A Bodipy-based derivative for selective fluorescence sensing of homocysteine and cysteine

Article abstract of DOI:10.1039/c0nj00720j

A method for the selective fluorescence sensing of homocysteine (Hcy) and cysteine (Cys) under neutral pH conditions, based on the reaction of the simple aldehyde containing Bodipy dye with Hcy/Cys, has been developed. The location of the aldehyde group at the Bodipy core is important and has an effect on the cyclization with Hcy/Cys. The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011.

Full text of DOI:10.1039/c0nj00720j

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LETTER
A Bodipy-based derivative for selective fluorescence sensing
of homocysteine and cysteinew
Ying Yue,ab Yong Guo,*a Jian Xua and Shijun Shao*ac
Received (in Montpellier, France) 21st September 2010, Accepted 28th October 2010
DOI: 10.1039/c0nj00720j
A method for the selective fluorescence sensing of homocysteine
(Hcy) and cysteine (Cys) under neutral pH conditions, based on
the reaction of the simple aldehyde containing Bodipy dye with
Hcy/Cys, has been developed. The location of the aldehyde
group at the Bodipy core is important and has an effect on the
cyclization with Hcy/Cys.
emission bandwidth, and thus good signal/noise ratio,
insensitivity to pH and solvent polarity, greater chemical
and photochemical stabilities in solution and in solid
state, and low biological toxicity.5 Applications of Bodipy
derivatives have been greatly enlarged in number and range,
they are used as biomolecular labels, protein probes, cation
sensors, anion chemosensors, etc.6 A probe based on the
Bodipy derivative aiming to selectively detect Hcy and Cys,
however, has not been developed to date.
The design and synthesis of systems which are capable to sense
analytes are of major interest for thiol compounds involved in
chemical and physiological processes in organisms. As a
particular interest, Hcy and Cys play crucial roles in plasma.
For instance, Hcy is the risk factor for human diseases such
as Alzheimer’s, Behcet’s, and cardiovascular disease;1 an
abnormal level of Cys links to the liver damage, skin lesions,
and slowed growth.2 Thus, the search for sensitive and
selective sensors for Hcy and Cys has aroused intense attention.
A variety of optical probes for thiols, utilizing the strong
nucleophilicity of thiols and their high binding affinity towards
metal ions, have been reported.3 Recently, a few fluorescent
and colorimetric methods for the selective detection of Hcy
and Cys have been reported, based on the dyes containing
aldehydes that afforded very stable thiazolidine or thiazinane
derivatives by the reaction with Hcy or Cys.4
By incorporating a Bodipy uorophore within the aldehyde
group, herein, we have developed two fluorescent compounds
to detect Hcy and Cys (Scheme 1). Compounds 1 and 2 were
designed to reveal the reactivity of aldehyde groups located on
different positions of Bodipy. The influence of the electronic
effect of the Bodipy core on the reactivity of aldehyde groups
in the two compounds was discussed.
Synthetic routes are shown in Scheme 1. 4-Hydroxymethyl-
benzaldehyde as the key reagent is obtained by the reduction
of dialdehyde.7a Compound 1 was synthesized according to
references.7 The second compound, 2, was obtained by the
Knoevenagel-type reaction of aldehyde with a known Bodipy
derivative, 1,3,5,7-tetramethyl-8-phenyl-Bodipy.
A fluorescent probe, which is composed of a fluorophore
and a receptor, used in cells or in vivo detection should be
sensitive, selective, stable, and reliable. Among the many
known fluorescent dyes, Bodipy dyes have gained recognition
and popularity with chemists and biochemists, because of their
high extinction coefficient, high quantum yield, narrow
a Key Laboratory of Chemistry of Northwestern Plant Resources and
Key Laboratory for Natural Medicine of Gansu Province,
Lanzhou Institute of Chemical Physics, Chinese Academy of
Sciences, Lanzhou 730000, P. R. China.
