A fluorescein-based fluorescence probe for the fast detection of thiol

Article abstract of DOI:10.1016/j.tetlet.2016.04.068

A new turn-on fluorescent probe for the selective detection of thiol over other amino acids was synthesized. Probe possesses the widely-used thiol-selective 2,4-dinitrobenzenesulfonyl (DNBS) group which can react with thiol and release the fluorescein whi

Full text of DOI:10.1016/j.tetlet.2016.04.068

Tetrahedron Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Tetrahedron Letters
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A fluorescein-based fluorescence probe for the fast detection of thiol
⇑
Yunchang Liu, Kaiqiang Xiang, Baozhu Tian, Jinlong Zhang
Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new turn-on fluorescent probe for the selective detection of thiol over other amino acids was synthe-
sized. Probe possesses the widely-used thiol-selective 2,4-dinitrobenzenesulfonyl (DNBS) group which
can react with thiol and release the fluorescein which has strong fluorescence. Fluorescein, a well known
xanthene fluorescent dye, has two states at different environment. Fluorescein is in the state of spirocy-
clization when connected with 2,4-dinitrobenzenesulfonyl (DNBS) group which has no fluorescence.
However, it is in the state of open form when it reacts with thiol which has a strong fluorescence. The
transition of the two states can be used to selectively detect thiol and the color can change from colorless
to yellow which can be differentiated by naked eyes. Upon the titration of thiol, the absorption band at
454 nm rises gradually and the fluorescence emerges at 521 nm and the detection limit can be as low as
Received 1 March 2016
Revised 14 April 2016
Accepted 19 April 2016
Available online xxxx
Keywords:
Fluorescein
2,4-Dinitrobenzenesulfonyl
Thiol
Fluorescent probe
0.16
lM. All of such good properties prove it to be a good sensor for the selective detection of thiol and it
shows a potential use in bioimaging applications.
Ó 2016 Elsevier Ltd. All rights reserved.
Introduction
especially popular for the detection of biomolecule20–22 in recent
years. Lin23 et al. designed a new NCL-mechanism-based ratiomet-
ric fluorescent probe with specificity toward aminothiols. The new
fluorescent probe is capable of ratiometric detecting of aminothiols
in newborn calf and human serum samples and is also suitable for
ratiometric fluorescent imaging of aminothiols in living cells. Zhu24
et al. applied fluorescein as fluorescent reporter, a 7-aminocou-
marin as photo-trigger and a thiol-removable energy acceptor.
Such doubly locked probes have bright prospect, promising bio-
chemically and optically labeling cells with spatial precision for
monitoring dynamic processes of cells in vitro or in vivo. Li25
et al. used naphthalimide as the fluorescence source for the detec-
tion of thiol. The synthesized two naphthalimide-based fluorescent
probes provided high on/off signal ratios and exhibited good selec-
tivity toward thiols over other analytes and they were identified as
good sensors for the detection of thiols both in living cells and in
rabbit plasma samples.
Fluorescein is widely used as fluorescent sensors in biological
systems.26–28 Its relatively high molar absorption coefficients and
quantum yields, high-intensity emission peaks, relative inertness
under physiologically relevant conditions, and relative nontoxicity
makes it an excellent dye for the design of sensors. On the other
hand, fluorescence spectroscopy has become a powerful method
for sensing and imaging trace amounts of samples because of its
simplicity,29,30 sensitivity, fast response times, and its application
for not only in vitro assays but also in vivo imaging studies.31,32
As we all know, fluorescein exhibits strong fluorescence when it
is in the state of open form while it has non-fluorescence when it is
in the spirocyclic form. The spirocyclic form of fluorescein is
Biological thiols such as cysteine (Cys), homocysteine (Hcy), and
glutathione (GSH), play essential roles in maintaining the appropri-
ate redox status in physiological and pathological processes.1,2
Generally, the abnormal level of cellular thiols has been related
with numerous diseases such as psoriasis, slowed growth, liver
damage, leukocyte loss, cancer, and AIDS.3,4 Cysteine (Cys), one
of the most abundant biothiols in living organisms, plays impor-
tant roles in biological systems5 and has been associated with neu-
rotoxicity. Its abnormal level can cause hair pigmentation,
retardation in growth, liver damage, and skin lesions.4 Elevated
level of Hcy is closely related to Alzheimer’s disease,6 cardiovascu-
lar,7 neural tube defects, complications during pregnancy, and
osteoporosis. GSH, the most abundant intracellular thiol with the
concentration of millimolar range,8 plays crucial roles in many cel-
lular functions such as xenobiotic metabolism, maintenance of
intracellular redox activity, and gene regulation.9–11 An abnormal
level of GSH is directly associated with cancer, aging, heart prob-
lems, and other ailments. Accordingly, the design and synthesis
of a sensor for the detection of thiol is of significant importance.
