and arylsulfonate agents that undergo metal-free C(sp2)-heteroatom bond formation at
micromolar concentrations. The underlying ipso-substitution click chemistry is irreversible and generates
stable and inherently fluorescent bioconjugates, and the broad selection of coumarin tags offers high
labeling flexibility and versatility. Different coumarins and arylsulfonates can be selectively attached to
amino and thiol groups in the small peptides glutathione and ornipressin, and both free as well as latent
thiols captured in disulfide bridges can be targeted if desired. The broad utility, ease of use, storage, and
preparation of 4-halocoumarins and arylsulfonates are very attractive features that extend currently
available dual bioconjugation capabilities.
Received 27th April 2021
Accepted 21st May 2021
DOI: 10.1039/d1ra03271b
rsc.li/rsc-advances
metal complexes29,30 or bioincompatible reaction conditions,
instability of the bioconjugate, uncontrolled formation of regio-
Introduction
and stereoisomeric products, or lack of functional group che-
moselectivity.31,32 In most cases, modular labeling of different
functional groups using the same class of bioconjugation agent
is not possible.
Biocompatible chemical modications of peptides and proteins
have received increasing attention in recent years due to the
tremendous value in the study of their biomolecular dynamics,
trafficking and biological functions. Site-selective bio-
conjugation with uorescent tags, affinity probes, polymers
such as PEG, or drugs provides an important tool set to inves-
tigate and modify protein mobility, distribution, biomolecular
interactions and biochemical reaction mechanisms, and it
holds considerable promise for the development of bio-
engineered materials, diagnostics or therapeutics. These
exciting prospects have led to considerable interest in chemo-
selective peptide modication strategies that have emerged as
powerful alternatives to genetically engineered proteins exhib-
iting unnatural amino acids specically incorporated for
chemical derivatization.1–4 Several methods that target an
endogenous amino acid in natural peptides and proteins, for
example lysine,5–7 histidine,8 methionine,9 tyrosine,10–12 trypto-
phan,13 serine14 and cysteine,15–23 or the use of chemoenzymatic
labeling strategies,24 disulde bridge modication chemistry25
and lysine–cysteine crosslinking26,27 have been reported.28
Despite the remarkable advance of this eld within the last 10
years, chemoselective bioligation remains a challenging task.
Persisting shortcomings of currently available methods include
the use of potentially toxic organometallic reagents, transition
We envisioned that coumarin click chemistry would provide
new effective bioconjugation venues that can address these
issues. Our laboratory has recently introduced small molecular
optical probes that achieve click chemistry sensing of the
enantiomeric composition and concentration of free amino
acids in aqueous solutions.33–36 The term ‘click chemistry’
generally refers to a small set of privileged reactions that
proceed smoothly with high yields and minimal byproduct
formation under mild conditions in environmentally benign
solvents. Additional characteristics include operationally
simple reaction protocols, the exclusive use of nonhazardous
materials and the elimination of cumbersome chromatographic
work-up steps, which altogether minimize necessary safety
precautions, waste production and cost.37 A wide variety of
analytical, synthetic and biomedical applications that display
all or at least some of the advantageous features of click
chemistry have been reported in recent years.38–43
Results and discussion
We now report chemoselective, modular thiol and amine
labeling via ipso-substitution with commercially available or
easily prepared, inherently uorescent 4-halocoumarin and
arylsulfonate bioconjugation agents (Fig. 1). Using glutathione
(GSH) as a small test peptide we have achieved high-yielding
conversion using stoichiometric amounts or minimal excess
Department of Chemistry, Georgetown University, 37th and O Streets, Washington, DC
20057, USA. E-mail: cw27@georgetown.edu
† Electronic supplementary information (ESI) available: Experimental details
including coumarin synthesis, bioconjugation optimization studies, ESI-MS
analysis. See DOI: 10.1039/d1ra03271b
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Scheme 1 Synthesis of the bioconjugation agents 2, 4 and 6.
halogenation, generating the 4-bromo-3-nitrocoumarin 4 in
three steps. As shown below, 4 can be used successfully in
chemoselective bioconjugation of GSH and one can imagine
that the cross-coupling chemistry provides a convenient entry to
the loading of a coumarin with a stapling agent or drug. Alter-
natively, coumarins are readily synthesized by well-known
condensation reactions44,45 which greatly facilitates the incor-
poration of additional functionalities into the fused benzene
ring distant from the carbon-halide bond if necessary. Finally,
we prepared phenyl 2,4-dinitrobenzenesulfonate, 6, in 86%
yield as described previously.33
Fig. 1 Chemoselective irreversible thiol and amine bioconjugation
based on metal-free coumarin and arylation click chemistry.
