Suzuki Cross-Coupling Reaction with Genetically Encoded Fluorosulfates for Fluorogenic Protein Labeling

Article abstract of DOI:10.1002/chem.202002037

A palladium-catalyzed cross-coupling reaction with aryl halide functionalities has recently emerged as a valuable tool for protein modification. Herein, a new fluorogenic modification methodology for proteins, with genetically encoded fluorosulfate-l-tyro

Full text of DOI:10.1002/chem.202002037

Accepted Article
Accepted Article
Title: Suzuki Cross-Coupling Reaction with Genetically Encoded
Fluorosulfates for Fluorogenic Protein Labeling
Authors: Xianxing Jiang, Qian Zhao, Guoying Guo, Weiwei Zhu, Liping
Zhu, Yifan Da, Ying Han, Hongjiao Xu, Shuohan Wu, Yaping
Cheng, Yani Zhou, and Xiaoqing Cai
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To be cited as: Chem. Eur. J. 10.1002/chem.202002037
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01/2020

10.1002/chem.202002037
Chemistry - A European Journal
FULL PAPER
Suzuki Cross-Coupling Reaction with Genetically Encoded
Fluorosulfates for Fluorogenic Protein Labeling
Qian Zhao,†[a] Guoying Guo,†[a] Weiwei Zhu,[a] Liping Zhu,[b] Yifan Da,[a] Ying Han,[a] Hongjiao Xu,[a]
Shuohan Wu,[a] Yaping Cheng,[a] Yani Zhou,[c] Xiaoqing Cai,*[a] and Xianxing Jiang*[a]
†These authors contributed equally to this work.
[a]
Dr. Q. Zhao,† G. Guo,† Dr. W. Zhu, Y. Da, Y. Han, Dr. H. Xu, S. Wu, Y. Cheng, Prof. Dr. X. Cai,* Prof. Dr. X. Jiang*
School of Pharmaceutical Sciences
Sun Yat-sen University
Guangzhou, 510006, China
E-mail: jiangxx5@mail.sysu.edu.cn, caixq7@mail.sysu.edu.cn
Dr. L. Zhu
[b]
School of Pharmaceutical Sciences
Guangzhou University of Chinese Medicine
Guangzhou 510006, China
Dr. Y. Zhou
[c]
School of Basic Medical Sciences
Lanzhou University
Lanzhou 730000, China
7]
Abstract: Palladium-catalyzed cross-coupling reaction with
aryl halide functionalities has recently emerged as a valuable
tool for protein modification. We herein describe a new protein
fluorogenic modification methodology using genetically encoded
fluorosulfate-L-tyrosine, which exhibited high efficiency and
biocompatibility in bacterial cells as well as in aqueous medium.
Furthermore, the cross-coupling of 4-cyanophenylboronic acid
on green fluorescent protein was shown to possess a unique
fluorogenic property, which may open up the possibility of a
responsive “off/on” with a great potential to enable spectroscopic
imaging of proteins with minimal background. Taken together, a
convenient and efficient catalytic system has been developed
firstly, which may provide broad utilities in vivo protein
visualization and live-cell imaging.
attractive approach for protein modification.[4b, In pioneering
work, the Davis group explored a convenient, water-soluble Pd
catalyst for Suzuki cross-coupling with a cysteine-linked aryl
halide as a reactive handle.[7b] Afterwards this methodology for
protein functionalization and cell-surface labeling was used in
genetically encoded aryl halides by the same group (Figure 1A).
Notably, Sonogashira reaction has been proved as
another important class of transition-metal-catalyzed cross-
coupling reactions for protein modification.[9] Lin and coworkers
[1b, f, 8]
discovered
a
robust aminopyrimidine-Pd(II) complex for
Sonogashira cross-coupling that enabled functionalization of an
alkyne-encoded proteins in both aqueous medium and in
bacterial cells (Figure 1B).[10] Alternatively, the Chen group
demonstrated
a ligand-free Sonogashira cross-coupling for
labeling proteins bearing an aliphatic alkyne or an iodophenyl
handle inside bacterial cells (Figure 1C).[11] Recently,
fluorosulfates have been known as
a “super sulfate” to
Introduction
participate in transition-metal-catalyzed cross-coupling reaction.
Sharpless and coworkers have recently applied fluorosulfates as
an alternative to halides and trifates in a Pd-catalyzed Suzuki
reaction in aqueous medium at room temperature.[12] These
advantages that make them ideal to address challenges
associated with current success of fluorosulfates-based Suzuki
reaction as a “bioorthogonal” ligation in this area. In this study,
we sought to capitalize on the potential specific reactivity of this
reaction to develop an effective and controllable fluorogenic
protein labeling modification methodology.