Considering the vital important roles in human body, great
efforts have been made for the detection of thiol.12,13 Fluorescent
probe, famous for its high sensitivity, relatively simple analysis
protocols,14 and far less expensive, has got much attention for
the detection of metal ions,15,16 hydrion,17 anion,18,19 and
⇑
Corresponding author. Tel./fax: +86 21 64252062.
E-mail address: jlzhang@ecust.edu.cn (J. Zhang).
http://dx.doi.org/10.1016/j.tetlet.2016.04.068
0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.
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2
Y. Liu et al. / Tetrahedron Letters xxx (2016) xxx–xxx
colorless and nonfluorescent due to the break of the
whereas the ring opening form shows strong spectroscopic signals
in both the absorption and fluorescence spectra because of the
p
-conjugation,
3.63 (d, J = 8 Hz, 3H), 1.47 (m, 3H) (Fig. S1). 13C NMR d 185.74,
165.59, 163.70, 159.17, 154.45, 151.16, 134.56, 132.71, 131.14,
130.39, 129.56, 128.93, 117.34, 114.66, 114.06, 106.58, 100.59,
77.40, 77.08, 76.76, 64.52, 52.42, 14.54 (Fig. S2). Yield 1.2 g.
p
-
conjugation. Using the transformation of the two states can
achieve the goal of testing different species. Here, we synthesized
an asymmetric fluorescein-based probe for the detection of thiol
using 2,4-dinitrobenzenesulfonyl (DNBS) group as the detection
group. When it is connected with DNBS, the fluorescent probe
shows either colorless in the absorption band nor non-fluorescence
in the emission band because of the formation of spirocyclic form.
However, it shows a color of yellow and a strong fluorescence
which can be differentiated by naked eyes when it is treated with
Preparation and characterization of F
Compound 3 was synthesized according to our previous work.36
Compound 2 (1 g) was dissolved in 15 mL methyl alcohol and
20 mL sodium hydroxide solution at the concentration of 2 M
was added to the above solution. The whole mixture was stirred
overnight in room temperature. After that, 20 mL water was added
and the pH was adjusted to 7 using HCl. The precipitates were fil-
tered and dried in vacuum. The targeted compound is isolated by
flash column chromatography on silica gel using dichloro-
methane/methanol (20:3, v/v) for elution. The compounds were
in a yellow and in the state of solids. 1H NMR (400 MHz, d6-DMSO)
d 10.17 (s, 1H), 8.03 (dd, 1H), 7.80 (m, 1H), 7.72 (t, J = 4 Hz, 1H),
7.28 (m, 1H), 6.92 (m, 1H), 6.68 (m, 3H), 6.57 (d, J = 8 Hz, 2H),
4.06 (m, 2H), 1.39 (m, 3H) (Fig. S3). 13C NMR d 168.64, 159.51,
151.78, 135.61, 130.10, 128.98, 126.02, 124.62, 123.97, 112.73,
110.80, 109.40, 102.17, 101.12, 82.71, 63.60, 39.88, 39.67, 39.46,
39.25, 39.05, 14.42 (Fig. S4). HRMS: 361.1082, calcd for:
301.1076 (Fig. S7). Yield 0.8 g.
thiol. The detection limit can be as low as 0.16 lM.
Experiment
Chemicals and instrumentals
Fluorescein was purchased from Energy Chemical and used
directly without any purification. The other chemicals were of
the highest grade available and were used without further purifica-
tion. All employed solvents were analytically pure and were
employed without any further drying or purification.