of a diverse set of bioconjugation agents at room temperature
and varying pH. The inexpensive agents are selectively and
irreversibly introduced to thiol and amino groups at micro-
molar concentrations, both free and latent thiols involved in
disulde bridges can be labeled if desired, and the bio-
conjugates are stable under mass spectrometry conditions and
across a wider pH range. The operational simplicity and effi-
ciency of this click chemistry approach allow practical chemo-
selective bioconjugation with increased labeling exibility and
versatility based on well-dened metal free carbon–heteroatom
bond formation which avoids complications that can arise from
the formation of regio- and stereoisomeric products when the
popular maleimide Michael acceptors are used.
At the onset of this study, we prepared several 4-hal-
ocoumarins and phenyl 2,4-dinitrobenzenesulfonate as
described in Scheme 1 to rst investigate the hydrolytic stability
and promise of these agents based on labeling experiments with
cysteine and lysine derivatives at room temperature in aqueous
solutions. The presence of the nitro group at C-3 in the coumarin
scaffold increases the reactivity toward ipso-substitution and was
deemed crucial for the envisioned quantitative attachment to
thiol and amine nucleophiles. The incorporation of different
halides at C-4 is straightforward and allows ne-tuning of the
electrophilicity and rate of nucleophilic displacement (Cl > Br > I)
if necessary. Treatment of 4-chloro-3-nitrocoumarin, 1, with NaI
gave the iodide 2 in quantitative yields.
Preliminary NMR studies with the 4-chlorocoumarin 1 and
the 2,4-dinitrobenzenesulfonate 6 showed fast and quantitative
reaction with the thiol group in Cys in aqueous solution at room
temperature. We also found that the 4-halo-3-nitrocoumarins
react rapidly with amino groups under similar conditions.
This ipso-substitution labeling approach is straightforward,
does not show side reactions, and displays straightforward click
chemistry features unlike bioconjugation methods that rely on
transition metal catalyzed arylation.46 We expected that che-
moselective tagging of thiol and amino residues with our 4-
halocoumarins and the arylsulfonate agent should be possible
through pH and buffer optimization. We were aware from
previous studies, however, that arylation of free cysteine with 6
is followed by thiol-to-amine migration which would be
a problem with a peptide carrying both functionalities in close
proximity as in glutathione.33 The complexity of undesired
byproducts resulting from incomplete monoconjugation, thiol-
to-amine tag-walking, tag replacement and undesired double
ligation that altogether need to be suppressed is shown in Fig. 1.
To test the feasibility of controlled dual bioconjugation we
began to investigate if thiol-selective tagging of glutathione with
chlorocoumarin 1 can be quantitatively achieved without
derivatization of the amino group by carefully screening the
effects of buffer concentration and pH (Fig. 2). Initial studies
using equimolar amounts of GSH and 1 at 5.0 mM and a citrate
buffer adjusted to pH 5.0 showed preferential formation of the
desired mono-conjugated product 12 but also substantial
amounts of the doubly labeled derivative 13 while GSH was
mostly consumed aer 90 minutes. Although the
The remote aryl bromide in commercially available 6-bromo-
4-hydroxycoumarin, 9, was appealing to us as this suggested the
possibility of coumarin modications without signicantly
affecting the bioconjugation chemistry. We therefore developed
a protocol for subsequent nitration, Suzuki cross-coupling and
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Fig. 2 Optimization of the thiol-selective bioconjugation of GSH with
1. (A) Desired and undesired reaction outcomes; (B) optimization of
buffer/GSH ratio using pH 5.0 citrate phosphate buffer for selective
monoconjugation of the thiol moiety in GSH; (C) optimization of buffer
type and pH: potassium phosphate buffer (a), sodium carbonate buffer
(b), TRIZMA (2-amino-2-(hydroxymethyl)-1,3-propanediol) (c); (D)
representative ESI-MS spectra of the reactions between 1 and GSH.
Reactions were carried out in acetonitrile–buffer (4 : 1) solution using
5.0 mM concentrations of 1 and GSH, 50.0 mM buffer concentration,
at room temperature for 1.5 hours. Relative conversions were calcu-
lated using ESI-MS peak intensities of m/z ¼ 495 and 684 corre-
sponding to the desired product and undesired byproduct,
respectively, see ESI† for details.