E-mail: guoyong@licp.cas.cn, sjshao@licp.cas.cn
b Graduate School of the Chinese Academy of Sciences,
Beijing 100039, P. R. China
c State Key Laboratory for Oxo Synthesis and Selective Oxidation,
Lanzhou Institute of Chemical Physics, Chinese Academy of
Sciences, Lanzhou 730000, P. R. China
w Electronic supplementary information (ESI) available: Instruments
and reagents. Spectral data of the two compounds; details concerning
the Benesi–Hildebrand analysis; spectra of titration experiments;
calculated structural figures and selected data. See DOI: 10.1039/
c0nj00720j
Scheme 1 Synthetic routes of target compounds 1 and 2.
c
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Fig.
2 Emission spectra (lex = 498 nm) of compound 1
Fig. 1 Emission spectra (lex = 498 nm) of compound 1 (2.0 ꢀ 10ꢁ6 M)
with or without various amino acids and GSH (100 equiv.) in
CH3CN/H2O (v/v, 3 : 2) at pH 7.4 buffered with 0.1 M HEPES at
ambient temperature. (Inset: fluorescence responses of 1 to Hcy/Cys in
the presence of various amino acids, GSH or all of them at 513 nm. White
bars represent the integrated emission of 1 in the presence of 100 equiv. of
other amino acids/GSH/all of them of interest; black and shadow bars
represent the changes in integrated emission that occur upon subsequent
addition of 100 equiv. of Hcy and Cys to solutions containing 1 and other
amino acids/GSH/all of them of interest, respectively.)
(1.0 ꢀ 10ꢁ6 M) upon the addition of increasing concentration
(0, 0.1, 0.2, 0.4, 0.5, 0.6, 0.8, 1 mM) of Hcy in CH3CN/H2O
(v/v, 3 : 2) at pH 7.4 buffered with 0.1 M HEPES at ambient
temperature. (Inset: the Benesi–Hildebrand plot of 1 with Hcy, in
which the fluorescence intensity (1/(I0 ꢁ I)) at 509 nm is plotted against
the concentration of 1/Hcy.)
Cys is added, the aldehyde group transforms into a stable ring
of thiazinane or thiazolidine (compound 10), which is formed
by thiol and amine together. This is a key element for the
selective recognition of Hcy and Cys.
High selectivity is a matter of necessity for an excellent
probe in the application of detecting amino acids. The
fluorescence response of compound 1 to various amino acids
and reduced glutathione (GSH) was investigated by emission
spectroscopy in a 0.1 M HEPES buffer of pH 7.4/CH3CN (v/v,
2 : 3) at ambient temperature (Fig. 1). Probe 1 (2.0 ꢀ 10ꢁ6 M)
was treated with 100 equiv. of a series of amino acids or GSH
separately. As shown in Fig. 1, the reaction of Hcy or Cys with
the probe results in a significant fluorescence enhancement
around 513 nm. In contrast, other amino acids, including Ala,
Thr, Arg, Gly and GSH, produce no discernible changes in
emission intensities for the probe 1 and do not interfere with
their Hcy/Cys response (inset graph in Fig. 1). The emission
profiles of 1–Hcy and 1–Cys are unchanged in the presence
of various amino acids, GSH or all of them, indicating
excellent selectivities for Hcy and Cys. A possible mechanism
is proposed to explain these results (Scheme 2). When Hcy or
The characteristics of the reaction between 1 and Hcy/Cys
were studied by titration experiments. Upon the addition of an
increasing concentration of Hcy (0–1000 equiv.) to the
solution of probe 1 (1.0 ꢀ 10ꢁ6 M), the intensities of
the emission bands around 513 nm increase gradually, and
the lmax of emission bands shows a hypsochromic shift of
about 4 nm to 509 nm (Fig. 2). At the same time, the
fluorescence quantum yields of 1 increase from 0.26 to 0.71,
which are determined by using fluorescein isothiocyanate as
the standard in a 0.1 M NaOH solution (j = 0.90).8 From the
fluorescence titration experiment, the binding constant (Ka) of
probe 1 with Hcy is obtained from the variation in the
fluorescence intensity at the appropriate wavelength 509 nm
(10) by plotting 1/(I0 ꢁ I) against 1/Hcy based on the
Benesi–Hildebrand analysis9 (ESIw). The binding constant
for probe 1 with Hcy is determined to be 1300 Mꢁ1. In
addition, the good linearity also indicates the presence of a
1 : 1 probe 1 to Hcy interaction (inset graph in Fig. 2). A
similar fluorescence enhancement is also found for Cys
(Fig. S2, ESIw). The fluorescence quantum yields of 1 increase
from 0.26 to 0.63. The binding constant for probe 1 with Cys is
determined to be 741 Mꢁ1. No visible changes, however, are
observed in the absorbance spectra of 1 + Hcy and 1 + Cys
(Fig. S3, ESIw).