Reactions were monitored by TLC using Merck Millipore DC
Kieselgel 60 F-254 aluminum sheets. 1H NMR and 13C NMR spectra
were recorded on Brucker AM-400 MHz instruments with tetram-
ethylsilane as internal standard. Low-resolution ESI mass analyses
were performed on a Waters LCT Premier XE spectrometer. UV–vis
absorption spectra were recorded on a SHIMADZU UV–Vis spec-
Preparation and characterization of PF
Probe PF was synthesized according to the methods of the
reported papers.37,38 Compound F (0.15 g) was dissolved in 15 mL
anhydrous dichloromethane and kept in ice bath under stirring.
trophotometer. Fluorescence spectra were measured on
SHMADZU RF-5301PC Fluorescence spectrophotometer.
a
Then, triethylamine (150 lL) was added to the above solution.
2,4-Dinitrobenzenesulfonyl chloride (0.133 g) was dissolved in
5 mL anhydrous dichloromethane and added to the above solution
dropwise in 30 min. The whole mixture was stirred in ice bath for
another 30 min and stirred in room temperature for 4 h. After that,
the solvents were evaporated and the product was purified by flash
column chromatography on silica gel using dichloromethane/alco-
hol (100:1, v/v) as the eluent. The probe was in the color of light
yellow and in the state of solids. 1H NMR (400 MHz, CDCl3) d
8.66 (d, J = 4 Hz, 1H), 8.52 (dd, 1H), 8.25 (d, J = 12 Hz, 1H), 8.02
(d, J = 8 Hz, 1H), 7.67 (m, 2H), 7.19 (d, J = 4 Hz,1H), 7.15 (d,
J = 8 Hz, 1H), 6.90 (m, 1H), 6.80 (d, J = 8 Hz, 1H), 6.75 (dd, 1H),
6.65 (m, 2H), 4.05 (q, 2H), 1.43 (t, J = 4 Hz, 3H) (Fig. S5). 13C NMR
d 169.02, 161.03, 152.55, 152.13, 151.88, 149.41, 135.38, 134.08,
133.23, 130.10, 128.96, 126.73, 126.35, 126.29, 123.89, 120.49,
119.47, 117.19, 112.81, 110.93, 110.26, 101.35, 81.87, 77.36,
77.05, 76.73, 63.99, 14.62 (Fig. S6). HRMS: 591.0715, calcd for:
591.0710 (Fig. S8). Yield 0.1 g.
Preparation and characterization of 2
Fluorescein methyl ester 1 was synthesized according to the
reported literature.33,34 Then, compound
was synthesized
2
according to the method of the reported Letter.35 Compound 1
(1.6 g) and K2CO3 (0.5 g) were dissolved in 15 mL DMF. 0.5 mL
iodoethane was added to the above solution. The whole mixture
was heated at 65 °C for 6 h under stirring. After being cooled to
room temperature, the solution was added to 80 mL 5% sodium
chloride solution under stirring. The precipitates were collected
and dried in vacuum. The targeted compound is isolated by flash
column chromatography on silica gel using dichloromethane/
methanol (20:3, v/v) for elution. The target products were in the
color of yellow and in the state of solids. 1H NMR (400 MHz, CDCl3)
d 8.25 (d, J = 8 Hz, 1H), 7.71 (m, 2H), 7.30 (m, 1H), 6.89 (m, 3H),
6.74 (dd, 1H), 6.58 (m, 1H), 6.51 (d, J = 2 Hz, 1H), 4.14 (m, 2H),
HO
O
O
HO
O
O
O
O
O
CH3OH
I
Reflux,30h
COOH
K2CO3/DMF
COOCH3
COOCH3
1
2
O
Cl
S
O
O
O
O
O
O
O
O
O
S
O
O2N
NO2
NaOH/H2O
6h
O2N
NO2
COOH
N(Et)3/CH2Cl2
O
PF
F
Scheme 1. The synthesis procedure of the probe PF.
