The interaction of Hg2+ and trivalent ions with two new fluorescein bio-inspired dual colorimetric/fluorimetric probes

Article abstract of DOI:10.1039/c6dt01180b

Two new luminescent compounds containing fluorescein-amino acid units have been designed and synthesized via an ester linkage between a fluorescein ethyl ester and Boc-Ser(TBDMS)-OH or Boc-Cys(4-MeBzl)-OH, and their photophysical properties have been expl

Full text of DOI:10.1039/c6dt01180b

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PAPER
2
+
The interaction of Hg and trivalent ions
with two new fluorescein bio-inspired dual
colorimetric/fluorimetric probes†
Cite this: DOI: 10.1039/c6dt01180b
a,b
b,c
b
d
b,e
A. C. Gonçalves, V. Pilla, E. Oliveira, S. M. Santos, J. L. Capelo,
a
b
A. A. Dos Santos* and C. Lodeiro*
Two new luminescent compounds containing fluorescein–amino acid units have been designed and syn-
thesized via an ester linkage between a fluorescein ethyl ester and Boc-Ser(TBDMS)-OH or Boc-Cys(4-
MeBzl)-OH, and their photophysical properties have been explored. The optical response of both com-
+
+
+
+
2+
2+
2+
2+
2+
2+
2+
pounds (2 and 3) towards the metal ions Na , K , Hg , Ag , Ca , Co , Ni , Cu , Zn , Cd , Pb
,
2+
3+
3+
3+
3+
Hg , Al , Fe , Ga and Cr was investigated in pure acetonitrile and in acetonitrile/water mixtures. A
strong CHEF (Chelation-Enhanced Fluorescence) effect was observed with all the trivalent metals and
2+
Hg ions in both solvents. UV-vis absorption, steady state and time resolved emission spectroscopy
3
+
3+
methods were employed. The results show the formation of mononuclear complexes with Al , Fe
,
3
+
3+
2+
Ga , Cr , and Hg . Theoretical calculation using Density Functional Theory was performed in order to
3
+
2+
Received 26th March 2016,
Accepted 29th April 2016
obtain atomistic insights into the coordination geometry of Al and Hg to the fluorescein 3, which is in
accordance with the experimental stoichiometry results obtained in the Job’s plot method. Among the
active cations, the minimum detectable amount is under 1 µM for most of the cases in both absorption
and fluorescence spectroscopy methods.
DOI: 10.1039/c6dt01180b
www.rsc.org/dalton
nucleophilic (phenolic) and electrophilic (carboxylate) func-
tional groups. However, the presence of oxygenated functional
Introduction
Fluorescein, and its derivatives, are a very interesting class of groups around the whole of its molecular structure makes fluo-
versatile green emissive dyes due to their potential application rescein poorly selective when seeking for a specific analyte or
in biological fields such as in cellular biology, cellular a class of analytes. The structural modification of the marginal
8
imaging, as molecular tools in biochemistry and biomedical functional groups of the xanthene moiety may allow the
1
,2
fields. Fluorescein-based derivatives are usually soluble in adjustment of the properties of the chromophore for specific
water, present good cell membrane penetration capacity, applications making this dye one of the most widely explored
exhibit pH-responsive variations, show high fluorescence probes. Having this concept in mind, the association of the
quantum yields, exhibit strong extinction coefficients, and are fluorescein core with endogenous biomolecules such as amino
3
–7
9,10
very useful for metal ion detection and quantification. In its acids or peptides
is a promissory field of investigation since
free-acid form, fluorescein is a very versatile fluorogenic build- biocompatible chemosensors for in vivo applications can be
2
+
3+
ing block for organic synthesis, due to the presence of both envisioned. Metals like mercury (Hg ) and chromium (Cr )
are considered to be the most impactful with respect to their
environmental contamination and toxicity to living organisms.
Hg exerts many toxic effects to living organisms including
2
+
a
Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, CxP.
6077, São Paulo, 05508-000, Brazil. E-mail: alcindo@iq.usp.br
2
b
oxidative damage, neuro-toxicological disorders, carcinogeni-
BIOSCOPE Group, UCIBIO-REQUIMTE, Departamento de Química, Faculdade de
Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Portugal.
