Flavin-Based Light-Driven Fluorescent Probe for the Detection of Antioxidant Amino Acids

Article abstract of DOI:10.1002/open.201700144

We have synthesized a flavin-N(5)-oxide derivative with a p-toluenesulfonyl (Ts-OF) group as a “turn-on” fluorescent probe for the detection of several antioxidant amino acids and biothiols. Oxidized flavin was synthesized by using dithiothreitol as the reducing agent. Ts-OF showed a light-driven fluorescence enhancement in the presence of several amino acids and biothiols such as histidine (His), methionine (Met), cysteine (Cys), glutathione (GSH), and homocysteine (Hcy). The ¹H NMR study indicated the reductive elimination of the p-toluenesulfonyl group from Ts-OF in the presence of antioxidants and photo-irradiation.

Full text of DOI:10.1002/open.201700144

DOI: 10.1002/open.201700144
Flavin-Based Light-Driven Fluorescent Probe for the
Detection of Antioxidant Amino Acids
Kyeong-Im Hong, Seong Min Lee, and Woo-Dong Jang*[a]
We have synthesized a flavin-N(5)-oxide derivative with a p-tol-
uenesulfonyl (Ts-OF) group as a “turn-on” fluorescent probe
for the detection of several antioxidant amino acids and bio-
thiols. Oxidized flavin was synthesized by using dithiothreitol
as the reducing agent. Ts-OF showed a light-driven fluores-
cence enhancement in the presence of several amino acids
and biothiols such as histidine (His), methionine (Met), cysteine
probe (Ts-OF) has an aza aromatic-N-oxide group. It is very
useful for photochemistry such as ring contraction, isomeriza-
tion of isoxazoles to oxazoles, ring expansion, ring fragmenta-
tion, and deoxygenation.[12] By reducing it using dithiothreitol
(DTT), the structure of the flavin-N(5)-oxide is converted to
flavin, which has a fully conjugated backbone structure and ex-
hibits strong fluorescence emission behavior.
1
(Cys), glutathione (GSH), and homocysteine (Hcy). The H NMR
The synthetic procedure of Ts-OF is summarized in
Scheme 1. Briefly, 6-chlorohexan-1-ol was reacted with 3,4-di-
methylaniline to obtain 1. Next, 6-chlorouracil was reacted
study indicated the reductive elimination of the p-toluenesul-
fonyl group from Ts-OF in the presence of antioxidants and
photo-irradiation.
It is known that several amino acids exhibit antioxidant ef-
fects.[1] Typically, sulfur-containing amino acids play crucial
roles as strong antioxidants.[2] Antioxidants are closely related
to the biological functions of cell proliferation, signal transduc-
tion, and tissue repair.[3] The disruption in the function of anti-
oxidants such as methionine (Met), cysteine (Cys), homocys-
teine (Hcy), and glutathione (GSH) is known to cause various
diseases such as liver damage, leukocytosis, psoriasis, Alzheim-
er’s and Parkinson’s diseases, and AIDS.[4] Therefore, the detec-
tion of antioxidant biomolecules is very important. To date,
various methods such as high-performance liquid chromatog-
raphy,[5] electrochemical detection,[6] capillary electrophoresis,[7]
optical analysis,[8] and mass spectroscopy[9] have been devel-
oped for the detection of antioxidant biomolecules. Among
them, optical detection techniques have attracted considerable
attention because they are convenient, simple, and precise, in
addition to having low detection limits.[10] Particularly, the tech-
niques based on “turn-on” change in fluorescence are very
useful for the detection of different biomolecules in vitro and
in vivo. By using the developed fluorescent probes, many sci-
entists have researched optical sensors that can image antioxi-
dants in cells. Specific analytes in cells could be easily visual-
ized and detected.[11]
Scheme 1. Synthesis of flavin-based fluorescent probe (Ts-OF). Reagents and
conditions: i) 6-chlorohexan-1-ol, triethylamine; ii) 6-chlorouracil, H2O/diox-
ane; iii) NaNO2, acetic acid; iv) p-toluenesulfonyl chloride, methanol; v) DTT,
ethanol.
with 1 to get 2. Subsequently, an oxidation reaction was car-
ried out under acidic conditions with NaNO2 to form the flavin
derivatives 3. Finally, the p-toluenesulfonyl group was intro-
duced in 3 to obtain a semiquinone flavin derivative flavin-
N(5)-oxide (Ts-OF). On the other hand, reduced flavine (OF)
was synthesized by the reduction of 3 using DTT and used as a
control molecule to compare its oxidation state with Ts-OF.