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Y. Liu et al. / Tetrahedron Letters xxx (2016) xxx–xxx
3
literature. Compounds 2 and F were synthesized in high yields
and F displays excellent fluorescence. Probe PF was conveniently
synthesized via the condensation of F with 2,4-dinitrobenzenesul-
fonyl chloride in dichloromethane, and its structure was confirmed
by 1H NMR, 13C NMR, HRMS spectra. Regulation of the spirocycliza-
tion of fluorescein dyes can achieve the goal of detecting thiol. PF
shows nonfluorescence because of the spirocyclization of
Results and discussion
Synthesis
The synthetic routine of probe PF is outlined in Scheme 1. We
can get PF with a relatively high yield by four steps. Fluorescein
methyl ester
1 was synthesized according to the reported
Figure 1. (a) Time-dependent absorption changes of probe PF with adding 5 equiv thiol. (b) The changes of absorption intensity at 454 nm in the time range of 0–10 min. (c)
Time-dependent fluorescence intensity changes of probe PF after adding 5 equiv thiol. (d) The fluorescence intensity changes at 521 nm excited by 454 nm in the time range
of 0–8 min. The spectroscopic properties of Probe PF were tested in aqueous phosphate buffered saline (PBS) solutions (10 mM, pH = 7.4) containing 30% DMSO in the
concentration of 10À5 M.
Figure 2. (a) Absorption of probe PF in the presence of different amino acids including Ala, Arg, Asp, Gln, Glu, Gly, His, Ile, Met, Phe, Pro, Ser, Thr, Trp, Cys, Hcy and GSH. The
inset picture is the color changes after adding different amino acids. (b) Fluorescence intensity of probe PF in the presence of different amino acids excited by 454 nm. The
tested amino acids are the same the absorption. Other amino acids are added 100 equiv to the probe and Cys, Hcy and GSH are added 5 equiv. The inset picture is the
fluorescence changes after adding different amino acids. The spectroscopic properties of Probe PF were tested in aqueous phosphate buffered saline (PBS) solutions (10 mM,
pH = 7.4) containing 30% DMSO in the concentration of 10À5 M.
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Y. Liu et al. / Tetrahedron Letters xxx (2016) xxx–xxx
fluorescein dyes while the strong fluorescence appears when it
react with thiol. The spectroscopic properties of Probe PF were
all tested in aqueous phosphate buffered saline (PBS) solutions
(10 mM, pH = 7.4) containing 30% DMSO in the concentration of
10À5 M.
time is collected and drawn in Figure 1b. It can be seen that the
probe gets to saturation in 10 min. Likewise, the fluorescence is
also tested. 5 equiv thiol is also added to the solution and the flu-
orescence is collected. The fluorescence has the same tendency
with the adding of the tested species. It can be found in Figure 1c
that the probe PF has nearly nonfluorescence before adding thiol.
However, the fluorescence intensity at 521 nm increases sharply
in one minute after the addition of thiol and continuing increasing
with the time going on. The relationship between fluorescence
intensity and time is shown in Figure 1d. It can be found that the
probe gets to saturated in 10 min. The color changes from colorless
to yellow which can be differentiated by naked eyes and the fluo-
rescence changes from none to strong yellow fluorescence.
The response time toward thiol
To evaluate when will the probe get saturated after adding
5 equiv thiol, the time-dependent absorption and fluorescence
intensity changes of probe PF after adding 5 equiv thiol is tested.
The probe is in the concentration of 10À5 M which is dissolved in
aqueous phosphate buffered saline (PBS) solutions (10 mM,
pH = 7.4) containing 30% DMSO. 5 equiv of thiol are added to the
solution and the absorption changes are shown in Figure 1. It can
be seen in Figure 1a that probe PF has nearly no absorption at
454 nm before adding thiol. However, the absorption at 454 nm
increases sharply in one minute with the adding of 5 equiv of thiol.
After that, the absorption intensity at 454 nm increases gradually
with the time going on. The absorption changes at 454 nm versus
The selectivity toward different amino acids
It is very important to selectively detect certain analytes for a
probe. The selectivity toward different analytes is tested and is
shown in Figure 2. Ala, Arg, Asp, Gln, Glu, Gly, His, Phe, Met, Ile,
Pro, Ser and Thr are used as the sources of other amino acids and
GSH, Cys and Hcy are used as the tested species. All the analytes
are dissolved in distilled water. The probe is in the concentration
of 10À5 M which is dissolved in aqueous phosphate buffered saline
(PBS) solutions (10 mM, pH = 7.4) containing 30% DMSO. 100 equiv
of other amino acids and 5 equiv of Cys, Hcy and GSH are added,
respectively. The spectra are collected after 10 min and the fluores-
cence spectra are collected with the excitation at 454 nm. It can be
seen from the absorption in Figure 2a that the absorption spectra
have little changes with the adding of 100 equiv other amino acids.