E-mail: cle@fct.unl.pt, clodeiro@bioscopegroup.org
11,12
city, etc.
Mercury may occur in metallic, ionic, organo-
13,14
2+
metallic and complex forms,
Hg being the most stable
c
oxidation state. In this form it can be easily bioaccumulated in
Instituto de Física, Universidade Federal de Uberlândia-UFU, Av. João Naves de
1
5
Ávila 2121, Uberlândia, MG 38400-902, Brazil
d
foods or directly in animals, humans or other living entities.
2+
CICECO - Aveiro Institute of Materials, Department of Chemistry,
Hg presents very high affinity for sulfur atoms, leading to
very stable complexes; indeed, it is able to bind very efficiently
to exogenous as well as endogenous sulfur-containing deriva-
tives and this is the main reason for accumulative intoxication
University of Aveiro, 3810-193 Aveiro, Portugal
e
Proteomass Scientific Society, Rua dos Inventores, Madan Park, 2829-516 Caparica,
Portugal
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
1
6,17
3+
c6dt01180b
in living organisms.
Concerning trivalent cations, Cr is
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an essential element playing an important role in nutrition
and some metabolic processes of humans but it is also a
1
8,19
major environmental pollutant.
The chromium oxidation
3
+
states can range from −2 to +6, wherein Cr is the far pre-
20
3+
dominant species of chromium in living organisms. Cr plays
essential roles in “trace amounts” (50–200 μg per day), necess-
ary for the maintenance of glucose metabolism. However, the
−
1
ingestion of doses up to 50 mg kg is lethal in human
2
1
3+
adults. Al is naturally abundant in silicates, cryolite and
bauxite rocks, this last mineral being the main raw material in
the production of metallic aluminum. It is even present in
some pharmaceutical formulations as aluminum hydroxide in
gastric pH regulators. Although not yet well understood, there
is some evidence pointing to the association of Al with brain
2
2
3
+
23,24
disorders like Parkinson’s and Alzheimer’s disease.
Con-
sidering the importance of these elements in life and conse-
quently the need for their detection as well as quantification
in different matrices, the development of selective and sensi-
ble analytical methods is still an interesting field of
Scheme 1 Synthesis of ligands 2 and 3.
2
5–28
29
investigation.
Xiaoxu Li et al. demonstrated the construc-
tion of bio-inspired compounds by the union of amino acids
and fluorescein from its phenolic group by a very simple strat-
egy. Marta Mameli et al. demonstrated the effect of the pres-
For compound 2, the H-methyl and H-tert-butyl attached to the
silane group are observed in the upfield region of the spectra,
due to the shielding effect by silicon.
Also, the signals due to the heterocyclic rings of the fluo-
rescein moieties were visible between 6.5 and 8.3 ppm. The
1
1
ence of the sulfur atom in anthracenylmethyl derivatives in
2
+
30
order to design probes to detect Hg . Barba-Bon et al.
reported the preparation of fluorescein ester derivatives used
as probes for trivalent metallic ions. In order to apply accessi-
ble reagents as fluorescein and amino acids in the detection of
metallic ions, in this work we present the synthesis, characteri-
zation and spectroscopic studies of two new fluorescein-based
amino acid derivatives and their application for the fluorescence
2 3
signals belonging to CH (4.1 ppm) and CH (1.0 ppm) from
ethyl benzoate of fluorescein were observed for compounds 2
1
3
and 3. Relative to the C NMR spectra (Fig. S4 and S6†), the
formation of the ester function was confirmed by the appear-
ance of the signal of the quaternary carbonyl group at about
3
+
3+
3+
3+
1
5
86 ppm, as well as by the α-carbon at 56.2 ppm (2) and
3.5 ppm (3). The IR spectra show some of the main signals,
detection of some trivalent ions (Al , Fe , Ga and Cr ) and
2+
Hg . The molecular probe structures and their photophysical
properties were characterized and their sensing ability towards a
range of ions in acetonitrile and aqueous acetonitrile solutions
was evaluated. DFT calculations were also performed to unveil
the complexation mode by varying the metal ion.
such as the carbonyl of the aliphatic ester that binds the
−
1
fluorescein to the amino acid residue at 1770 cm and the
−
1
carbonyl of the ethyl benzoate at 1720 cm . The broad band
around 3300 cm− corresponds to the secondary N–H carb-
1
3
1
amate stretching present in both compounds 2 and 3. More-
over, from the MALDI-TOF-MS analysis it is possible to
+
identify the protonated molecular ion peak species [2 + H ] at
Results and discussion
Synthetic approach
+
662.28 m/z and [3 + H ] at 668.23 m/z (Fig. S12 and S13†).