We speculate that the enhancement of fluorescence intensity
upon the addition of Hcy and Cys is due to the transformation
from F–A system 1 (F—Bodipy fluorophore; A—electron
acceptor, formylphenyl) to F–D system 10 (D—electron
donator, alkyl substituent with sulfur and amino group) by
the reaction of Hcy or Cys with the aldehyde group. The
mutation of the pull–push effect leads to the enhancement of
the fluorescence yield.4c Besides, the optimized geometries of 1
Scheme 2 Reaction of compound 1 with Hcy or Cys to form
thiazinane (n = 2) or thiazolidine (n = 1).
c
62 New J. Chem., 2011, 35, 61–64
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and 10 were investigated to reveal the reason why the emission
intensity of 1–Hcy is higher than that of 1–Cys. Calculated
structures and relevant geometrical parameters are shown
in ESIw (Fig. S4 and Table S1). As for 1 or 1–Cys, the
formylphenyl group is nearly perpendicular to the Bodipy
core, excluding any significant conjugation between
8-phenyl and the Bodipy core. While the dihedral angle
C(9)–C(8)–C(11)–C(12) of 1–Hcy is 66.61, indicating that the
planarity and conjugated relationship between 8-phenyl and
the Bodipy core of 1–Hcy is better than those of 1 and 1–Cys,
so the molecular rigidity and fluorescence intensity of 1–Hcy
increase and are higher than those of 1–Cys.
Compound 2 was synthesized with the expectation that the
aldehyde group will generate higher reactivity than that in
compound 1. Surprisingly, no significant changes were
observed in the fluorescence emission spectra (lex = 560 nm)
and absorption spectra of 2 in the presence of Hcy or Cys. In
order to determine whether or not the reaction has taken place,
HPLC-FLD and HPLC-UV/vis were used to compare two
sample solutions of 2, one with and one without Hcy. The results
showed that no difference appeared in the chromatogram signals.
In other words, the reaction of 2 with Hcy or Cys does not take
place. To boost the reaction, we tried to increase pH from 3 to 11
and raise the reaction temperature to 60 1C, but without success.
These facts indicate that the exact location of the aldehyde group
in a Bodipy derivative affects its reactivity.
Fig. 3 Chromatogram of a mixture of Hcy and Cys using probe 1 as
the pre-column reagent. Mobile phase: methanol–water (AcOH,
pH = 3.5), 70 : 30 (v/v). Detection: UV/Vis (500 nm) and MS
detector. Flow rate: 1 mL minꢁ1. T: 20 1C. Injection volume: 20 mL.
Standard thiols concentration: 0.2 mM for Hcy and Cys. The
concentration of 1 is 12 mM. 1 and two amino acids in the mixed
solution of CH3CN : H2O (3 : 2). Peaks: a: 1–Cys (11.1 min (a)),
b: compound 1 (13.5 min (b)) and c: 1–Hcy (16.6 min (c)).
is the aldehyde group in 1, while the aldehyde group in 2 shifts
upfield to 10.01 ppm. Moreover, infrared spectra show that
1699 cmꢁ1 band, assigned to the CQO stretching of the
aldehyde group of 1, shifts to higher wave number as
compared with that of 2. These data illustrate that carbon of
aldehyde in 2 has higher electronic density than that of 1. So,
we hold that the electrophilic reactivity of aldehyde in 1 is
higher than that in 2.
