O,o-Difluorination of aromatic azide yields a fast-response fluorescent probe for H2S detection and for improved bioorthogonal reactions

Article abstract of DOI:10.1039/c7ob00830a

The development of efficient bioorthogonal reactions for sensing of endogenous biomolecules and for bioconjugation should be of paramount importance in the field of chemical biology. In this work, the o,o′-difluorinated aromatic azide was firstly employed to develop a new fast-response fluorescent probe 1 for H₂S detection and for bioorthogonal reactions. Compared with non- and mono-fluorinated probes, 1 showed faster reaction toward H₂S, the third gasotransmitter, in buffer (pH 7.4), implying that the reaction rate could be enhanced by the dual-fluorine groups. Furthermore, such enhanced reaction rates of 1 were also observed in the Staudinger reaction and strain-promoted azide-alkyne cycloaddition (SPAAC) based on the comparison studies of the non-fluorinated probe. Our results firstly highlight that the o,o′-difluorinated aromatic azide group should be useful for fast bioorthogonal reactions and H₂S detection.

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Takeharu Haino et al.
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ARTICLE
o,o-Difluorination of aromatic azide yields fast-response fluorescent
†
probe for H S detection and for improved bioorthogonal reactions
2
a
a
a
a
b
c
a
Jie Zhang, Yasi Gao, Xueying Kang, Zhentao Zhu, Zhiqian Wang,* Zhen Xi and Long Yi*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
7
Development of efficient bioorthogonal reactions for sensing
of endogenous biomolecules and for bioconjugation should be
paramount importance in the field of chemical biology. In this
work, the o,o‘-difluorinated aromatic azide was firstly
employed to develop a new fast-response fluorescent probe 1
workers in 2000. Shortly after this, they reported a more
attractive ligation named traceless Staudinger ligation, in which
the triaryl phosphine oxide moiety is cleaved by hydrolysis and
phosphorus-containing moieties are not appended to the final
8
5
5
6
6
0
5
0
5
product. Their further works on the mechanistic study of the
Staudinger ligation demonstrated the overall rate of the reaction
is dependent on the electronic properties of both the phosphine
1
1
2
0
5
0
for H S detection and for bioorthogonal reactions. Compared
2
with non- and mono-fluorinated probes, 1 showed faster
9
and the azide. Electron-donating substituent on the phosphine
reaction toward H S, the third gasotransmitter, in buffer (pH
2
could accelerate the overall reaction. While the o,o‘-difluorinated
aromatic azide in Staudinger ligation was not clear. Alternatively,
the first example of SPAAC was also reported by Bertozzi and
co-workers in 2004 when they used cyclooctyne as an active
reaction partner with azide. Numerous cyclooctyne derivatives
displaying improved rates were developed in the following years,
including difluorocyclooctyne,
dibenzocyclooctyne ,
biarylazacyclooctynone,
However, little research about rate enhancement by tuning the
structure of azide was carried out for the SPAAC ligation.
Therefore, it should be deserved to explore the o,o‘-difluorinated
aromatic azide group in these bioorthogonal reactions.
7
.4), implying that the reaction rate could be enhanced by the
dual-fluorine groups. Furthermore, such enhanced reaction
rates of 1 were also observed in Staudinger reaction and
strain-promoted azide-alkyne cycloaddition (SPAAC) based
on comparison studies of the non-fluorinated probe. Our
results firstly highlight the o,o‘-difluorinated aromatic azide
group should be useful for fast bioorthogonal reactions and
1
0
11
12
dibenzocyclooctyne,
aza-
13
14
bicyclo[6.1.0]nonyne,
H S detection.
2
15
16
difluorobenzocyclooctyne,
et, al.
Introduction
Hydrogen sulfide (H S) is an important endogenous signalling
2
1,2
2
3
3
4
4
5
0
5
0
5
molecule with multi-biological functions. Fluorescence-based
methods have recently been emerged as a highly desirable and
sensitive approach for in situ and real-time visualization of H S in
2
living biological systems because of its non-destructive sensing
3
of bio-targets with readily available detection. Azide-reduction is
a commonly used strategy for the design of H S fluorescent
2
3
e,4
probes.
However, most of H S probes based on reduction of
2
aromatic azide showed a delayed response (typically > 20 min)
3
e,4
toward H S.