Photophysical characterization
The first step of the synthetic route started with Fischer’s ester-
ification of fluorescein, followed by coupling with the amino The fluorescein derivatives 1, 2 and 3 were characterized in
acid derivatives Boc-ser(TMS)-OH and Boc-cys(4-methylbenzyl)- acetonitrile at 298 K and the main characterization data were
OH, using N,N′-dicyclohexylcarbodiimide (DCC) as a carboxylic gathered and are given in Table 1. Fig. 1 presents the absorp-
activator group, which resulted in the esters 2 and 3 in 86% tion, emission and excitation spectra of all compounds.
and 90% yields, respectively as presented in Scheme 1. Both Indeed, the perfect match between the absorption and exci-
1
13
the compounds were characterized by H and C NMR, tation spectra rules out the absence of emissive impurities.
elemental analysis, mass spectrometry (MALDI-TOF-MS), UV- The absorption spectra revealed peaks at 452 nm for 1, and
vis absorption, and emission spectroscopy, and lifetime 428 nm for 2 and 3 attributed to π–π* transitions of the fluor-
1
measurements of the excited state were performed. The
H
escein chromophore. Considering the emission spectra charac-
NMR spectra of 2 and 3 (Fig. S3 and S5†) present the singlet teristic bands at 519 nm for 1 and 546 nm for 2 and 3 are
signal of the t-butyl from the N-Boc protective group and the visualized. The coupling of the amino-acids to compound 1
characteristic signals of the amino acid backbone NH carb- leads to a bathochromic shift of 24 and 27 nm in the absorp-
amate, α-CH and β-CH from serine and cysteine side chains. tion and emission maximum bands, and an increase in the
2
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Table 1 UV-vis and fluorescence data for 1, 2 and 3 (1 × 10− M) in acetonitrile
UV-vis Fluorescence
Paper
5
λ
exc
λ
em
λem Solid
Stokes’ shift
Quantum
Lifetime τ
Compounds
(nm)
log ε
(nm)
(nm)
(nm)
yield ϕ
(ns)
1
2
3
452
428
428
4.30
4.22
4.16
519
546
546
—
576
572
67
118
118
0.024
0.002
0.004
3.8 ± 0.1
2.3 ± 0.1
2.5 ± 0.1
Fig. 1 Room temperature absorption (full line), normalized emission (dashed line, λexc = 452 nm in part A; λexc = 428 nm in parts B and C) and exci-
−5
tation spectra (dotted line, λem = 519 nm in part A; λem = 546 nm in parts B and C) of compounds 1 (A), 2 (B) and 3 (C) in acetonitrile (1.10 M). Emis-
sion spectra in the solid state (dotted dashed line, λexc = 428 nm, in parts B and C) of compounds 2 (B) and 3 (C).