Fluorogenic protein labeling has provided a powerful tool to
visualize the statics and dynamics of protein in live cells with
excellent signal-to-noise ratio, such as Cu(I)-catalyzed azide-
alkyne cycloaddition[13] and strain-promoted azide-alkyne
cycloadditions.[14] In addition to azide-alkyne chemistry, other
biorthogonal reaction could also been allowed for the site-
specific fluorogenic labeling of proteins, including Staudinger
ligation,[15] and tetrazine-alkyne,[16] tetrazine-alkene[17] or light-
inducible tetrazole-alkene cycloadditions.[18] On the basis of our
recent efforts to develop the fluorogenic bioorthogonal reaction
that enabled effective and controllable bioimaging in our
group,[19] we hoped to expand our studies to develop a novel
Chemical modification of proteins has emerged as an invaluable
tool for detection and quantification of proteins, creating
therapeutic proteins and generating novel protein constructs.[1]
Traditional protein chemical modification has been involved with
the use of native cysteine and lysine residues,[2] which is limited
for its low selectivity and precision.[3] In the past two decades, a
variety of chemical methods have been developed for the mild
and site-specific protein modification through introducing
bioorthogonal chemical handles into the protein structure.[4] One
of the key driving forces of this approach has been the
emergence of robust methods that allow for reprograming the
genetic code and enable efficient incorporation of unnatural
amino acids (UAA) into proteins.[4a,
UAA containing
5]
bioorthogonal functionalities such as azide, terminal alkyne and
terminal alkene have been widely applied for site-specific protein
labeling.[6] Despite encouraging progress, the repertoire of
bioorthogonal reactions remains to be further explored to
expand the toolkit for protein modification.
Transition-metal-catalyzed cross-coupling reactions, which
has been well established as indispensably important tools in
modern organic synthesis, have recently emerged as an
1
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10.1002/chem.202002037
Chemistry - A European Journal
FULL PAPER
Suzuki reaction with genetically encoded chemical handles for
protein fluorogenic labeling. Herein, we describe a novel protein
mg/L) and high fidelity (Supplementary Fig S1A, no protein
expression was observed in the absence of FSY).
modification
methodology
using
genetically
encoded
Our initial study was focused on the Suzuki cross-coupling
between phenylboronic acid 1a and GFP-Y151FSY using the
previously reported 2-amino-4,6-dihydroxypyrimidine (L1)[7b] as
the palladium ligand. We began our investigation by incubating
GFP-Y151FSY with 1a (45 mM, 1500 equiv) and (L1)2Pd(OAc)2
(4.5 mM, 150 equiv) in 90 mM sodium hydrogen phosphate
buffer, pH 8.0 at 37 °C. A reaction kinetic study was performed,
which indicated that the coupling reaction reached completion in
approximately 24 h as monitored by LC-MS (Table 1, entries
1−5). Notably, to achieve precise reaction monitoring by mass-
spectrometry, 1,4-dithiothreitol (DTT) was used as a scavenger
to prevent the non-specific Pd binding to proteins
fluorosulfate-L-tyrosine (FSY), which exhibited high efficiency
and biocompatibility in aqueous medium and in bacterial cells.
We also demonstrate that this protein modification strategy can
be further used for protein fluorogenic labeling that enables in
vitro and in vivo imaging of proteins with minimal background
(Figure 1D).
(Supplementary Fig S2). Next, a variety of previously reported
11]
ligands for Pd-catalyzed cross-coupling (L2−L5)[1f,
were
further examined, but the results were unsatisfactory (Table 1,
entries 6−9). In addition, control experiments showed that the
reaction did not proceed in the absence of ligands (Table1, entry
10). Lastly, we reduced the concentration of (L1)2Pd(OAc)2 from
4.5 mM to 3.0 mM or 1.5 mM. However, using the reduced
amount of ligand and catalyst only afforded the desired product
in lower yields (Table 1, entries 11−12).
Table 1. Reaction optimization for Suzuki cross-coupling on GFP-Y151FSY.
Figure 1. Reaction schemes of Pd-mediated protein cross-coupling in living
bacterial cells. (A) Suzuki cross-coupling was conducted in pIPhe-containing
OmpC protein on E. coli cell membrane. (B) Sonogashira cross-coupling was
performed on HPG functionalized ubiquitin protein in bacterial cells. (C)
Ligand-free Sonogashira cross-coupling was employed inside gram-negative
bacterial pathogens on PenK-containing OspF protein. (D) Suzuki cross-
coupling was conducted in fluorosulfate-L-tyrosine (FSY)-containing GFP
inside E. coli cells.