However, the absorption at 454 nm increases obviously with the
adding of Cys, Hcy and GSH and the color changes from colorless
to yellow which can be differentiated by naked eyes (Fig. 2a). Sim-
ilarly, the fluorescence spectra have the similar trend as the
absorption spectra. It can be found in Figure 2b that nearly no flu-
orescence can be seen after the addition of other amino acids when
excited by 454 nm while probe PF shows strong yellow fluores-
cence after the addition Cys, Hcy and GSH. It has the emission band
at 521 nm. All these data demonstrate that the probe PF has a good
selectivity toward thiols over other amino acids. Thus, the probe
has thiols specificity.
Figure 3. Effect of amino acids. The fluorescence intensity changes of probe PF in
the presence of 100 equiv of various of amino acids. The short bar in the left is the
fluorescence intensity of probe PF with other amino acids in the concentration of
1 lM. The amino acids including Ala, Arg, Asp, Gln, Glu, Gly, His, Ile, Met, Phe, Pro,
The effect to the probe of different amino acids
Ser, Thr and Trp. The high bar in the right is the fluorescence intensity of probe PF
with the adding of 5 equiv thiol to the above solutions. The spectroscopic properties
of probe PF were measured in aqueous phosphate buffered saline (PBS) solutions
(10 mM, pH = 7.4) containing 30% DMSO in the concentration of 10À5 M.
However, only thiols specificity for
a good probe is not
enough. Many amino acids are mixed together whether they are
Figure 4. (a) Fluorescence intensity changes with adding different equivalent of thiol with excitation at 454 nm taken after 10 min. (b) Fluorescence intensity changes versus
concentration of thiol at 521 nm with excitation at 454 nm taken after 10 min The spectroscopic properties of Probe PF were tested in aqueous phosphate buffered saline
(PBS) solutions (10 mM, pH = 7.4) containing 30% DMSO in the concentration of 10À5 M.
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Y. Liu et al. / Tetrahedron Letters xxx (2016) xxx–xxx
5
in human bodies or in other environments. Therefore, it is very
important for the detection of thiols without the interference of
other amino acids, especially when the other amino acids are in
high quantity. Then, the effect of other amino acids is investi-
gated. First, the probe PF is dissolved in phosphate buffered saline
(PBS) solution (10 mM, pH = 7.4) containing 30% DMSO in the
concentration of 10À5 M. 100 equiv of different amino acids are
added to the above solution respectively. The fluorescence spec-
trum is tested and collected after 10 min, which is shown in
Figure 3. The fluorescence intensity after adding 100 equiv of
other amino acids is corresponding to the left bar in the figure.
All the solutions show nearly nonfluorescence. 5 equiv of thiols
are added to the above solutions respectively. The fluorescence
is investigated and collected after 10 min as shown in Figure 3.
The fluorescence intensity after adding 5 equiv of thiol is corre-
sponding to the right bar in the figure. It can be seen that the
probe PF can still detect thiol even with adding quantity of other
amino acids. All this proved that this sensing for thiols is hardly
interfered by other amino acids as can be seen in Figure 3. There’s
little influence to the detection of thiols even with quantity of
other amino acids.
Figure 5. pH effect on the fluorescence behavior of probe PF (black line) and probe
PF with Cys (red line). The fluorescence spectrum is collected with the excitation at
454 nm taken after 30 min. The spectroscopic properties of Probe PF were tested in
aqueous phosphate buffered saline (PBS) solutions (10 mM, pH = 7.4) containing
30% DMSO in the concentration of 10À5 M.
Determination of the detection limit
pH effect to the probe and probe PF
To calculate the detection limit, titration of thiols (0–6 lmol) to
the probe PF solution is tested. Probe PF is dissolved in phosphate
As we all know, it is very important for a fluorescent probe to be
stable in a wide pH range. At least, it must be stable in physiolog-
ical environment because the pH is varied in a wide range in nature
and it also has slight variation in human bodies. Ester group is sus-
ceptible to pH change, both acidic and basic media can promote the
hydrolysis of the ester group. pH effects on the fluorescence behav-
ior of Probe (PF) and Probe PF with Cys are shown in Figure 5. A
wide pH range from 2 to 12 is adjusted using phosphate buffered
saline (PBS) solutions. Then, probe PF is dissolved in the prepared
phosphate buffered saline (PBS) solutions using 30% DMSO as good
solvent and then 5 equiv of Cys is added to the above solutions. The
spectra of probe and probe PF with Cys are recorded after 10 min
and the fluorescence spectra are collected as shown in Figure 5.