Therefore, we developed o-fluorinated-azido-
2
5
based probes for fast detection of H S, where the electronic-
2
drawing fluorine group on the o-position of the aromatic azide
could help improve the reduction rate by H S. We hypothesized
2
that addition of dual-fluorine groups on the aromatic azide should
further enhance the reactivity of azide toward H S. Herein we
2
employed the o,o‘-difluorinated aromatic azide for development
of new fluorescent H S probe for the first time.
2
On the other hand, azide is also an important group in
bioorthogonal chemistry, including the famous Staudinger
Scheme 1. a) The azido-involved reaction types. b) The reactivity of
fluorinated aromatic azides toward the reactions in a).
6
ligation, click reaction and copper-free click reaction (SPAAC).
70
In this work, we rationally designed and synthesized a new
fluorescence probe 1 based on the o,o‘-difluorinated aromatic
azide group as sensing moiety for proof-of-concept studies of
Staudinger ligation was firstly introduced as a mild reaction for
the metabolic engineering of cell surfaces by Bertozzi and co-
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H2S detection and bioorthogonal reactions (Scheme 1a).
the reactivity of 1 was the highest among these probes and
Comparing with the control nonfluorinated probe, 1 showed 50 indicated the reaction rate could be enhanced by increasing
faster reaction rate in H S-mediated reduction, SPAAC and
fluorine groups. The addition of a fluorine group on the o-
2
Staudinger reactions. For the first time, these results highlight the
position of the aromatic azide can improve about 2-fold reaction
5
0
5
0
using of o,o‘-difluorinated aromatic azide group in fast
rate for H S-mediated reduction.
2
bioorthogonal reactions and H S detection.
2
Results and discussion
1
1
2
The synthesis route for probe 1 was shown in Scheme 2.
Compound 5 was obtained from demethylation of commerically
available 2,6-difluoro-3-methoxyaniline under BBr . We could
3
not obtain aminocoumarin 4 by using transitional ZnCl catalysis
2
5
as descript in our previous paper. The synthsis of 4 was achieved
by using a new catalysis ZrOCl /SiO under neat condition.
1
7
2
2
Then 4 was transformed into azidocoumarin 1 by means of a
Sandmeyer reaction. The structure of probe 1 was confirmed by
1
19
13
H, F, and C NMR spectra and high-resolution mass
spectrometry (HRMS). The non- and single-fluorinated
5
azidocoumarins 2 and 3 were employed from our previous work.
5
6
5
0
Scheme 2. Synthesis of difluorinated fluorescent probe 1 and the
structure of the reference probes 2 and 3. Yeilds: 5, 77%; 4, 52%; 1, 61%.
2
5
Fig. 1 Probe 1 could react with H
spectra of probe 1 (1 μM) in the presence of H
photographs show fluorescence changes of 1 upon treatment with
2
S fastest among 1-3. (a) Emission
S (100 μM). The inset
2
H S under a UV lamp (excitation = 365 nm). (b) Reaction kinetics
2
Firstly, we investigated the absorbance spectra of 1 and its
of 1, 2 and 3 (1 μM) toward H
2
S (100 μM) in PBS (50 mM, pH 7.4)
reaction with H S (using Na S as an equivalent) in PBS buffer
2
2
containing 10% CH CN at room temperature.
3
(50 mM, pH 7.4). The absorbance of probe 1 at 327 nm exhibited
3
3
4
4
0
5
0
5
a wide linear range in PBS (Fig. S1), indicating that the probe had
good water-solubility up to over 20 µM. The probe 1 exhibited
maximal absorbance at 325 nm, which was shift to 350 nm after
treatment with H S (Fig. S2). We next investigated the
2
fluorescent spectra of probe 1 and its reaction with H S (Fig. 1).
2
Probe 1 (1 μM) was non-fluorescence in PBS buffer (pH 7.4).
After treatment with Na S (100 μM), more than 160-fold
2
fluorescent enhancement was observed at 436 nm emission,
implying that the azide groups in 1 were reduced by H S to
2
generate strongly turn-on fluorescence. Such fluorescence change
from colorless to blue could be clearly observed by naked eye
under UV lamp (Fig. 1a).