Concentration
(mM)
Conversion
(%) [a]
Entry
Time (h)
Pd catalyst
Results and Discussion
1
2
3
4
5
6
7
8
9
1
6
(L1)2Pd(OAc)2
(L1)2Pd(OAc)2
(L1)2Pd(OAc)2
(L1)2Pd(OAc)2
(L1)2Pd(OAc)2
(L2)2Pd(OAc)2
(L3)2Pd(OAc)2
(L4)2Pd(OAc)2
(L5)2Pd(OAc)2
Pd(OAc)2
4.5
4.5
4.5
4.5
4.5
4.5
4.5
4.5
4.5
4.5
3.0
1.5
7
16
69
> 95
> 95
19
< 5
48
-
We started by introducing the desired aryl fluorosulfate handle
into proteins via genetic-code expansion with fluorosulfate-L-
tyrosine (FSY). We selected green fluorescence protein (GFP)
as a protein model based on the following considerations: (1)
The GFP-based system could facilitate us to investigate the
potential denaturation effects caused by different reagents
during the chemical modification process. (2) Hypothetically, the
incorporation of a biphenyl moiety into GFP could be used to
probe protein structure, which could possibly enhance or alter its
fluorescence activity. The genetic encoding of FSY has been
recently reported by the Wang group.[20] Accordingly, we
mutated five residues (Ala302, Asn346, Cys348, Tyr384,
Trp417) in the active sites of Methanosarcina mazei PylRS to
prepare the evolved pyrrolysyl-tRNA synthetase (FSYRS). Next,
12
24
36
24
24
24
24
24
24
24
10
11
12
-
62
58
(L1)2Pd(OAc)2
(L1)2Pd(OAc)2
General conditions: GFP-Y151FSY (30 μM); Ar-B(OH)2 (45 mM); Pd reagents
for indicated concentration; sodium hydrogen phosphate buffer (pH 8.0); air;
37 °C for indicated time.
[a]
Conversions were determined based on LC-MS analysis of the reaction
mixtures based on the equation: conversion %
= Iproduct / (IGFP-Y151FSY +
Iproduct),[10] where IGFP-Y151FSY and Iproduct represent the ion counts of the
remaining GFP-Y151FSY and product.
we constructed
a
pUltra-FSYRS plasmid encoding the
With the optimal conditions in hand, we then investigated
tRNAꢃꢀꢄꢁꢅꢂ /FSYRS pair. Finally, C-terminally His6-tagged GFP
containing a TAG codon at position 151 (GFP-Y151TAG) was
expressed in the presence of 1 mM FSY using pUltra-FSYRS.
After purification by Ni-NTA affinity chromatography, the product
(GFP-Y151FSY) was analyzed by SDS-PAGE, and then further
characterized by ESI-MS (Supplementary Figure S1B&C).
Gratifyingly, GFP-Y151FSY was obtained in good yield (14
the generality of this method for
a variety of aryl- and
vinylboronic acids (Table 2). We first examined a range of 4-
substituted phenylboronic acid substrates containing various
functional groups, i.e., cyan (1b), methoxy (1c), fluorine (1d),
and methylsulfonyl (1e) groups. As shown in Table 2, boronic
acids 1b, 1c and 1d were found to deliver the desired modified
proteins in excellent yields (> 95%). However, cross-coupling
with 4-(methylsulfonyl)phenylboronic acid (1e) proceeded much
2
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10.1002/chem.202002037
Chemistry - A European Journal
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more slowly and afforded the desired product in a lower yield
(Table 2, entry 4). Replacement of the phenyl ring with a
biphenyl group was well tolerated providing the corresponding
product 2g in 62% yield, while replacement with a thiphene (1f)
or a naphthyl group (1h or 1i) only resulted in lower yields. In
addition, crossing coupling with vinylboronic acid 1j was tested,
which gave the modified protein in 54% yield (Table 2, entry 9).
Lastly, two carbohydrate boronic acid substrates (1k and 1l)
were synthesized (see Supporting Information for details) and
tested for the cross-coupling with GFP-Y151FSY (Table 2,
entries 10−11). Gratifyingly, the reaction proceeded smoothly,
giving the corresponding glycoprotein with good yields (2k: 45%
yield; 2l: 64% yield).
indicated the successful coupling of the rhodamine probe 1m.