Fig. 3 Monoconjugated derivatives of GSH obtained with various
agents. (A) Structures of bioconjugated GSH products and relative
conversions. All reactions were carried out in acetonitrile–buffer (4 : 1)
solution, at 5.0 mM concentrations of the bioconjugation agent and
GSH in pH 9.0 TRIZMA buffer (50.0 mM), at 25 ꢀC for 2 hours. Relative
conversions were calculated using ESI-MS peak intensities of the
bioconjugated product and GSH (m/z ¼ 307). (B) Representative MS
spectra of products 14 and 15 (see ESI† for details). The undesired
byproducts carrying two aryl rings were not detected. (C) Fluores-
cence emission spectra of increasing concentrations of mono-
conjugated product 12 (excitation wavelength was 335 nm). (D) The
fluorescence intensity at 441 nm plotted against the GSH/1 ratio, see
ESI† for details.
chemoselectivity toward 12 increased at higher buffer concen-
trations, we still observed free glutathione while more than 5%
of 13 was formed according to ESI-MS analysis (Fig. 2B).
Attempts to improve results with the less reactive but presum-
ably more chemoselective 4-iodocoumarin 2 were unsuccessful
(see ESI†). We therefore decided to test phosphate, carbonate
and TRIZMA buffers at higher pH. Unsatisfactory chemo-
selectivities were obtained with the inorganic buffers but we
were pleased to nd that MS analysis indicates that the
conversion of GSH to 12 is quantitative while the formation of
the undesired byproduct 13 is less than 2% when the bio-
conjugation is conducted in TRIZMA at pH 9.0 (Fig. 2C and D).
The optimized protocol for chemoselective mono-
conjugation of GSH was then applied to 4-chloro-6-uoro-3-
aer 24 hours, proving the highly chemoselective thiol ligation
outcome with GSH (see ESI†).
With these results in hand, we continued exploring the
possibility of modular chemoselective thiol and amino group
bioconjugation (Fig. 4). We rst screened the effect of the
reaction time, equivalents of the bioconjugation agent and pH
to optimize the amine ligation step and found that an increase
in reaction time is mostly sufficient (see ESI†). We then attached
either a 3-nitrocoumarin or a 2,4-dinitrophenyl ring at the thiol
site in GSH, which proceeded according to MS analysis with
more than 99% conversion to the desired structures 12 and 16,
respectively, thus setting the stage for in situ amine ligation. To
these solutions was added another 4-halocoumarin carrying
a uoride, methoxy-phenyl or bromide in the fused benzene
ring, dansyl chloride, 7, or the biotin N-succinimidyl ester 8.
Aer 12 hours, the desired orthogonally ligated peptide
derivatives 17–23 were produced in excellent yields ranging
from 93–99%. While we achieved high conversions under mild
reaction conditions, we were able to effectively suppress the
competing amination with 1 or 6 as well as tag-walking and tag
substitution processes. The results show that different couma-
rins can be chemoselectively introduced to thiol and amino
residues or combined with other arylating agents such as
phenyl 2,4-dinitrobenzenesulfonate.
nitrocoumarin,
3,
4-bromo-6-(4-methoxyphenyl)-3-nitro-
coumarin, 4, and to the benzenesulfonate 6 (Fig. 3). In accor-
dance with the high-yielding formation of 12, we observed
quantitative conversion of the 4-halocoumarins to products 14
and 15 without detectable amounts of the undesired double
bioconjugation products. Interestingly, the same high degree of
chemoselective transformation of GSH to 16 was observed with
6. In order to conrm that the monoconjugation takes place at
the thiol group in GSH, we subjected the corresponding disul-
de GSSG, which only carries free amino groups, to equimolar
amounts of chlorocoumarin 1 in TRIZMA buffer at pH 8 and 9.
In both cases, no sign of C–N bond formation was observed
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achieved with equal control and efficiency at much lower peptide
concentrations. For example, we observed quantitative conversion
of GSH to 22 when the reaction was scaled down to 25 mM (see ESI†).
Finally, we applied our method to GSSG and Ornipressin
(Fig. 5). As expected, these disulde peptides do not react with 1
and 6 under the thiol-selective monobioconjugation conditions
optimized as described above. However, upon addition of
dithiothreitol (DTT), a well-known disulde reducing agent, the
desired thiol labeling occurs with high conversion and selec-
tivity based on MS analysis. The bioconjugates are stable even in
the presence of reducing agents and no sign of cleavage of the
aryl–sulfur bond in 16 was observed aer addition of another
equivalent of DTT. These results further prove that the mono-
conjugation occurs selectively at the free thiol function in the
peptide and that the bioconjugation is irreversible. Moreover,
Fig. 4 (A) Structures of the site-selectively diconjugated GSH deriv-
atives using various reagents. All reactions were carried out in pH 9.0
TRIZMA buffer (50.0 mM) : acetonitrile (1 : 4) solutions at 5.0 mM GSH
concentrations with 1 to 2 equivalents of the bioconjugation agent.