Stokes’ shift is also observed. The relative fluorescence Metallic ion sensing
quantum yields (Φ) of compounds 1 to 3 were calculated using
3
2
+
acridine yellow as the standard (Φ = 0.47 in ethanol), where The sensing ability of compounds 2 and 3 towards alkali (Na ,
+
2+
2+
2+
2+
2+
values of 0.023, 0.002 and 0.004 were determined, respectively. K ), alkaline earth (Ca , Ba ), transition (Co , Ni , Cu ,
3
+
3+
+
2+
+
2+
2+
As can be noted, the insertion of substituted amino acids in Fe , Cr , Ag , Cd , Hg and Hg ), and post-transition (Zn ,
2
+
3+
3+
the precursor 1 at position 6 of the xanthene moiety led to a Pb , Al , Ga ) metal ions were evaluated by ligand titrations
decrease of 10 times in the fluorescence quantum yield. In with increasing additions of the metal salts. The titrations
turn, when compared the quantum yield of 1 (Φ = 0.024) with were followed by absorption and fluorescence emission spec-
4
the free acid fluorescein in its neutral form (Φ = 0.30), there troscopy. Both compounds were silent in the presence of alka-
was a decrease of 13 times in the emission of fluorescence. line and alkaline earth metal ions. Concerning transition and
2
+
3+
The decrease in the quantum yield of the free-acid fluorescein post-transition metal ions, only Hg and the trivalent Fe ,
3
+
3+
3+
compared to 1 can be attributed to the steric hindrance Cr , Al and Ga metal ions were able to coordinate with the
imposed by the ethyl group, which results in a perpendicular fluorescein derivatives 2 and 3. Fig. 2 shows the absorption
arrangement between xanthene and benzoic moieties, making and emission titrations of compounds 2 and 3 with the
the π orbital overlap impaired, a condition for the π–π* elec- Hg metal ion. A similar spectral behavior was observed in
tronic transitions. It is known that the methyl ester of fluor- both compounds, whereas, in the absorption spectra, an
2
+
3
3
escein, where both hydroxyl groups are modified, is highly increase at 431 nm and 434 nm in the absorbance with a con-
33
fluorescent. However, herein two asymmetric probes (2 and 3) comitant disappearance of the band at 490 nm was detected,
34
are reported, they can be compared with rhodols. In the case for compounds 2 and 3, respectively. Probes 2 and 3
of rhodols the fluorescence quantum yields decrease in accord- practically exhibit no fluorescence, however, surprisingly, a
ance with the length of the linker. Fluorescence decays were strong chelation-enhanced fluorescence (CHEF) at 470 nm (2)
2
+
measured in acetonitrile at 298 K, with excitation at 460 nm. All and 468 nm (3) was observed with Hg . Due to its heavy metal
fluorescence decays were well fitted with a double exponential ion nature, which usually leads to non-radiative decays,
law, with decay times of 3.8, 2.3 and 2.5 ns, which are directly most systems using fluorescence spectroscopy for detecting
3
2+
proportional to the quantum yield. The emission of compounds Hg are based on the complexation enhancement of the fluo-
1
1,35–37
2
and 3 was measured in the solid state using a fiber optic rescence quenching (CHEQ) effect.
On the other hand,
system connected to the HORIBA Scientific Fluoromax-4. In the only a few fluorescence chemosensors are based on the CHEF
3
8–42
solid state both compounds show the maximum emission peaks effect.
to be red shifted, Δλ = 26–30 nm, in relation to the ones Thus, this fact makes probes 2 and 3 very appealing for
obtained in solution, the maximum wavelength being located at detection of toxic Hg metal ions. Probes 2 and 3 also showed
2
+
576 nm and 572 nm for compounds 2 and 3, respectively.
immediate turn-on upon the addition of trivalent metal ions,
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2+
Fig. 2 Spectrophotometric titrations of 2 (A) and 3 (C), with increasing amounts of Hg in acetonitrile solution. Inset: Absorption at 434 nm as a
2
+
2+
−5
function of [Hg ]/[2] and [Hg ]/[3] (T = 298 K; [2] = [3] = 1.10 M). Spectrofluorometric titrations of 2 (B) and 3 (D), with increasing amounts of
2+
2+
2+
−5
Hg in acetonitrile solution. Inset: Emission at 470 nm as a function of [Hg ]/[2] and [Hg ]/[3] (T = 298 K; [2] = [3] = 1.10 M, λexc = 362 nm).
3
+
3+
3+
3+
3+
3+
such as Fe , Cr , Al , and Ga , despite Cr and Fe being (Fig. 3A), the cation is tetrahedrally coordinated to the receptor
3
+
3+
known as fluorescence quenchers. Trivalent Cr and Fe are (dAl⋯OvC = 1.77 Å and dAl⋯S = 2.30 Å) and a water molecule
very effective fluorescence quenchers due to their paramag- (dAl⋯OH2 = 1.83 Å). Similarly, and in the case of 3–Hg
netic nature, for this reason there are few turn-on sensors for (Fig. 3B), the cation is coordinated to the receptor (dAl⋯OvC
2
+
=
4
3,44
3+
these trivalent metal ions.
