Selective and competitive cysteine chemosensing: Resettable fluorescent turn on aqueous detection via Cu2+ displacement and salicylaldimine hydrolysis

Article abstract of DOI:10.1039/c2nj40387k

A novel rechargeable meso-aryl Bodipy-based Cys sensing ensemble has been developed ( turn on, Φ_F = <0.05 → 0.72, limit of detection = 6.0 × 10^-6 M) with exceptional selectivity over homocysteine, N-acetylcysteine, glutathione (GSH) and methionine.

Full text of DOI:10.1039/c2nj40387k

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LETTER
Selective and competitive cysteine chemosensing: resettable fluorescent
‘‘turn on’’ aqueous detection via Cu2+ displacement and salicylaldimine
hydrolysisw
Olga G. Tsay, Kang Mun Lee and David G. Churchill*
Received (in Gainesville, FL, USA) 11th May 2012, Accepted 25th July 2012
DOI: 10.1039/c2nj40387k
A novel rechargeable meso-aryl Bodipy-based Cys sensing ensemble
has been developed (‘‘turn on,’’ UF = o0.05 - 0.72, limit of
detection = 6.0 ꢀ 10ꢁ6 M) with exceptional selectivity over
homocysteine, N-acetylcysteine, glutathione (GSH) and methionine.
ensemble for selective detection of sulphur-containing amino
acids under biologically relevant conditions. It is important to have
analyte discrimination in the presence of other thiol-containing
amino acids and peptides, such as Hcs, N-acetyl-L-Cys, GSH, and
Met. For ‘‘turn-on’’ fluorescence, the fluorescence-quenched
Cu2+ complex of Bodipy is considered. Due to the strong
affinity of thiols for coordinatively accessible copper, Cu2+
biothiol competition can be effected and fluorescence intensity
recovery for potential probing can be studied.
Recently, detection and chemosensing of sulphur-containing
amino acids and peptides have attracted considerable attention
as they play essential and unique roles in biological systems.1
Abnormal levels of biological thiols can lead to several health
problems. For example, elevated concentration of homocysteine
(Hcs) in human plasma is associated with human diseases such
as Alzheimer’s disease and cardiovascular disease;2 cellular
glutathione (GSH) deficiency leads to oxidative stress and is
linked to several diseases including certain neurodegenerative
disorders, cancers and AIDS.3 The deficiency of cysteine (Cys)
may be involved in symptoms as wide ranging as liver damage,
edema, hair depigmentation, lethargy, muscle and fat loss,
and skin lesions.4 Thus, selective detection of biological thiols
continues to be of great interest. Various detection methods
include HPLC, capillary electrophoresis, electrochemical methods,
mass spectrometry, and UV-Vis and fluorescence spectroscopy. In
particular, advances in molecular fluorescence chemosensing are
widely sought due to its simplicity, high sensitivity and compatibility
for thiol detection5 in real biological samples.6
Bodipy 2 with a Schiff base functionality allows for [ONO]
Cu2+ binding. It was prepared by the condensation of 1 with
2-aminophenol. Bodipy 1 was synthesized by the condensation
of aldehyde 39 with 2,4-dimethylpyrrole, and then by cleavage
of the isopropylidene acetal with the action of a catalytic amount
of HCl and oxidation with MnO2 (Scheme 1 and ESIw).7e
Compounds were characterized by NMR spectroscopy and
HR-MS; compounds 1 and 4 were structurally characterized.
Detailed synthetic procedures and characterization data are
provided in the ESI.w
Importantly, copper-mediated Cys ‘‘turn-on’’ fluorescence
sensing was determined (Scheme 2). To enable this, time-
dependent fluorescence spectra of free 2 (1.0 mM), and 2 in
the presence of Cu2+ (1.0 mM), under aqueous conditions
Several Bodipy fluorophores have been developed for thiol
sensing; however, their detection mode is often based on a
chemical reaction between fluorophore and analyte.7 Although
systems utilizing non-covalent interactions between fluorophore
and thiol have been reported,8 Bodipy-based ensembles have
not yet been explored in this context. Herein, we report a
new ‘‘turn-on’’ fluorescence Bodipy-based Cu2+ chemosensing
Department of Chemistry, Korea Advanced Institute of Science and
Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon,
305-701, Republic of Korea. E-mail: dchurchill@kaist.ac.kr;
Fax: +82-42-350-2810; Tel: +82-42-350-2845
w Electronic supplementary information (ESI) available: Synthesis,
characterization and spectroscopic and binding studies of compound
2 and 2ꢂCu2+ with natural amino acids and related compounds.