The time-dependent emission of probes 1, 2 and 3 in the
presence of H S could be recorded for kinetic studies. The results
2
65
could be obtained through building a correlation between the
emission signal and the corresponding time (Fig. 1b). The
Fig. 2 Selective test of probe 1 (1 μM) to various species (Cys, Hcy, 1
mM; GSH, 5 mM; others, 100 μM) in PBS (50 mM, pH 7.4) containing
-
4
pseudo-first-order rate, k , was found to be 20.5 × 10 , 9.03 ×
obs
10% CH CN at room temperature for 60 min.
3
-
4
-4 -1
1
0
and 4.60 × 10 s for 1, 2 and 3, respectively, by fitting the
data with single exponential function. This results revealed that
70
Inspiring with the above results, we further examined the
2
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selectivity of probe 1, because the fast-response molecular probe
should also keep good selectivity. Various biological relevant
species were used to incubate with probe 1 in PBS buffer and
their fluorescence response were measured seperately (Fig. 2).
These biological relevant species included reactive sulfur species
the reaction of b) 1 (100 μM) or c) 3 (100 μM) and 6 (1 mM) in PBS (50
mM, pH 7.4) containing 70% CH CN at room temperature. (d) Reaction
kinetics of 1 or 3 (10 μM) toward 6 (1 mM) in PBS (50 mM, pH 7.4)
containing 50% CH CN at room temperature. The solid lines represent
the best first-order fitting. The kobs for the SPAAC of 1 and 3 were 0.10
3
3
0
5
3
3
-1
-1
5
min and 0.053 min , respectively.
(
(
glutathione (GSH, 5 mM), cysteine (Cys, 1 mM), homocysteine
Hcy, 1 mM)), reactive oxygen species, anions and cations. The
To gain more insight into the reaction kinetics, we used
fluorescence change in buffer to monitor the progress of SPAAC
fluorescence increases of the tested molecules were far lower than
(Fig. 3d), since the SPAAC product was fluorescence. The
that of H S. Thus, probe 1 is highly selective toward H S over
2
2
pseudo-first-order rate, kobs was determined by fitting the
fluorescence intensity data with single exponential function. The
results indicated that 1 worked more efficient (about 1.9 fold
larger in kobs) than that of 3, and therefore showed fluorination of
azide in enhanced SPAAC reaction. To obtain the reaction rate of
1
1
2
2
0
5
0
5
other biologically relevant species including millimolar biothiols.
The results indicated that probe 1 maintains high selectivity with
4
4
5
0
5
0
fast reaction rate toward H S.
2
Encouraged by the above results in H S detection, we further
2
studied the using of difluorinated probe 1 for bioorthongal
reactions in aqueous buffer (pH 7.4). As shown in Fig. 3, we
firstly tested the SPAAC reaction based on 1 and 6. The
measurements were performed under pseudo first-order
conditions using an excess of cycloalkyne 6. We monitored the
reaction by HPLC, HRMS, absorbance and fluorescence spectra.
As shown in Fig. 3b and 3c, probe 1 could react with alkyne
within 5 h, and the reaction for difluorinated probe 1 is faster than
that for non-fluorinated 3. The formation of cycloaddition 7 was
identified by HRMS (see ESI). A well defined isosbestic point at
1
in SPAAC, the time-dependent fluorescence signal at 430 nm
was recorded for data analysis, and the linear fitting between kobs
and 6 concentrations gives the reaction rate (k ) (Fig. 4). The
2
-
1 -1
second order rate constant based on 1 was k = 1.69 ± 0.02 M s .
2
This value is slightly larger than that in the reported SPAAC
-
1
-1
reactions (0.0024 to 0.96 M s ) that was explored for protein
6
g
labelling.
2
80 nm in the absorbance spectra implied the clean SPAAC
reaction (Fig. S3).
Fig. 4 The fluorescence signal of probe 1 (10 μM) at 430 nm in the
presence of different concentrations of 6 in PBS (50 mM, pH 7.4)
5
6
5
0
containing 50% CH
3
CN at room temperature. The inset shows the linear-
was calculated as
ship plot of the concentration of 6 versus kobs. The k
2
-1 -1
1
.69 ± 0.02 M S .