In contrast Al is diamagnetic, 2.45 and 2.46 Å and dAl⋯S = 2.74 Å) and a water molecule
whereas its binding to sensors usually enhances the fluo- (dAl⋯OH2 = 2.31 Å) in a highly distorted tetrahedral geometry
rescence signal. Fig. S7–S10† show the absorption and emis- (the water molecule was included in the calculation as a way to
3
+
3+
3+
sion titration of both compounds with Al , Fe , Cr and provide an anchor point for stabilizing the coordination
3
+
Ga metal ions. Upon exposition of the probes to these metal sphere of the cation).
ions, the absorbance at 490 nm decreases gradually and simul-
On one hand, both binding arrangements suggest that the
taneously a new significant band at 431 nm and 434 nm was prearrangement of the two carbonyl groups and the thioether
developed. In the range of 450–600 nm, compounds 2 and 3 adequately provide a coordination pocket for accommodating
presented a weak emission, whereas a significant enhance- the cation. On the other hand, the ionic radius of the metallic
ment of the emission signal was detected at 500 nm and cation suggests that it plays an important role in the overall con-
3
0
4
93 nm for 2 and 3, respectively. Barba-Bon and co-workers
formation of the association since coordination to larger
published a similar selective turn-on chromo-fluorogenic cations can lead to the involvement of the ester oxygens from
3
+
3+
3+
probe for Fe , Cr and Al , whereas they showed by NMR the fluorescein moiety. These results support the experimentally
studies the eventual involvement of the xanthene moiety in the inferred coordination mode of the metal to the receptor. The
4
5
metal coordination. Atomistic insights into the coordination association constants using the HypSpec program corroborate
3
+
2+
geometry of Al and Hg to the fluorescein 3 were obtained the theoretical model complexation of one ligand per metal ion
from density functional theory calculations with the B3LYP (L : M 1 : 1), and the main results are summarized in Table 2.
functional. The lowest energy complex conformations found
The values obtained are almost of the same order (log β ≈
3
+
2+
2+
for both 3–Al and 3–Hg (Fig. 3) involve the coordination of 5), with the exception of Hg whereas a higher value of log β ≈
the metal to two cabonylic oxygens (from the ester moieties) 6.5 was observed for compound 3. This fact is due to the pres-
3
+
and the sulphur from the thioether. In the case of 3–Al
ence of the sulphur atom in the cysteine amino acid in
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3+
Fig. 3 (A) Top, side and front views of the lowest energy conformation of the 3–Al complex, indicating the distances between the receptor’s
interaction sites and cation (color code: C – teal; O – pink; H – white; S – yellow; N – blue, Al – light blue sphere). (B) Top, side and front views of
2+
the lowest energy conformation of the 3–Hg complex, indicating the distances between the receptor’s interaction sites and cation (color code as
in (A); Hg – red sphere).
2
+
2+
2+
2+
2+
+
Table 2 Stability constants log β obtained from absorbance (A) and alkaline earth (Ca , Ba ), transition (Co , Cu , Ni , Ag ,
fluorescence (F) titration data. The HypSpec program was applied con-
2+
+
2+
3+
3+
2+
2+
Cd , Hg , Hg , Fe , Cr ) and post-transition (Zn , Pb ,
Al , Ga ) metal ions were recorded. Careful inspection of
sidering 1 : 1 interaction
3+
3+
Fig. 4 leads to the conclusion that 2 and 3 show a turn-on and
an unprecedented selectivity for Hg , Al , Fe , Cr and Ga
metal ions producing intense emissive green-blue colours
Compounds
Metal ions
2
3
2
+
3+
3+
3+
3+
log β (A)
log β (F)
log β (A)
log β (F)
2
+
upon complexation by these metals. The difference in the
5.8 ± 0.3 coordination mode observed in the DFT calculations can be
Hg
Ga
5.1 ± 0.1
5.0 ± 0.2
5.2 ± 0.3
5.3 ± 0.3
5.0 ± 0.3
5.4 ± 0.1
4.8 ± 0.3
4.9 ± 0.2
5.1 ± 0.3
5.2 ± 0.2
6.5 ± 0.2
5.4 ± 0.3
5.3 ± 0.1
5.5 ± 0.1
4.9 ± 0.3
6.2 ± 0.2
3
+
3
+
Cr
5.1 ± 0.3
5.1 ± 0.3
5.5 ± 0.1
extended to the other trivalent ion complexes, and is reflected
3
+
Al
3+
3
+
directly in the emission wavelengths: green for X (around
Fe
2
+
5
00 nm) and blue for Hg (470 nm), as shown at the bottom
of Fig. 4B. In order to study the properties of probes 1, 2 and
compound 3, which confers a higher affinity to the Hg metal 3, the photophysical characterization in a mixture of aceto-
ion. The final stoichiometry of 2 and 3 with each of the ions nitrile/water (1 : 1) was carried out (Fig. 5). The addition of
was confirmed by the Job’s plot method and the results are water induces a red shift in the maximum of absorption, from
compiled in Fig. S14.† Fig. 4 shows a general overview of the 428 nm to 460 nm in compounds 2 and 3.