CCDC reference numbers 875567 and 881381. For ESI and crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/
c2nj40387k
Scheme 1 Depiction of the transformations required to obtain
compounds 1 and 2.
c
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Scheme 2 Loss of Cu2+ ion and 2-aminophenol from compound 1 to
give selective Cys recognition.
(CH3OH : HEPES buffer, 30 : 70, pH 6.5) were obtained which
revealed predictably that Cu2+ addition/binding immediately
quenches fluorescence (Fig. 1).8 No significant changes were
observed over time, whereas compound 2 (FF = 0.05 ꢃ 0.009)10
shows a gradual increase in fluorescence intensity (Fig. S9, ESIw).
The Schiff base imine bond is easily hydrolyzed under aqueous
conditions:11 2, in aqueous media, reverts to 1 (FF = 0.72 ꢃ
0.043); at 30 minutes the probe emission intensity reaches 90%
of that determined for 1 (Fig. S9, ESIw). Importantly, the
Fig. 2 (a, top) Emission spectra of 2ꢂCu2+ (1.0 mM) with various
amino acids and GSH (10.0 mM), and (b, top) fluorescence titrations
of 2ꢂCu2+ (1.0 mM) with Cys (0–50.0 mM) in aqueous solution
(CH3OH: HEPES buffer, 30: 70, pH 6.5). Excitation: 499 nm (slit =
1.5/1.5). Inset: plot of emission intensity at 510 nm vs. [Cys]. All spectra
were acquired after 30 min of analyte addition to ensure equilibration.
(bottom) Photograph of 2ꢂCu2+ (A, 1.0 mM) and (B) 2ꢂCu2+ + Cys
(50.0 mM) under UV excitation (365 nm) after 30 min.
fluorescence quantum yield difference is significant: FF
=
Fluorescence titration experiments for 2 (1.0 mM) with
different concentrations of Cu2+ show gradual emission
intensity decreases; saturation is reached with 1.0 equiv. of
Cu2+ (Fig. 1b) in forming 2ꢂCu2+ (decrease by B80% in
fluorescence intensity). A 1 : 1 binding stoichiometry for
2ꢂCu2+ was established; a binding constant of Ka = 6.0 ꢀ 105
was obtained using Benesi–Hildebrand analysis (Fig. S11, ESIw).12
In addition, competitive experiments revealed that copper
complexation is predominant in the presence of other metal
ions, including Fe2+ (Fig. S12, ESIw).
o0.05 [2ꢂCu2+] - 0.72 [1]. Until its decomplexation, Cu2+
inhibits hydrolysis of 2; hydrolytic loss of 2-aminophenol as a
secondary step enhances the fluorescence turn-on response
(Fig. 2). To verify selective action of fluorophore 2ꢂCu2+ over
fluorophore 1, fluorescence responses with 1 in the presence of
10 equiv. of Cu2+ or amino acids were measured and found to
give negligible fluorescence chemosensing. Emission spectra
of 2 (1.0 mM) in the presence of other metal ions (e.g., Na+,
K+, Cs+, Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Zn2+
,
Hg2+, Pb2+) were also obtained. As shown in Fig. 1a and
Fig. S9 (ESIw), 2 alone, or in the presence of 10 equiv. of various
metal ions (except for [Fe2+]), undergoes rapid hydrolysis; its
fluorescence intensity reaches close to the same value as that for 1
after B25 min. A similar tendency was observed by Kim and
coworkers for an iminocoumarinꢂCu2+ complex.11b For probe 2,
Fe2+ could also be used analogously in Cys sensing in the absence
of free Cu2+. Hydrolytic loss of aminophenol from 2 is also tracked
by 1H NMR spectroscopy which clearly indicates the reappearance
of the formyl group (d 9.87, CHCl3) and the concomitant diminu-
tion of the imine proton signal (Fig. S10, ESIw).
Based on the above observation, loss of Cu2+ from 2ꢂCu2+
leads to imine hydrolysis and fluorescence enhancement
through production of 1. Enhancement is a result of a
combination of electronic and steric effects (aryl dihedral angle
in compound 1 = 76.81). Due to the strong affinity of Cu2+ to
thiols, the ability of 2ꢂCu2+ to participate in the detection of
various biological thiols was examined. Signal changes are
displayed in emission spectra of 2ꢂCu2+ (1.0 mM) generated
in situ in the presence of thiol-containing amino acids and
peptides (L-Cys, Hcs, N-acetyl-L-Cys, Met, GSH) and the
remaining non-S-containing amino acids (Ala, Arg, Asn,
Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr,
Trp, Tyr, Val) in aqueous solution at 10.0 mM (CH3OH :
HEPES buffer, 30 : 70, pH 6.5) (Fig. 2a and Fig. S13, ESIw).