The absorption spectra of Ts-OF and OF were measured in a
mixture of ethanol and H2O (v/v=1:1, 100 mm). Both Ts-OF and
OF showed multiple absorption bands in the visible region.
The wavelengths of the absorption maxima for Ts-OF and OF
were 460 and 440 nm, respectively (Figure 1a). We could esti-
mate that the molecular absorption coefficient of Ts-OF was
larger than that of the oxidized flavin (OF). However, the fluo-
rescence emission intensity of Ts-OF was significantly smaller
than that of OF. Therefore, it was concluded that these proper-
ties would be useful for the design of turn-on fluorescence
probes, because the reduction of flavin-N(5)-oxide to oxidized
flavin resulted in fluorescence enhancement.
To investigate the fluorescence probes, we have designed a
flavin-based fluorescent probe, which showed an enhance-
ment in its fluorescence emission upon photoirradiation in the
presence of several antioxidant amino acids. The synthesized
[a] K.-I. Hong, S. M. Lee, Prof. W.-D. Jang
Department of Chemistry, Yonsei University
50 Yonsei-ro, Seodaemu-gu, Seoul 03722 (Korea)
E-mail: wdjang@yonsei.ac.kr
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Figure 1. a) Absorption and fluorescence spectra of OF and Ts-OF, b) time-
dependent fluorescence measurement of Ts-OF and 3 with 100 equivalents
of DTT.
It is well known that flavin derivatives exhibit redox-sensitive
properties.[13] Therefore, the electronic structure of flavin deriv-
atives can be changed by electrochemical treatment and pho-
toexcitation. Generally, oxidized flavin derivatives tend to be
reduced by an intermolecular electron transfer from reducing
agents.[14] The flavin derivative 3 was also reduced to OF upon
treatment with DTT. However, the reduction of 3 with DTT re-
quired high-temperature conditions (608C). Although 3 re-
sponded to DTT, it was not possible to utilize it as a fluores-
cent probe because of the harsh reaction conditions.[15] There-
fore, we introduced the p-toluenesulfonyl group, which is an
excellent leaving group, to 3 in order to enhance the reduction
reaction. As a result, Ts-OF (100 mm) dissolved in a mixture of
ethanol and H2O (v/v=1:1) showed strong fluorescence en-
hancement upon treatment with 100 equivalents of DTT at
room temperature. We also conducted the time-dependent
measurement of fluorescence emission to monitor the reaction
rate of Ts-OF and 3 with DTT. As shown in Figure 1b, 3 shows
a very slow increase in its fluorescence intensity when mixed
with DTT at room temperature, whereas Ts-OF shows remark-
able enhancement in fluorescence because the p-toluenesul-
fonyl group can be readily lost.
Figure 2. a) Changes in the emission intensity and fluorescence of Ts-OF on
addition of various amino acids and b) under dark conditions; c) the time-
dependent change in the emission intensity of Ts-OF monitored at 544 nm
with 100 equivalents of Cys; d) emission intensity before and after UV irradi-
ation for 11 min.
ous amino acids, GSH, and Hcy were added separately and in-
cubated for 11 min in the dark. Although there were no spec-
tral changes under the dark conditions, changes in the absorp-
tion and emission of these solutions of Ts-OF with amino acids
were evident when they were subjected to irradiation by using
a UV hand lamp (VILBER Lourmat). The emission intensity of
the solutions containing methionine, Cys, GSH, Hcy, and histi-
dine increased significantly upon UV irradiation (Figure 2). It is
known that methionine, Cys, GSH, Hcy, and histidine have a
strong antioxidant effect, because of the sulfur- and imidazole-
bearing side groups. Therefore, the reaction was initiated by
the photoexcitation of Ts-OF and reductive elimination of the
p-toluenesulfonyl group during its interaction with antioxidant
molecules. Also, we conducted the fluorescent titration with
antioxidants such as Cys, GSH, Hcy, Met, and His. They required
different amounts to reach the maxima of emission intensity.
For example, the emission intensity of the solutions containing
Cys or GSH was significantly enhanced at the amount of
10 equivalents. Met, Hcy, and His showed enhancement of the
emission intensity above 3 equivalents, which is lower than
Cys and GSH (Figure 3).