As a control, labeling reaction in the absence of Pd-catalyst did
not exhibit any fluorescent band. Encouraged by this result, we
went to assess the Pd-mediated Suzuki reaction for protein
labeling in a cellular environment. Prior to the study, the catalyst
complex (L1)2Pd(OAc)2 and phenylboronic acid were evaluated,
respectively, for their compatibility within living bacterial cells,
and the corresponding cell viability assay confirmed that both
(L1)2Pd(OAc)2 and phenylboronic acid exhibited negligible
toxicity to bacterial cells at a concentration equal to or below 512
μM within an incubation period of 24 h (Supplementary Figure
S3). Following this, BL21 (DE3) cells expressing GFP-Y151FSY
were treated with 2.2 μM 1m followed by 0.22 μM (L1)2Pd(OAc)2,
and then incubated at 37 °C for 12 h. Confocal fluorescence
imaging study was subsequently carried out to determine the
labeling efficiency. As shown in Figure 2C, GFP maintained its
intrinsic fluorescence property during the Pd-mediated labeling
process (green channel, Figure 2C). Clearly, the red fluorescent
labeled GFP was visible upon the treatment of both 1m and
(L1)2Pd(OAc)2 (red channel, Figure 2C). As control experiments,
E. coli cells expressing GFP-Y151FSY without the
supplementation of 1m or (L1)2Pd(OAc)2 did not give detectable
red fluorescent signals.
Table 2. Boronic acid substrates for Suzuki reaction with GFP-Y151FSY.
Mass
calculated
Mass
observed
Conversion
(%) [a]
Entry
1
R-B(OH)2
Finally, to extend the utilities of this modification strategy for
protein labeling and imaging, we examined the fluorescence and
overall effect on the protein with the cross-coupling of various
boronic acids. Position 151 was selected in the study because it
is at the terminus of a β-barrel of the protein and is relatively
rigid. Therefore, we envision that Suzuki cross-coupling at
position 151 with various boronic acid substrates may resulted in
biphenyl-moiety incorporation on the protein, which might be a
valuable tool to tune protein fluorescence and probe
environmental or structural alteration. Accordingly, a variety of
boronic acids from Table 2 were subjected to Pd-mediated
Suzuki coupling with GFP-Y151FSY using the standard
condition. Interestingly, we found that the cross-coupling with 4-
cyanophenylboronic acid (1b) resulted in a dramatic alteration in
the fluorescence activity of GFP. As shown in Figure 3B, under
315 nm excitation, a unique fluorescence peak around 410 nm
was detected within the protein only when cross-coupling with
1b. By contrast, merely background signals were detected for
the wild-type GFP, GFP-Y151FSY, 1b or the mixture of GFP-
Y151FSY and (L1)2Pd(OAc)2. These encouraging results
prompted us to employ 1b for protein labeling in E. coli. We
expressed GFP-Y151FSY within E. coli cells, followed by the
Pd-mediated Suzuki modification with 1b. As shown in Figure
3C, we observed a distinct blue fluorogenic signal only for the
successful cross-coupling of GFP-Y151FSY with 1b (blue
channel, excitation at 405 nm). Meanwhile, GFP-Y151FSY was
found to maintain its intrinsic fluorescence upon this Pd-
mediated modification process (green channel; Figure 3C).
Notably, cells expressing GFP-Y151FSY treated without
(L1)2Pd(OAc)2 or both 1b and (L1)2Pd(OAc)2 did not give
detectable blue fluorescence signals under 405 nm excitation,
which further confirmed that the fluorogenic labeling is attributed
to the Suzuki protein modification with 1b promoted by
(L1)2Pd(OAc)2. Taken together, the incorporation of a new
chromophore, i.e., 4-cyanobiphenyl structural moiety into GFP
opens up the possibility of a responsive “off/on”, which may have
a great potential to enable in vitro and in vivo fluorogenic
imaging of proteins.
28227.02
28227.65
> 95
2
3
4
28232.04
28220.02
28280.00
28232.17
28223.14
28248.22
> 95
> 95
24
5
6
28210.04
28279.12
28218.48
28245.49
38
62
7
28254.11
28254.87
34
8
9
28254.11
28194.06
28347.49
28191.25
33
54
10
11
28485.12
28647.18
28485.34
28642.17
45
64
General conditions: GFP-Y151FSY (30 μM); R-B(OH)2 (45 mM); L12Pd(OAc)2
(4.5 mM); sodium hydrogen phosphate buffer (pH 8.0); air; 37 °C , 24h.