It can be seen that Probe PF is stable in a wide pH range from 2
to 12. This proves that Probe PF can be used in physiological
buffered saline (PBS) solutions (10 mM, pH = 7.4) containing 30%
DMSO in the concentration of 10À5 M. 0.4
lmol, 0.8
lmol,
1.0 lmol, 1.5 lmol, 2.0 lmol, 4.0 lmol and 6.0 lmol thiols are
added to the solution respectively. Upon the titration of thiols,
the fluorescence intensity at 521 nm increases gradually as shown
in Figure 4. The detection limit was calculated based on the
method reported in the literatures.37,39 The detection limit is calcu-
lated by the equation: Detection limit = 3r/k. Where r is the stan-
dard deviation of blank measurement, k is the slop between the
fluorescence intensity versus thiols concentration. The fluores-
cence emission spectrum of PF was measured ten times to obtain
the standard deviation
r of blank measurement. To get the plot,
the fluorescence intensity at 521 nm is plotted as a concentration
of thiols as shown in Figure 4b. And the detection limit is calcu-
lated to be 0.16 lM.
Figure 6. Proposed response mechanism of Probe PF to thiol.
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Y. Liu et al. / Tetrahedron Letters xxx (2016) xxx–xxx
environment. What’s more, Probe PF with Cys in a wide pH range is
also tested. In a pH range of 7–12, it can detect Cys, which can be
used in physiological environment. The detection of thiol is a pro-
cess of nucleophilic aromatic substitution and nucleophilic reac-
tion is affected by pH. In a pH range of 2–6, the nucleophilic
reaction is prevented. As a result, the probe cannot detect thiol.
References and notes
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The proposed mechanism for the detection of thiols
It has been reported by Maeda’s group40 and others41,42 that thi-
ols could selectively cleave sulfonate ester which was a process of
nucleophilic aromatic substitution as shown in Figure 6. To verify
our design principle of probe PF for thiols, the reaction mechanism
was studied by mass spectra. The fluorescein exhibits no fluores-
cence when connected with DNBS because the fluorescein is in
the state of spirocyclic form which breaks the
p-conjugation,
whereas the ring opening form shows strong spectroscopic signals
in the fluorescence spectra by reacting with thiols as shown in Fig-
ure 6. To further prove the mechanism of the reaction, the reaction
of probe PF with Cys was conducted under the same conditions and
the products were subjected to mass spectral analysis and 1H NMR
analysis. The peak at m/z 361.1082 corresponding to compound 1
was observed which proved the proposed mechanism (Fig. S9).
The 1H NMR has further proved the reaction mechanism as shown
in Figure S10.
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Conclusions
In summary, we report a turn-on fluorescent probe for the
detection of thiols which has visible color changes that can be dif-
ferentiated by naked eyes. Probe PF has good selectivity toward
thiols over other amino acids. The probe shows nonfluorescence
when connected with 2,4-dinitrobenzensulfonyl (DNBS) group
because the probe is in the state of spirocyclization. When the
probe reacted with thiols, 2,4-dinitrobenzenesulfonyl (DNBS)
group left and the fluorescin is exposed which had strong fluores-
cence. The detection limit of probe PF reaches to be as low as
0.16 lM. All good properties prove it to be a good sensor for the
selective detection of thiol and it shows a potential use in bioimag-
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This work has been supported by the National Natural Science
Foundation of China (21277046, 21173077, 21377038), the
Shanghai Committee of Science and Technology (13NM1401000),
the Research Fund of Education Minister of the People’s Republic
of China (JPPT-125-4-076).
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Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2016.04.
068.
Please cite this article in press as: Liu, Y.; et al. Tetrahedron Lett. (2016), http://dx.doi.org/10.1016/j.tetlet.2016.04.068