As the solution of Ts-OF and DTT showed strong fluores-
cence enhancement, we tested the fluorescence response of
Ts-OF with Cys as the representative antioxidant in the biosys-
tem. It was found that the fluorescence emission of Ts-OF solu-
tion (100 mm) was greatly enhanced by the addition of 1 mm
Cys, and the time-dependent emission intensity changes
showed complete saturation within 11 min (Figure 2c). Also, it
was faster than the treatment of DTT. Based on the time-de-
pendent fluorescence emission measurement, continuous exci-
tation at 450 nm was applied to the Ts-OF solution for the
monitoring of fluorescence emission. As mentioned before, the
electronic structure of flavin derivatives can be changed by
electrochemical treatment and also by photoexcitation. There-
fore, we expected that light irradiation would influence the
rate of the reaction between Ts-OF and Cys. To confirm the in-
fluence of light irradiation, the Ts-OF solution containing Cys
was incubated in the dark for 11 min. It was found that the
emission of Ts-OF almost maintained its original intensity, indi-
cating that the reaction is only promoted by light irradiation
(Figure 2).
To confirm the reductive elimination of the p-toluenesulfonyl
1
group from Ts-OF, H NMR studies were carried out in D2O. As
shown in Figure 4, the aromatic proton signals of Ts-OF
appear at 8.71 and 8.19 ppm. When an excess amount
(80 equiv.) of Cys was added and the Ts-OF solution was irradi-
ated with light, a new set of aromatic proton signals appeared
at 8.43 and 8.16 ppm, which are consistent with the aromatic
proton signals of OF and indicate that the p-toluenesulfonyl
group is eliminated from Ts-OF. The intensity of the new peaks
gradually increased with increasing exposure time to UV light,
indicating a photoinduced reaction.
The fluorescence response of Ts-OF to various amino acids
and biothiols is shown in Figure 2. To a 100 mm solution of Ts-
OF in 10 mm PBS (pH 7.4, 150 mm NaCl), 10 equivalents of vari-
In summary, we have synthesized a flavin-based fluorescent
probe, which showed light-driven fluorescence enhancement
1
in the presence of several amino acids and biothiols. H NMR
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Experimental Section
All commercially available chemicals were of reagent grade and
used as-received without further purification. Dichloromethane
(DCM) was freshly distilled before use. UV/Vis absorption spectra
were recorded on a JASCO model V-660 spectrometer. Fluores-
cence spectra were measured by using a JASCO FP-6300 spectro-
photometer. All spectral measurements were carried out in a
quartz cuvette with a path length of 1 cm. 1H NMR spectra were re-
corded on Bruker Advance PDX 250 and DPX 400 spectrometers at
258C in either CDCl3, [D6]DMSO, CD3OD, or D2O. MALDI-TOF-MS ex-
periments were performed on a Bruker Daltonics LRF20 with di-
thranol (1,8,9-trihydroxyanthracene) as the matrix.
1: To a solution of 3,4-dimethylaniline (123 mmol, 15 g) in triethyla-
mine (30 mL), 6-chlorohexanol (82.5 mmol, 11.2 g) was added and
the reaction mixture was refluxed for 6 h. Subsequently, it was ex-
tracted with H2O/CH2Cl2, and the organic layer was concentrated
and purified by silica column chromatography using 5% MeOH/
1
CH2Cl2 as an eluent to give 1 (10 g, 57%). H NMR (400 MHz, CDCl3)
d=6.94–6.92 (d, J=8 Hz, 1H), 6.44 (s, 1H), 6.40–6.39 (d, J=4 Hz,
1H), 6.38–6.37 (d, J=4 Hz, 1H), 3.67–3.64 (t, J=12 Hz, 2H), 3.10–
3.07 (t, J=12 Hz, 2H), 2.19 (s, 3H), 2.15 (s, 3H), 1.64–1.57 (m, 4H),
1.48–1.40 ppm (m, 4H).
2: To a solution of 1 (22 mmol, 5.0 g) in a mixture of H2O and 1,4-
dioxane (v/v=50%, 30 mL), 6-chlorouracil (7.5 mmol, 1.1 g) was
added and the reaction mixture was refluxed for 15 h. Subsequent-
ly, the solvents were removed under reduced pressure using a
rotary evaporator and the residue was purified by silica column
chromatography using 6% MeOH/CH2Cl2 as an eluent to give 2
Figure 3. The spectra of fluorescence titration with various antioxidants:
a) cysteine, c) histidine, d) methionine, e) homocysteine, and e) glutathione;
b) the change of emission intensity according to equivalents of cysteine
monitored at 525 nm.