The reaction times were 2 hours for the monoconjugation and 18–24
hours for the second bioconjugation step, respectively. Conversions
were determined using the relative ESI-MS intensities of product peaks
in comparison to starting materials or monoconjugated intermediates,
see ESI† for details. (B) Fluorescence spectra of the bioconjugation
products 16 and 22 and of the agents 1 and 6 were collected using an
excitation wavelength of 275 nm. Two major emission peaks were
observed at 300 nm and 591 nm for the monoconjugated product 16.
Fluorescence quenching was observed when 16 was converted to the
diconjugated derivative 22.
The presence of the uoride in agent 3 provides an opportunity
to track the labeled peptide 20 by 19F NMR spectroscopy while the
aryl-bromide bond in the double bioconjugation product 21 could
be used for late-stage functionalization purposes. The successful use
of 4-bromocoumarin 4 shows that modication at C-6 prior to the
bioconjugation step is a viable alternative which underscores the
versatility of chemoselective amine and thiol bioconjugation with
modular coumarin click chemistry. As expected, the placement of
two uorophores in close proximity in 22 results in signicant
uorescent quenching47 of the major emission peaks at 300 nm and
591 nm. Importantly, the modular bioconjugation can also be
Fig.
5 Chemoselective thiol ligation upon reductive disulfide
cleavage. (A) GSSG (5.0 mM) was exposed to the bioconjugation agent
6 (10.0 mM) in acetonitrile : TRIZMA pH 9.0 buffer (4 : 1) solution for 24
hours. The formation of any bioconjugated product was not observed
by ESI-MS under these conditions. Instead, a strong signal corre-
sponding to GSSG (m/z ¼ 612) was obtained. An equimolar amount of
DTT was added to the above reaction mixture and after 24 hours MS
analysis showed formation of the monoconjugated products 16. (B)
This protocol was then successfully extended to Ornipressin and for
modular GSSG bioconjugation with 6 and either 1 or 8. Conversions
were determined using the relative MS intensities of product peaks in
comparison to starting materials or monoconjugated intermediates,
see ESI.†
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addition of 4-chlorocoumarin 1 or the biotin derivative 8 to the was ltered, washed with hexanes (4 ꢁ 10 mL) and dried under
solution containing 16 generated in situ from GSSG gave the vacuum to afford 256.0 mg (0.90 mmol, 90%) of a yellow crys-
1
desired products 22 and 23, respectively, with excellent selec- talline solid, which was used without further purication. H
tivity and conversion (see ESI† for details).
NMR (399 MHz, DMSO-d6) d 7.92 (d, J ¼ 2.5 Hz, 1H), 7.66 (dd, J
¼ 8.7, 2.6 Hz, 1H), 7.17 (d, J ¼ 8.7 Hz, 1H), 4.33 (bs, 1H). 13C
NMR (100 MHz, DMSO-d6) d 166.4, 157.6, 151.9, 135.1, 128.1,
124.5, 120.8, 119.2, 115.5.
Conclusions
In conclusion, we have demonstrated chemoselective thiol and
amine bioconjugation using stoichiometric amounts of readily
available 4-halocoumarin and arylsulfonate agents that undergo
C(sp2)-heteroatom bond formation without the common need
for transition metal assistance. The peptide modication is
high-yielding and occurs at micromolar concentrations at room
temperature and varying pH. The underlying ipso-substitution
click chemistry is irreversible, generates stable products and the
broad selection of coumarin tags offers high labeling exibility
and versatility. Different coumarins can be selectively attached
to amino and thiol groups, and both free as well as latent thiols
captured in disulde bridges can be labeled if desired. The 4-
halocoumarins and arylsulfonates can also be used in combi-
nation with biotin affinity tags or dansyl chloride. Altogether,
the broad utility, ease of operation, storage, and preparation of
4-halocoumarins and arylsulfonates make these very attractive
bioconjugation agents. We note that the coumarins are inher-
ently uorescent and can be loaded with drugs or stapling units
if desired. The advantageous features embodied in the hal-
ocoumarin and arylsulfonate agents are of far-reaching scope
and expected to nd late-stage functionalization and dual
labeling applications in chemical biology, bioengineering, bio-
sensing, and in the biopharmaceutical and materials sciences.