We further studied the using of fluorinated probe in Staudinger
reaction, and found that 1 also should improved reaction
performance than that of 3. As shown in Fig. 5, the reaction of 1
and PPh led to strong fluorescence enhancement (larger than
3
6
5
100-fold). Such significant fluorescent turn-on could be
obviously observed by naked eye under 365 nm UV lamp,
implying that the probe was also sensor for the bioorthongal
reactions. To gain insight into the reaction kinetics, the time-
dependent emission at 450 nm was recorded and fitted with single
exponential function. As expected, the difluorinated probe 1
showed 2.9-fold faster Staudinger reaction than that of probe 3
1
8
70
(Fig. 5b, S4), further supporting fluoride in accelerating the
bioorthogonal reactions. It is noted that emission of the
Staudinger reaction was around 450 nm, which is about 20 nm
Fig. 3 (a) The reaction of probe 1 and 6 to give 7. (b,c) HPLC analysis of
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Page 4 of 7
red-shifted comparing with the H S reduction. These results
2
imply that the Staudinger reaction of 1 gave product with larger
To further investigate the Staudinger reaction, we also used
conjugate system.
20 PPh PhCOOMe 8, and found that the reaction rate was similar
2
with that of PPh (Fig. S5). Such reaction was further monitored
3
by absorbance spectra and time-dependent HRMS tests. The
HRMS results indicated the quantitative formation of coupling
production 9 was stable for at least 48 h in buffer, and not
Staudinger-Bertozzi ligation product was observed in HRMS and
spectra (Fig. 6). The time-dependent absorption spectra of the
Staudinger reaction showed a new peak at 370 nm, which is
25
larger than that of H S-mediated reduction and SPAAC reaction
2
(Fig. S6). 9 had larger conjugate system than 4 and 7 to give
30
longer wavelength in absorbance and emission.
Conclusions
35
In summary, a new o,o‘-difluorinated azido-based probe 1 was
rationally designed and synthesized for proof-of-concept studies
of H S detection and bioorthogonal reactions. Comparing with
2
non-fluorinated reference probe, 1 has four-fold faster reaction
rate towrad H S, 1.9-fold and 2.9-fold faster ligation rate in
2
4
4
5
5
6
6
7
0
5
0
5
0
5
0
SPAAC and Staudinger reactions, respectively. Therefore, the
introduction of fluoride should be a useful strategy for faster H2S
probe and for enhanced bioorthogonal reactions. Our results
firstly highlight the o,o‘-difluorinated aromatic azide group for
fast bioorthogonal reactions and H S detection. We propose that
2
efficient bioorthogonal reactions based on the difluorinated and
even multifluorinated aromatic azides could be explored in future
for better bioconjugation.
5
0
Fig. 5 a) Emission spectra of probe 1 (10 μM) in the presence of PPh
3
(
200 μM). b) Reaction kinetics of 1 and 3 (10 μM) toward PPh
3
(200
Experimental section
General
μM) in PBS (50 mM, pH 7.4) containing 50% CH
3
CN at room
temperature. The kobs for the Staudinger reaction of 1 and 3 were 0.086
-1
-1
The coumarin-based probes 2 and 3 were synthesized based on
min and 0.030 min , respectively. The inset photographs show
5
1
fluorescence changes of 1 upon treatment with PPh under a UV
our previous work. All other chemicals and solvents used for
3
lamp (excitation = 365 nm).
synthesis were purchased from commercial suppliers and applied
directly in the experiment without further purification. The
progress of the organic synthesis reaction was monitored by TLC
on pre-coated silica plates (Merck 60F-254, 250 μm in thickness),
and spots were visualized by UV light. Merck silica gel 60 (70-
2
00 mesh) was used for general column chromatography
1
13
purification. H NMR and C NMR spectra were recorded on a
Bruker 400 spectrometer. Chemical shifts are reported in parts
per million relative to internal standard tetramethylsilane
1
(
Si(CH3)4 = 0.00 ppm) or residual solvent peaks. H NMR
coupling constants (J) are reported in Hertz (Hz), and multiplicity
is indicated as the following: s (singlet), d (doublet), dd (double
doublet), m (multi). High-resolution mass spectra (HRMS) were
obtained on a Varian 7.0 T FTICR-MS. Analytical HPLC was
performed using an Agilent HPLC system equipped with a
ZORBAX SB-C18 column (5 µm, 4.6 × 150 mm) with a flow
rate of 1.0 mL/min. The buffer system for the analyses was buffer
A H O (containing 0.1% TFA) and buffer B MeOH (containing
2
0
.1% TFA). The UV-visible spectra were recorded on a UV-3600
UV-VIS-NIR
Fluorescence study was carried out using
spectrophotometer
(SHIMADZU,
Japan).