[a] For entries 1−3, conversions were determined based on ESI-MS. For entries
4−11, conversions were determined based on LC-MS analysis of the reaction
mixtures based on the equation: conversion %
= Iproduct / (IGFP-Y151FSY +
Iproduct),[10] where IGFP-Y151FSY and Iproduct represent the ion counts of the
remaining GFP-Y151FSY and product.
Next, we employed this Suzuki reaction for protein labeling
within living bacteria cells. We started by examining the
conjugation efficiency between our fluorosulfate-containing GFP
template (GFP-Y151FSY) and a rhodamine fluorescent probe
(1m) bearing a boronic acid group (Figure 2A). Accordingly,
GFP-Y151FSY (30 μM) was treated with 45 mM 1m and 4.5 mM
(L1)2Pd(OAc)2 at 37 °C for 24 h. As shown in Figure 2B, SDS-
PAGE presented us with a unique fluorescent band, which
3
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Figure 2. Fluorescent labeling of GFP-Y151FSY by rhodamine boronic acid 1m in vitro or in E. coli via (L1)2Pd(OAc)2-mediated cross-coupling. (A) Reaction
scheme of GFP-Y151FSY with 1m. (B) Fluorescent imaging and coomassie blue staining of SDS-PAGE gel showing the generation of rhodamine-labeled GFP-
Y151FSY. Lane 1 and 3: in the absence of (L1)2Pd(OAc)2; lane 2 and 4: in the presence of (L1)2Pd(OAc)2. (C) Confocal microscopic imaging of Suzuki cross-
coupling with GFP-Y151FSY in E. coli. A 100 µL aliquot (OD600=0.2) was cultured in the presence of 2.2 µM 1m and treated with or without 0.22 µM of the
(L1)2Pd(OAc)2 in PBS buffer (pH 8.0) at 37 °C for 12 h. Scale bars: 10 μm.
Figure 3. Protein modification of GFP-Y151FSY by 1b via (L1)2Pd(OAc)2-mediated cross-coupling. (A) Reaction scheme of GFP-Y151FSY with 1b. (B)
Fluorescence spectra of GFP, GFP-Y151FSY, 1b, GFP-Y151FSY+ (L1)2Pd(OAc)2 and 2b; samples were excited at 315 nm. (C) Confocal microscopic imaging of
Suzuki cross-coupling with GFP-Y151FSY in E. coli. A 100 µL aliquot (OD600=0.2) was cultured in the presence of 2.2 µM 1b and treated with or without 0.22 µM
of the (L1)2Pd(OAc)2 in PBS buffer (pH 8.0) at 37 °C for 12 h (2b was excited at 405 nm). Scale bars: 10 μm.
encoded aryl fluorosulfates. The ligand applied for this reaction
is of low toxicity, water-soluble and commercially available. To
the best of our knowledge, this is the first example of applying
Conclusion
bioorthogonal Suzuki cross-coupling reaction with aryl
fluorosulfates for protein modification in vitro and in bacterial
In conclusion, we have disclosed a convenient and efficient
Suzuki reaction for protein modification using genetically
4
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10.1002/chem.202002037
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biphenyl chromophore into the protein resulted from Suzuki
protein modification opens up the possibility of a responsive
“off/on”, which has a great potential to enable in vitro and in vivo
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Future work is underway towards screening more boronic acids
with improved fluorescent properties and exploring this strategy
for other protein systems with more biological applications.
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Experimental Section
Synthetic schemes and characterization for all compounds,
protein expression and purification, experimental procedures for
Suzuki protein modification with genetically encoded
fluorosulfates in vitro and in vivo can be found in the
Supplementary Information.
Acknowledgements
We gratefully acknowledge the financial support from Major
Research Program of National Natural Science Foundation of
China (No. 91853106 to X.J.), the National Natural Science
Foundation of China (No. 21708051 to X.C.), and the Program
for Guangdong Introducing Innovative and Entrepreneurial
Teams (2016ZT06Y337). X.J. thanks the Thousand Young
Talents Program and Fundamental Research Funds from the
Central Universities (No. 19ykzd25) for financial support.
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Keywords:
Bioorthgonal reaction • Cross-coupling • Fluorogenic •
Fluorosulfates • Protein modification
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Entry for the Table of Contents
A new protein fluorogenic modification methodology was developed using genetically encoded fluorosulfate-L-tyrosine, which
exhibited high efficiency and biocompatibility in bacterial cells as well as in aqueous medium. We also demonstrate that this protein
modification strategy can be further used for protein fluorogenic labeling that enables in vitro and in vivo imaging of proteins with
minimal background.
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