Significant fluorescence enhancement was observed upon
addition of Cys only; other amino acids resulted in very minor
changes in emission intensity (Fig. 2a). Interestingly, 2ꢂCu2+
can selectively recognize cysteine over other biothiols (Hcs,
N-acetyl-L-Cys, Met, GSH) (Fig. 2a). To our knowledge, this
is the first example of a Cys-selective Bodipy-containing
system. By the mechanism proposed herein, 2-aminophenol
is liberated and may also chelate Cu2+; it is an amphoteric and
hydrophilic species with internal hydrogen bonding capability,
which may play a part in its hydrolysis.
Fig. 1 Emission spectra of 2 (1.0 mM) in the presence of (a) different
metal ions (Na+, K+, Cs+, Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Zn2+
,
Hg2+, Pb2+) after 30 min, and (b) upon addition of various concentrations
of Cu2+ after 10 min in aqueous solution (CH3OH : HEPES buffer, 30 : 70,
pH 6.5). Excitation: 499 nm (slit = 1.5/1.5). Inset (b, top right): plot of
emission intensity at 510 nm vs. [Cu2+]/[2].
Fluorescence titration profiles (Fig. 2b) of 2ꢂCu2+ (1.0 mM)
with different concentrations of Cys (0–50.0 mM) indicate that
c
1950 New J. Chem., 2012, 36, 1949–1952
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fluorescence intensity increases gradually with increasing [Cys]
until saturation is reached at 30 mM, constituting 80% of
the fluorescence signal for compound 1 (a 7-fold increase from
2ꢂCu2+). The binding constant (Ka) for the 1 : 1 interaction of
2ꢂCu2+ with cysteine was determined as 3.4 ꢀ 104 using
Benesi–Hildebrand analysis (Fig. S15, ESIw). From titrations
(Fig. 2b, inset) the detection limit for Cys (Fig. S16, ESIw) was
determined as 6.0 ꢀ 10ꢁ6 M (CH3OH : HEPES buffer, 30 : 70,
pH 6.5). Under these conditions, fluorescence intensity changes
are linear with [Cys] in the range of 6–30 mM. Additionally,
2ꢂCu2+ is stable under aqueous conditions within a pH range
of 6–8, underscoring potential practical use under physiological
conditions (Fig. S18, ESIw). Competitive experiments also
confirm that Cys sensing is possible in the presence of significant
concentrations of other amino acids (Fig. S14, ESIw). Since the
addition of Cys gives recovery of fluorescence identical to
that for 1, and the time course of fluorescence enhancement is
the same as for free 2 (Fig. S17, ESIw), we proposed that
the binding of Cys to copper ion promotes [CQN] bond
hydrolysis and release of 1, giving ‘‘turn-on’’ fluorescence
signalling (Scheme 2).
used again after treatment with 2-aminophenol. Also, the
facile synthesis of compound 1, bearing the 5-salicylaldehyde
moiety, provides an example of the ease and variation possible
for emergent selective biological probes.
Experimental section
Synthesis
Compound 4. 20 mL of trifluoroacetic acid was added to
a stirred solution of aldehyde 39 (0.8 g, 4.17 mmol) and
2,4-dimethylpyrrole (863 mL) in dry CH2Cl2 under an inert
atmosphere at 0 1C. The reaction mixture was stirred for
B15 min whereupon pyrrole was removed by high vacuum
desiccation. The crude mixture was then subjected to silica
gel column chromatography (hexane : CH2Cl2, 1.0 : 0.5). The
dipyrromethane fraction was collected and the solvent was
evaporated (yield of dipyrromethane; 0.98 g, 63%). The dipyrro-
methane was dissolved in methylene chloride; 1.0 equiv. of DDQ
(0.590 g, 2.61 mmol), relative to dipyrromethane, in CH2Cl2 was
added. The mixture was stirred for 15 min at room temperature.