2
+
4
6
fluorescence properties of the probes 2 and 3 upon the
As is known, due to the high affinity of the studied metallic
addition of all the metal ions studied. The emission spectra of ions for water, the addition of an excess of water to the system
+
+
2
and 3 upon the addition of 4 equivalents of alkali (Na , K ), involving the probes/analytes in acetonitrile solution induces
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+
+
2+
2+
2+
2+
2+
2+
+
2+
+
2+
Fig. 4 Relative emission intensity of 2 (A) and 3 (B) upon addition of 4 equiv. of Na , K , Ca , Ba , Zn , Co , Cu , Ni , Ag , Cd , Hg , Hg
,
3+
3+
3+
3+
2+
−5
Fe , Cr , Al , Ga and Pb in acetonitrile ([A] = [B] = 1 × 10 M; λexc = 362 nm). Inset: Normalized emission displacement between the com-
plexes of Hg and Ga for 2 (A) and 3 (C). Bottom: Visual changes of 2 (left) and 3 (right) under UV light after addition of metallic ions (λexc
65 nm).
2
+
3+
=
3
2
+
3+
Table 3 Minimal detectable and quantified amount (µM) for Hg , Fe
,
3+
3+
3+
Cr , Al and Ga metal ions in acetonitrile. Relative standard deviation
(RSD) of the values measured was below 10%, n = 3
Minimal
detectable
amount (μM)
Minimal
quantified
amount (μM)
Compounds
Metal ions
Abs
Em
Abs
Em
2
+
Hg
1.04
0.99
1.08
0.95
1.09
0.97
0.91
0.88
0.82
0.99
9.76
0.28
0.23
0.20
0.34
0.86
0.40
0.24
0.22
0.31
1.15
1.10
1.18
1.05
1.20
1.07
1.00
0.97
0.91
1.10
26.7
0.76
0.64
0.55
0.93
1.43
0.67
0.40
0.37
0.52
3
+
Fe
Cr
3
+
2
3
+
Al
3
+
Ga
2
+
Hg
3
+
Fe
Cr
3
+
3
3
+
Al
3
+
Ga
2+
Fig. 5 Progress of emission spectra of the complex 3–Hg (4 equiv.) in
solutions with different amounts of water in acetonitrile ([3] = 1 × 10−
M; λexc = 423 nm).
5
2
+
3+
3+
3+
3+
Hg , Fe , Cr , Al and Ga was determined according to
the procedure described in the Experimental section. The
quenching of the fluorescence emission, due to that the resulting values were gathered and are given in Table 3. The
coordination sphere of the ions is substituted by water mole- limit of detection by absorption for compounds 2 and 3 was
2
+
cules. The presence of water in the metal complex 3–Hg was obtained applying eq. (1), LODabs = 0.16 ± 0.01; and by emis-
studied by fluorescence analysis in different percentages of sion the results are LODem2 = 0.04 ± 0.01 and LODem3 = 0.16 ±
water (4.8%, 9.1%, 33% and 50%) in acetonitrile. The emission 0.01. Regarding the limit of quantification using eq. (2), the
2
+
band at 470 nm (black solid line), related to complex 3–Hg is LOQabs = 0.18 ± 0.02 for both compounds and LOQem2 = 0.10 ±
completely quenched up to 9.1% of water. Up to 4.8% of water, 0.03, and LOQem3 = 0.18 ± 0.02 were obtained by absorption
the main emission band is not that observed for the complex and emission, respectively. The minimal detectable amount
2+
3
–Hg at 470 nm, but at 525 nm, which belongs to the species obtained by absorption for the metal ions studied ranges from
between compound 3 bounded to water molecules through 0.95–1.09 µM and 0.88–0.99 µM for compounds 2 and 3,
hydrogen bonds, as shown in Fig. S15.† respectively. Through emission spectra, the value ranges
In order to shed more light on the sensing ability of these between 0.20–9.76 µM (2) and 0.31–0.86 µM (3) were
systems, the minimal detectable and quantified amount of determined.