F-280
a
Fig. 6 a) The reaction of probe 1 and 8. b) The HRMS spectrum of the
product of probe 1 (1 mM) treated with 8 (10 mM) in PBS (50 mM, pH
spectrophotometer (Tianjin Gangdong Sci & Tech., Development.
1
5
7
.4) containing 70% CH
3
CN at room temperature for 2 days. The reaction 75 Co., Ltd).
mixture was submitted into ESI-MS without purification.
4
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Organic & Biomolecular Chemistry
Synthesis of 5
Measurements were performed in phosphate-buffered saline
2
,6-Difluoro-3-methoxyaniline (2 g, 12.6 mmol) was dissolved in 60 buffer (PBS, 50 mM, pH 7.4, containing 10%, 50% or 70%
CH Cl . The solution was cooled to 0 °C and protected by Ar gas.
CH CN). Compounds 1, 2, 3 and 6 were dissolved into DMSO
2
2
3
Then BBr was added dropwisely within 30 min. After stirred at
and PPh and 8 were dissolved into CH CN to prepare their stock
3
3
3
5
0 °C for 1 h and at room temperature for another 2.5 h, the
resulting mixture was poured into ice water slowly and was
solutions with a concentration of 10 mM for probes and 100 mM
for the other molecules. Stock solutions of Na S in degassed PBS
2
extracted by CH Cl twice. The water layer was collected, 65 buffer were used as H S source. Probes were diluted in PBS
2
2
2
adjusted the pH to 8-9 by concentrated ammonia water and
extracted by EtOAc twice. The organic layer was dried by
anhydrous Na SO and the solvent was removed under reduced
buffer to afford the final concentration of 1-10 µM. For the
selectivity experiment, different biologically relevant molecules
(100 mM) were prepared as stock solutions in PBS buffer.
Appropriate amount of biologically relevant species were added
1
1
2
2
3
3
4
4
5
5
0
5
0
5
0
5
0
5
0
5
2
4
1
pressure to give light yellow solid 5 (1.4 g, 77%). H NMR (400
MHz, DMSO-d ) δ 9.31 (bs, 1H), 6.67-6.62 (m, 1H), 6.11-6.06 70 to separate portions of the probe solution and mixed thoroughly.
6
(m, 1H), 5.02 (bs, 2H).
All measurements were performed in a 3 ml corvette with 2 ml
solution. The reaction mixture was shaken uniformly before
emission spectra were measured. If it is not stated specially, the
excitation wavelength is 350 nm and the emission 360-600 nm
Synthesis of 4
(50 mg, 0.35 mmol), ethyl acetoacetate (50 µL, 0.35 mmol) and
catalyst ZrOCl .8H O.SiO (95 mg) were mixed in 2 mL 75 was recorded.
5
2
2
2
eppendorf tube. The mixture was uniformly mixed and heated to
°C in thermostat metal bath overnight. Column
9
0
chromatography (petroleum ether: EtOAc = 4:1) of the crude
Acknowledgements
product over silica gel gave 4 (38 mg, 52%). R = 0.3 (petroleum
f
1
This work was supported by NSFC (21302010, 21402007,
21572019), 111 project (B14004).
ether:EtOAc = 2:1). H NMR (400 MHz, MeOD-d ) δ 7.26 (dd, J
11.3, 2.0 Hz, 1H), 6.13 (s, 1H), 2.40 (d, J = 1.1 Hz, 3H).
4
1
9
=
F
NMR (376 MHz, MeOD-d ) δ -137.75, -137.78. HRMS (ESI):
4
+
80 Notes and references
m/z [M+H] calcd. for C H F NO : 212.0518; found: 212.0521.