After that time, the reaction mixture was cooled to 0 1C, and
B10.0 equiv. of triethylamine and 10.0 equiv. of BF3ꢂOEt2
were added, subsequently; the reaction mixture was stirred for
B3 hours at 0 1C. The mixture was filtered through a silica
pad (CH2Cl2); the solvent was then evaporated. The residue
was dissolved in CH2Cl2; the organic phase was washed
with water 2 times, dried with Na2SO4 and concentrated.
The product was purified through silica gel column chromato-
graphy (silica, hexane: CH2Cl2, 1.0 : 1.0) to afford a red powder
(0.260 g) in 25% yield. 1H NMR (300 MHz, CDCl3: 7.24 ppm):
Selective fluorimetric detection of cysteine over other biothiols
was reported in several recent papers. Only a few systems show
high detection limits for Cys (10ꢁ7 M);6m,8d the others have
values similar to our system. Bodipy systems for Cys sensing with
higher detection limits were also reported,7b but the probe gives a
positive response for homocysteine as well.
Lastly, since it is known that GSH may reduce Cu2+ to
Cu+,8c,13 the stability of 2ꢂCu2+ towards reduction needs to be
addressed. Closely-related systems were studied electrochemically
wherein most cases, the Cu2+/+ redox couple behaviour is
quasi-reversible.14 Kulkarni and coworkers studied related
structural analogues of the binding pocket of probe 2; these
coumarin-based frameworks also bore quasi-reversible electro-
chemical waves (e.g., DEp values of 0.546 and 0.458; at
1.0 V).14a Nagashri and coworkers studied hydroxyflavone
compounds; here also, a quasi-reversible Cu2+/+ couple was
found (e.g., 0.154 and 0.140 at 100 mV sꢁ1).14b Labisbal and
coworkers reported an electrochemical synthesis of a closely-
related complex of the 2ꢂCu2+ binding site; it was prepared
from Cu0. They produced this like the Cu2+[O,N,O] complex
in which phenanthroline was also bound, demonstrating how
Cu2+ availability allows for facile ligand binding.14c Here, the
Cu+ complex was not isolated. These reports show that, by
extension, glutathione reductive action might be complicated.
But, straight-forward chemical GSH extraction of Cu2+ for
now is a fair proposed pathway considering the chemical
action of related sulfhydryl species such as Cys.
3
4
d 7.02 (dd, JH–H = 10.45 Hz, JH–H = 2.28 Hz, 1H), 6.91
(d, 3JH–H = 8.28 Hz, 1H), 6.85 (d, 4JH–H = 2.25 Hz, 1H), 5.95
(s, 2H), 4.84 (s, 2H), 2.52 (s, 6H), 1.56 (s, 6H), 1.43 (s, 6H).
13C NMR (100 MHz, CDCl3: 77 ppm): d 155.4, 151.8, 142.9,
141.4, 131.7, 127.8, 126.8, 124.2, 121.1, 120.4, 117.9, 99.9, 60.6,
24.6, 14.6, 14.5.
Compound 1. 100 mL of concentrated HCl were added to a
solution of 4 (0.10 g, 0.24 mmol) in 20 mL of CH3CN–H2O
(7 : 3) and the reaction mixture was heated at 40 1C for 10 min.
The reaction was monitored by TLC (silica; CH2Cl2 : CH3OH,
9 : 1); addition of hydrochloric acid was repeated until full
deprotection was effected. The reaction mixture was added
into a methylene chloride and water mixture. The aqueous
layer was extracted with CH2Cl2 three times. The combined
organic layers were then dried with Na2SO4 and the solvent
was removed under reduced pressure. Column chromatography
(silica gel; CH2Cl2 : CH3OH, 1.0 : 0.3) allowed for the isolation
of the dicarbinol (0.08 g) in 88% yield. To a solution of this
dicarbinol in CH2Cl2 (5 mL), MnO2 (activated, 0.19 g,
2.2 mmol) was added in portions, and the mixture was refluxed
until the total consumption of dicarbinol was determined
(monitoring by TLC, silica, CH2Cl2). The mixture was filtered
through a silica pad (CH2Cl2); the filtrate was evaporated and
the residue was further purified by column chromatography
(silica gel; hexane: CH2Cl2, 1.0 : 1.0) to yield compound 1.