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It is clearly notable that compound 3 is more sensitive and tion and emission, in comparison with the amounts of 2.5 and
3
0
reactive to the metal ions studied than 2, showing lower detect- 0.5 µM published previously.
2
+
able amounts. This fact is evident for Hg metal ions,
whereas a remarkable decrease from 9.76 to 0.97 µM (10
times) in the detectable amount was determined (Table 3).
Lifetime and fluorescence quantum yield of the metal
complexes
Once more, this is attributed to the presence of the amino acid Fluorescence decays of the ligands and complexes were
cysteine, which contains a sulphur atom with high affinity to measured in acetonitrile. The main data are depicted in
4
6
this heavy metal ion. Regarding the minimal quantified Table 4. As a representative example, Fig. 6A shows the fluo-
2
+
3+
3+
3+
3+
2+
amount for Hg , Fe , Cr , Al and Ga metal ions in aceto- rescence decay of the 2–Hg complex and also the correlation
nitrile, the values between 0.91 and 1.20 µM were calculated by between the lifetime and quantum yield of 2, 3 and the com-
absorption; 0.55–26.7 µM for 2 and 0.52–1.43 µM for 3 were plexes (Fig. 6B and C). The fluorescence decays of both com-
calculated by emission. This clearly makes compound 3 the pounds and their metallic complexes were fitted with a double
best probe to be used as an analytical tool for trivalent and exponential law, suggesting two different conformers in the
2
+
33,47
Hg metal ion detection. Our systems show similar detection excited state.
However, in the case of the free ligands, the
results to the one published previously by Barba-Bon and co- relative amplitudes (B ∼ 3% and C ∼ 97%, Table 4) indicate a
3
0
3+
workers. However for Cr ions our probes showed to be predominant species in solution of a short lifetime and
more effective since the detectable amounts by absorption and low quantum yield. On the other hand, by the analysis of the
emission are lower. In our systems, the lowest values of complexes, the higher values of amplitudes follow the higher
0
.88 µM and 0.23 µM are determined respectively, by absorp- lifetimes, indicating an effective complex formation. These
Table 4 Fluorescence quantum yield and lifetime for compounds 2 and 3. Fluorescence decays for 2 and 3 were fitted using A + B Exp(−t/τ) +
C Exp(−t/τ*). The values presented are average of six independent measurements
2
3
τ
τ*
τ
τ*
(
ns)
(ns)
B
C
B
C
Compounds
Free ligand
Φ
[Rel. amp. %]
Φ
[Rel. amp. %]
0.002 ± 0.001
0.14 ± 0.01
0.11 ± 0.01
0.11 ± 0.01
0.12 ± 0.01
0.12 ± 0.01
2.3
0.13
[97.1]
0.5
0.005 ± 0.001
0.16 ± 0.01
0.12 ± 0.02
0.12 ± 0.03
0.12 ± 0.02
0.12 ± 0.02
2.5
[4.1]
3.4
[91]
3.2
[50]
3.2
[70]
3.1
[60]
3.2
0.37
[95.9]
0.6
[
2.9]
3.9
96]
3.8
44]
3.8
79]
3.8
5.4]
3.8
80]
2
+
Hg
[
[4]
[9]
3
+
Fe
0.06
[56]
0.11
[21]
0.072
[46]
0.09
[20]
0.10
[50]
0.24
[30]
0.13
[40]
0.31
[20]
[
3
+
Al
[
3
+
Cr
[
3
+
Ga
[
[80]
2+
Fig. 6 (A) Fluorescence decays and global analysis of 2 (Flu-TMS) with 4 equivalents of Hg and the Ludox reference. The decay time values
obtained for fitting with a sum of two exponentials are 0.49 and 3.87 ns, Chisq 1.06. Decay time and quantum yield averages for (B) 2 and (C) 3 com-
plexes are presented.