1
0
7
2
2
a
State Key Laboratory of Organic-Inorganic Composites and Beijing Key
Synthesis of probe 1
(38 mg，0.18 mmol) was dissolved in 5 mL water and 10 mL
Laboratory of Energy Environmental Catalysis, Beijing University of
Chemical Technology, Beijing 100029, China. E-mail:
4
yilong@mail.buct.edu.cn
concentrated HCl and cooled down to 0 °C. Then 2 mL NaNO2
(100 mg, 1.4 mmol) aqueous was slowly added to the mixture at
b
8
9
9
5
0
5
Public Hatching Platform for Recruited Talents, College of Science,
Beijing University of Chemical Technology, China. E-mail:
wangzhq@mail.buct.edu.cn
0
°C. After 0.5 h reaction, 2 mL NaN (100 mg, 1.5 mmol)
3
c
aqueous was slowly added at 0 °C and stirred for another 1 h. The
mixture was extracted by EtOAc for three times and the organic
layer was dried by anhydrous Na SO . After removed the solvent
State Key Laboratory of Elemento-Organic Chemistry and Department
of Chemical Biology, National Pesticide Engineering Research Center
(Tianjin), Collaborative Innovation Center of Chemical Science and
Engineering (Tianjin), Nankai University, Tianjin, 300071, China.
2
4
under reduced pressure, column chromatography (petroleum
ether: EtOAc = 15:1) of the crude product over silica gel gave 1
† Electronic Supplementary Information (ESI) available: spectra data and
additional figures. See DOI: 10.1039/b000000x/
1
(
26 mg, 61%). R = 0.6 (petroleum ether:EtOAc = 2:1). H NMR
f
(
2
400 MHz, CDCl ) δ 7.12 (dd, J = 10.8, 2.2 Hz, 1H), 6.32 (s, 1H),
.39 (d, J = 1.2 Hz, 3H). C NMR (101 MHz, CDCl ) δ 158.58,
3
3
1
3
1
(a) C. Szabó, Nat. Rev. Drug Discov., 2007, 6, 917; (b) L. Li, P.
Rose and P. K. Moore, Annu. Rev. Pharmacol. Toxicol., 2011, 51,
169; (c) B. Predmore, D. Lefer and G. Gojon, Antioxid. Redox
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152.56, 152.52, 151.12, 151.09, 151.06, 150.09, 150.06, 115.92,
15.67, 109.33, 109.10, 106.12, 106.08, 105.90, 105.86, 18.82.
1
1
9
F NMR (376 MHz, CDCl ) δ -127.02, -127.27. HRMS (ESI): 100
3
+
m/z [M+H] calcd. for C H F N O : 238.0423; found: 238.0418.
1
0
5
2
3
2
8
33; (f) L. Yi, L. Wei, R. Wang, C. Zhang, J. Zhang, T. Tan and Z.
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A. A. Untereiner, Z. Altaany, L. Wu, C. Szabó and R. Wang,
Pharmacol. Res., 2016, 113, 116.
Synthesis of 8
-(diphenylphosphino)benzoic acid (612.6 mg, 2 mmol), DMAP
4-dimethylaminopyridine, 244.3 mg, 2 mmol), and EDCI (1-(3-
2
(
1
1
05
10
2
3
(a) G. D. Yang, L. Y. Wu, B. Jiang, W. Yang, J. S. Qi, K. Cao, Q. H.
Meng, A. K. Mustafa, W. T. Mu, S. M. Zhang, S. H. Snyder and R.
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Dimethylaminopropyl)-3-ethylcarbodiimide, 1151.6 mg, 6 mmol)
were dissolved in MeOH. The mixture was stirred for 22 h at
room temperature under N gas. After removed the solvent under
2
reduced pressure, column chromatography (pure CH Cl ) of the
2
2
1
crude product over silica gel gave 8 (526 mg, 82%). H NMR
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.31 (m, 6H), 7.31-7.26 (m, 4H), 6.97-6.89 (m, 1H), 3.74 (s, 3H).
+
HRMS (ESI): m/z [M+H] calcd. for C H O P: 321.1039; found: 115
2
0
18
2
3
21.1037.
General Procedure for Spectroscopic Studies
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| Journal Name, [year], [vol], 00–00
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Organic & Biomolecular Chemistry
Graphic abstract
Based on the o,o‘-difluorinated aromatic azide, a new
fluorescent probe was developed for fast detection of
H S and for improved copper-free click and Staudinger
2
5
reactions.
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