Crystallization from a hexane–CH2Cl2 mixture gave 1 (0.06 g)
in 68% yield. 1H NMR (300 MHz, CDCl3: 7.24 ppm): d 11.16
(s, 1H, OH), 9.87 (s, 1H, CHO), 7.47 (d, 4JH–H = 2.25 Hz, 1H),
In conclusion, we report a new metallo-Bodipy-based ensemble
for selective ‘‘turn-on’’ fluorescence sensing with cysteine (Cys) in
aqueous media. Molecular sensing is based on (i) demetalla-
tion of the weakly fluorescent 2ꢂCu2+ complex with cysteine,
and subsequent (ii) hydrolysis of the pendent Schiff base
moiety resulting in fluorescence enhancement. The probe
shows remarkable competitive selectivity for Cys over homo-
cysteine, N-acetyl-cysteine, methionine and glutathione. The
cysteine detection limit was estimated at 6.0 ꢀ 10ꢁ6 M,
comparable with the concentration of Cys in biological media.3a
Moreover, the probe is rechargeable: 1 is produced and can be
c
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3
7.40 (dd, JH–H = 9.8 Hz, JH–H = 2.49 Hz, 1H) 7.12
4
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(d, JH–H = 8.46 Hz, 1H), 5.98 (s, 2H), 2.52 (s, 6H), 1.43
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156.0, 142.6, 139.0, 136.6, 133.0, 131.5, 126.5, 121.5, 120.8,
118.8, 14.9, 14.5. 11B NMR (128.4 MHz, CDCl3): d 5.43
(t, JB–F = 33.8 Hz). HRMS (ESI): calcd for C20H19BF2N2O2:
368.151, found: m/z 391.1408 (M + Na)+.z
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10 Average molecular fluorescence quantum yields are provided from
triplicate measurements from determinations in CH3OH.
Compound 2. To a solution of 1 (0.09 g, 0.25 mmol) in THF
(5 mL), 2-aminophenol (0.027 g, 0.25 mmol) was added. The
reaction mixture was stirred at room temperature for 1 hour.
The solvent was removed under reduced pressure; the solid
was washed with a small amount of CH2Cl2 and methanol.
Recrystallization from CH2Cl2 gave 2 (0.06 g) as a red powder
in 53% yield. 1H NMR (300 MHz, CD2Cl2: 5.32 ppm): d 12.67
4
(br s, 1H, OH), 8.71 (s, 1H, HCQN), 7.40 (d, JH–H = 2.22
3
4
Hz, 1H) 7.33 (dd, JH–H = 8.37 Hz, JH–H = 2.22 Hz, 1H),
7.26–7.16 (m, 3H), 7.02–6.96 (m, 2H), 6.04 (s, 2H), 5.79 (br s,
1H), 2.52 (s, 6H), 1.52 (s, 6H), 13C NMR (100 MHz, CD2Cl2:
54 ppm): d 163.5, 161.6, 156, 150.3, 143.6, 140.9, 135.8, 133.7,
132.5, 132.1, 129.3, 126.3, 121.7, 121.6, 120.3, 118.9, 118.6,
116.3, 15.1, 14.7. 11B NMR (128.4 MHz, CDCl3): d 5.43
(t, JB–F = 33.7 Hz). HRMS (ESI): calcd for C26H24BF2N3O2:
459.193, found: m/z 460.1993 (M + H)+.
The Molecular Logic Gate Laboratory (D.G.C.) acknowledges
research support from the National Research Foundation
(NRF) (Grant # 2011-0017280, 2010-0013660, 2009-0070330).
Mr. Hack Soo Shin and Prof. Youngkyu Do are acknowledged,
respectively, for facilitating the acquisition of NMR spectro-
scopic and crystallographic data. The research support staff at
KAIST facilitated the acquisition of MS data.
Notes and references
z Crystallographic data of compound
1
(CCDC 875567):
C20H19BF2N2O2, FW 368.18. Monoclinic, P2(1)/c, Z = 4, a =
11.408(3) A, b = 8.848(2) A, c = 18.193(5) A, V = 1820.5(8) A3,
T = 296(2), no. of reflections = 19 941, no. of independent reflections =
2572 [Rint = 0.0685], R1 = 0.0504, wR2 = 0.1167, largest diff. peak and
hole = 0.201 and ꢁ0.118 e Aꢁ3. Crystallographic data of compound 4
(CCDC 881381): C23H25BF2N2O2, FW 410.26. Monoclinic, P2(1)/n,
Z = 4, a = 13.996(2) A, b = 11.0508(17) A, c = 14.095(2) A, V =
2144.4(6) A3, T = 296(2), no. of reflections = 23 730, no. of independent
reflections = 2935 [Rint = 0.0633], R1 = 0.0844, wR2 = 0.2186, largest
diff. peak and hole = 0.766 and ꢁ0.237 e Aꢁ3
.
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