1
(2.35 g, 94%). H NMR (250 MHz, [D6]DMSO) d=10.36 (s, 1H), 10.15
(s, 1H), 7.23–7.20 (d, J=7.5 Hz, 1H), 7.03 (s, 1H), 6.98–6.94 (m, 1H),
4.40–4.36 (t, J=10 Hz, 1H), 4.06–4.05 (d, J=2.5 Hz, 1H), 3.60–3.54
(t, J=15 Hz, 2H), 2.23 (s, 6H), 2.08 (s, 3H), 1.48–1.20 ppm (m, 7H)
3: To a solution of 2 (0.31 mmol, 0.1 g) in glacial acetic acid
(1.6 mL), sodium nitrite (1.47 mmol, 0.1 g) was added and the reac-
tion mixture was further stirred for 3 h in the dark. Subsequently,
H2O was added and the solvents of the reaction mixture were re-
moved. The residue was purified by silica column chromatography
using a mixture of MeOH, ethylacetate, and CH2Cl2 (MeOH:E-
1
tOAc:CH2Cl2 =5:15:75) as an eluent to give 3 (0.1 g, 97%). H NMR
(400 MHz, [D6]DMSO) d=9.5 (s, 1H), 8.09 (s, 1H), 7.80 (s, 1H), 4.54–
4.42 (m, 4H), 2.47 (s, 3H), 2.39 (s, 3H), 1.72–1.68 (m, 4H), 1.47–
1.48 ppm (m, 4H); MALDI-TOF-MS m/z: cald. for C18H22N4O4: 358.16
[M+]; found: 357.88.
OF: To a solution of 3 (0.03 mmol, 0.01 g) in ethanol (20 mL), DTT
(0.014 mmol, 0.02 g) was added under an inert N2 atmosphere and
refluxed for 4 h. Subsequently, the solvent was removed and the
residue was recrystallized with ethanol to give OF (8 g, 79%).
1H NMR (250 MHz, CD3OD) d=7.97 (s 1H), 7.77 (s, 1H), 3.61–3.57 (t,
J=10 Hz, 2H), 2.59 (s, 3H), 2.47 (s, 3H), 1.94–1.75 (m, 4H), 1.67–
1.52 ppm (m, 6H); MALDI-TOF-MS m/z: cald. for C18H22N4O3: 342.17
[M+]; found: 343.83.
Figure 4. 1H NMR experiments of Ts-OF with 80 equivalents of Cys according
to exposure UV irradiation time (0, 5, 10, 60 min) in D2O.
studies revealed that OF is generated from Ts-OF through re-
ductive elimination of the p-toluenesulfonyl group. As the fluo-
rescence response requires both chemo- and photo-stimuli,
this methodology makes it possible to reduce the misreading
of research results by undesirable fluorescence “turn on” pro-
cesses upon the detection of specific chemicals.
Ts-OF: To a solution of 3 (0.09 mmol, 0.03 g) in CH2Cl2, p-toluene-
sulfonyl chloride (0.13 mmol, 24.8 mg) was added and the mixture
solution was stirred for 12 h at room temperature. Subsequently,
the solvent was removed and the residue was purified by silica
column chromatography with 25% MeOH/CH2Cl2 as an eluent to
give Ts-OF (3 mg, 7%). 1H NMR (400 MHz, D2O) d=8.71 (s, 1H),
8.19 (s, 1H), 8.12–8.10 (d, J=8 Hz, 2H), 7.78–7.75 (d, J=8 Hz, 2H),
4.02–3.99 (t, J=12 Hz, 2H), 2.99 (s, 3H), 2.91 (s. 3H), 2.84 (s, 3H),
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cald. for C25H29N4O6S: 513.18 [M+]; found: 514.89.
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Acknowledgements
This work was supported by the Mid-Career Researcher Program
(2014R1A2A1A10051083) funded by the National Research Foun-
dation (NRF) of Korea, Ministry of Science, ICT & Future, Korea.
Conflict of Interest
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Keywords: biothiols · flavin · photoexcitation · reductive
elimination · turn-on sensor
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Received: August 26, 2017
Revised manuscript received: September 25, 2017
Version of record online && &&, 2017
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COMMUNICATIONS
Driving light: Light-driven fluorescence
enhancement is reported in the pres-
ence of antioxidant amino acids. A
flavin derivative with a p-toluenesulfon-
yl group is prepared as a “turn-on” fluo-
rescent probe for the detection of sev-
eral antioxidant amino acids and bio-
thiols.
K.-I. Hong, S. M. Lee, W.-D. Jang*
&& – &&
Flavin-Based Light-Driven Fluorescent
Probe for the Detection of Antioxidant
Amino Acids
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These are not the final page numbers! ÞÞ