To find the influence of electronic effect on the reactivity of
aldehyde group with Hcy, a comparative experiment was
carried out using benzaldehyde, p-hydroxybenzaldehyde and
p-nitrobenzaldehyde. Under the same conditions as Hcy with
the probe 1, Hcy only reacts with p-nitrobenzaldehyde,
indicating that the electron-withdrawing effect could enhance
the electropositivity of the aldehyde group and raises the
reactivity. Thus, this result illustrates that the Bodipy core shows
an electron-withdrawing effect to the aldehyde group in 1.
The conformational properties of compounds 1 and 2
were studied by molecular mechanics and semi-empirical
calculations to investigate the influence of the location of the
aldehyde group on the reactivity. As shown in Fig. S5 (ESIw),
the geometries of 1 and 2 illustrate the almost vertical relation-
ship between 8-formylphenyl and the Bodipy core owing to the
presence of the methyl groups at C-1 and C-7 positions, while
formylphenyl through ethylene linked at 5-methyl is almost in
the same plane as the Bodipy core. Relevant geometrical
parameters are compiled in Table S2, ESIw. These data indicate
that the Bodipy cores of 1 and 2 have planar geometries. As for
1, the formylphenyl group is nearly perpendicular to the
Bodipy core (dihedral angle C(9)–C(8)–C(11)–C(12) 90.21),
excluding any significant conjugation between aldehyde-
phenyl and the Bodipy core. The role of the electron-
withdrawing effect of the Bodipy core appears and becomes
a dominant factor that promotes the reaction of the aldehyde
group in 1. While in the structure of 2, formylphenyl is fully
conjugated with the Bodipy core. Because of the conjugative
effect, the averaging of the electron cloud density depresses the
electropositivity of the carbon in the aldehyde group, which
results in the failure of the reaction between 2 and Hcy/Cys.
1H-NMR spectra and infrared spectra of compounds 1 and 2
can also confirm this deduction. The singlet peak at 10.10 ppm
Compound 1 (1.2 ꢀ 10ꢁ5 M) is used as the derivative
reagent to the simultaneous detection of Hcy and Cys
(2.0 ꢀ 10ꢁ4 M, respectively) in a mixed solution by the
separation of HPLC-UV/Vis-MS. As shown in Fig. 3, the
three peaks are separated absolutely. The mass spectra results
indicate that the m/z of peaks a and c, 456.2 and 470.2,
attributes to 1–Cys and 1–Hcy, respectively, while m/z of peak
b is 353.3, corresponding to probe 1. The result also confirms
that thiazinane or thiazolidine is formed by the interaction of
aldehyde with Hcy or Cys. However, at present this method
cannot be used quantitatively to determine Hcy and Cys
because the linear ranges of Hcy and Cys have not been found.
Thus, in this work, we have shown the utility of the Bodipy
chemistry in the design and synthesis of a determination
system for Hcy and Cys. The sensors are based on the reaction
of the aldehyde group with Hcy or Cys to form a rather stable
thiazinane or thiazolidine derivative. The exact location of the
aldehyde-phenly group is important, if it is at the meso
position, upon the addition of Hcy and Cys, the fluorescence
intensities enhance by the mutation of the pull–push effect; if it
is in full conjugation with the Bodipy core, however, the
averaging of the electron cloud density depresses the electro-
positivity of carbon in aldehyde and results in a failure of the
reaction with Hcy and Cys. These provide a theoretical basis for
the development of probes based on Bodipy derivatives and for
the application of the reaction between aldehyde and Hcy/Cys.
c
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New J. Chem., 2011, 35, 61–64 63

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This work was supported by the National Natural Science
Foundation of China (Grant No. 20972170 to S.-J. Shao), the
open fund of State Key Laboratory of Oxo Synthesis &
Selective Oxidation (Grant No. OSSO2008kjk6 to S.-J. Shao)
and by the Natural Science Foundation of Gansu province
(No. 096RJ2A033 to Y. Guo).