4-Hydroxy-6-(4-methoxyphenyl)-3-nitrocoumarin (11)
A mixture of compound 10 (145.0 mg, 0.51 mmol), 4-methox-
yphenylboronic acid (152.1 mg, 1.01 mmol), Pd(dppf)Cl2$CH2-
Cl2 (41.6 mg, 0.05 mmol), aq. Na2CO3 (2.0 mL, 1.4 M) and DME
(2.5 mL) were stirred at 90–100 ꢀC. Aer 72 hours, the reaction
was acidied with 1.0 M HCl and extracted with ethyl acetate.
The combined organic layers were dried and concentrated
under vacuum. Column purication using 0–30% ethyl acetate
in hexanes afforded 70.0 mg (0.22 mmol, 44%) of a yellow
crystalline solid. 1H NMR: (399 MHz, chloroform-d) d 8.25 (d, J
¼ 1.7 Hz, 1H), 7.99 (d, J ¼ 8.7 Hz, 1H), 7.55 (d, J ¼ 8.3 Hz, 2H),
7.42 (d, J ¼ 8.7 Hz, 1H), 7.03 (d, J ¼ 8.3 Hz, 2H), 3.88 (s, 3H). 13
C
NMR (100 MHz, chloroform-d) d 169.5, 160.0, 152.5, 152.3,
138.6, 136.3, 130.6, 128.1, 123.2, 117.7, 117.5, 114.6, 113.3, 55.4.
HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C16H11NO6Na:
336.0484; found: 336.0479.
4-Bromo-6-(4-methoxyphenyl)-3-nitrocoumarin (4)
A mixture of 11 (70.0 mg, 0.22 mmol), TBAB (386.9 mg, 1.2
mmol) and P4O10 (340.6 mg, 2.4 mmol) in toluene (3 mL) was
stirred at 90–95 ꢀC overnight. The mixture was allowed to cool to
room temperature and washed with water, sat. NaHCO3 and
extracted with dichloromethane. The combined organic layers
were dried over MgSO4 and concentrated in vacuo. Purication
by ash column chromatography on silica gel (0–50% ethyl
acetate in hexanes) afforded 36.1 mg (0.09 mmol, 42%) of
a greenish yellow solid. 1H NMR (399 MHz, chloroform-d):
d 8.05 (d, J ¼ 2.2 Hz, 1H), 7.91 (dd, J ¼ 8.7, 2.1 Hz, 1H), 7.55
(d, J ¼ 8.7 Hz, 2H), 7.48 (d, J ¼ 8.7 Hz, 1H), 7.04 (d, J ¼ 8.6, 2H),
3.88 (s, 3H). 13C NMR (100 MHz, chloroform-d): d 160.1, 151.6,
150.1, 139.6, 133.8, 133.8, 130.7, 128.3, 127.0, 117.8, 117.3,
Experimental section
General information
All reagents and solvents were commercially available and used
without further purication. Flash chromatography was per-
1
formed on silica gel, particle size 40–63 mm. H NMR and 13C
NMR spectra were obtained at 400 MHz and 100 MHz, respec-
tively, using deuterated DMSO and chloroform as solvents.
Chemical shis are reported in ppm relative to TMS or to the
solvent peak. 4-Chloro-3-nitrocoumarin, 1, 4-chloro-6-uoro-3-
nitrocoumarin, 3, 6-bromo-4-chloro-3-formylcoumarin, 5, dan-
syl chloride, 7 and NHS-biotin, 8, are commercially available
and were used without additional purication. 4-Iodo-3-
nitrocoumarin, 2, and phenyl 2,4-dinitrobenzenesulfonate, 6,
were synthesized using literature procedures.33,34 4-Bromo-6-(4-
methoxyphenyl)-3-nitrocoumarin, 4, was synthesized from 9
by following modied literature protocols, Scheme 1.48,49
114.7, 55.4. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C16H10
BrNO5Na: 397.9640; found: 397.9636.
-
Conflicts of interest
There are no conicts to declare.
Acknowledgements
6-Bromo-4-hydroxy-3-nitrocoumarin (10)50
We are grateful for nancial support from the U. S. National
Science Foundation (CHE-1764135).
Acetic acid (1.0 mL) was added to a mixture of sodium nitrite
(2.8 mg, 0.04 mmol) and 6-bromo-4-hydroxycoumarin,
9
(241.0 mg, 1.00 mmol) in a round bottomed ask and immersed
to a pre-heated oil bath of 60 ꢀC. Nitric acid (140.0 mL, 70%) was
added to the mixture. Aer 15 minutes, the reaction was
allowed to cool to room temperature. The resultant precipitate
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© 2021 The Author(s). Published by the Royal Society of Chemistry
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