127.7, 122.4, 122.1, 117.6, 14.5. IR n: 1692 (CQO), 1617, 1595,
1546, 1497 (CQC) cmꢁ1. MS (ESI): calcd for C27H23BF2N2O:
440.2, found: m/z 441.4 (M + H)+.
Notes and references
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aldehyde7a (5.0 mmol, 0.68 g) and 2,4-dimethylpyrrole
(10.0 mmol, 0.80 g) in deoxygenated CH2Cl2 (150 mL), one
drop of TFA was added and the mixture was stirred overnight
under N2 at rt. The red solution was treated with DDQ
(5.0 mmol, 1.14 g), stirring was continued for 30 min followed
by the addition of Et3N (15 mL). After 15 min, BF3ꢂEt2O
(15 mL) was added at 0 1C, and the mixture was stirred at rt
for further 3 h. After washing with satd aq. NaHCO3, the
organic phase was separated, dried with MgSO4, filtered, and
concentrated. The residue was purified by silica gel column
chromatography (CH2Cl2/hexane) to afford 4,4-difluoro-8-(40-
hydroxymethylphenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-
indacene. This alcohol was oxidized by MnO2 to afford
compound 17c (0.55 g, 1.6 mmol). Orange solid. mp 174–176 1C,
1H-NMR (CDCl3, 400 MHz): d = 10.10 (s, 1H, CHO), 8.01
(d, 2H, ArH), 7.49 (d, 2H, ArH), 5.98 (s, 2H, pyrrole H), 2.54
(s, 6H, CH3), 1.33 (s, 6H, CH3). IR n: 1699 (CQO), 1618,
1599, 1541, 1496 (CQC) cmꢁ1
. MS (ESI): calcd for
C20H19BF2N2O: 352.2, found: m/z 353.3 (M + H)+.
5-{20-(400-Formylphenyl)ethenyl}-4,4-difluoro-8-phenyl-1,3,5,7-
tetramethyl-4-bora-3a,4a-diaza-s-indacene (2). 4-Hydroxy-
methylbenzaldehyde7a (2.0 mmol, 272 mg) and 1,3,5,7-tetra-
methyl-8-phenyl-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene
(1.5 mmol, 800 mg) were refluxed in a mixture of toluene
(100 mL) and piperidine (4 mL). Any water formed during the
reaction was removed by heating overnight in a Dean–Stark
apparatus. The crude product was evaporated to dryness and
purified by silica gel column chromatography (CH2Cl2/hexane) to
afford 5-{20-(400-hydroxymethylphenyl)ethenyl}-4,4-difluoro-
8-phenyl-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene.
To a solution of this alcohol (0.4 mmol, 177 mg) in CH2Cl2
(40 mL), MnO2 (10.0 mmol, 869 mg) was added and the
mixture was refluxed until the consumption of alcohol was
complete (6 h, as monitored by TLC). The mixture was filtered
through Celite, the filtrate was evaporated, and then the solid
residue was further purified by silica gel column chromato-
graphy (EtOAc/hexane) to yield compound 2 (179 mg,
0.41 mmol). Black red powder. mp 239–241 1C. 1H-NMR
(CDCl3, 400 MHz), d = 10.01 (s, 1H, CHO), 7.89–7.87
(d, 2H, ArH), 7.83–7.78 (d, J = 16.4 Hz, 1H, vinylic),
7.73–7.71 (d, 2H, ArH), 7.52–7.50 (m, 3H, ArH), 7.32–7.30
(m, 2H, ArH), 7.24–7.20 (d, J = 16.4 Hz, 1H, vinylic), 6.63
(s, 1H, pyrrole-H), 6.06 (s, 1H, pyrrole-H), 2.62
(s, 3H, CH3), 1.44 (s, 3H, CH3), 1.41 (s, 3H, CH3). 13C-NMR
(CDCl3, 100 MHz), d = 191.5, 133.3, 130.2, 129.2, 128.0,
c
64 New J. Chem., 2011, 35, 61–64
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011