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results are in accordance with the literature, where multiple 9H
2
O, Cr(NO
3
)
3
·9H
2
O, Fe(NO
3
)
3
·9H
2
3 3 2
O, Ga(NO ) ·9H O,
(
with two lifetimes of ∼4 ns and ∼0.5–2 ns) and single decays Zn(NO ) ·6H O, Ca(BF ) ·xH O, Cd(ClO ) ·6H O, Co(NO ) ·6H O,
3
2
2
4 2
2
4 2
2
3 2
2
4
7
have been reported to ligand coupled fluorescein structures.
Ni(NO )
3 2
·6H
, KNO
xanthene unit in the precursor 1 leads to a decrease in the and used as received. Boc-Ser(TBDMS)-OH was prepared as
2
O, Cu(NO
3
)
2
·6H
2
O, Pb(ClO
4
)
2
·3H
2
O, Hg(ClO
4
)·4H
2
O,
The insertion of amino acid derivatives at the backbone of the NaNO
3
3
, AgNO
3
) were purchased from Sigma-Aldrich
4
8
fluorescence quantum yield from ϕ1 = 0.024 ± 0.001 to ϕ2 = reported in the literature.
.002 ± 0.001 and ϕ3 = 0.005 ± 0.001 (ca. 10-times lower).
However, upon complexation a strong CHEF takes place,
0
Instruments
resulting in values of fluorescence quantum yield between 0.11 Elemental analysis was performed using a Thermo-Finnigan
and 0.16, which represents a huge increase ranging from 24 to CE Flash-EA 1112-CHNS instrument provided by the Chemical
7
0 times, compared to the free ligands. The same behavior was Analysis Service of the REQUIMTE, DQ, Universidade Nova de
observed in lifetime measurements, whereas higher values Lisboa, Monte da Caparica, Portugal. Mass spectrometry
were obtained for the metal complexes (τ = 3.1–3.9 ns) in com- characterization of compounds 1 and 2 was carried out in an
parison with the free ligands (τ = 2.3–2.5 ns).
Ultraflex II MALDI-TOF/TOF instrument from Bruker Daltonics
The lifetime values obtained showed no significant difference (BIOSCOPE-PROTEOMASS Research Lab) and high-resolution
between compounds 2 and 3. These results are in agreement mass spectrometry spectrum of compound 3 was recorded in
with the ones obtained for the fluorescence quantum yield, once an Electron Spray Ionization Time of Flight (ESI-TOF)
generally, emissive systems with higher fluorescence quantum micrOTOF-QII Bruker spectrometer (IQ-Universidade de São
1
13
yields are more stable in the excited state.
Paulo). H and C NMR spectra were recorded on a Bruker
1
13
Avance III 200 (200 MHz, H; 50 MHz, C) spectrometer at
Universidade de São Paulo, São Paulo, Brazil. The lifetime of
the excited state was measured using a Horiba Jobin-Yvon
Tempro equipped with the light source NanoLED at 460 nm,
Conclusions
Two new bio-inspired fluorescein derivatives were successfully PROTEOMASS Scientific Society, Portugal. The infra-red
2
+
synthesized and applied as probes for Hg and trivalent met- spectra were recorded on a Spectrum BX Perkin-Elmer FT-IR
allic ions in acetonitrile solution. The complexation process System.
promoted huge changes in the photophysical properties of
free ligands. A strong CHEF, an absorption blue shift, an
Spectrophotometric and spectrofluorometric measurements
increase in extinction coefficient, longer lifetimes of the Absorption spectra were recorded on a Jasco V-650 spectro-
excited states and higher quantum yields of fluorescence were photometer and fluorescence emission on a Horiba-Yvon-Spex
observed as result of the complex formation. DFT calculations Fluoromax-4 spectrofluorometer. A correction for the absorbed
3
+
suggested different models of interaction between the 3–Al
light was applied when necessary. All spectrofluorometric titra-
2
+
and 3–Hg complexes, which were reflected in the green emis- tions were performed as follows: the stock solutions of the
sion of the trivalent ion complexes and blue emission of the ligands (ca. 5 × 10− M) were prepared by dissolving an appro-
Hg complex. The application of compounds 2 and 3 as priate amount of the ligands in 25 mL volumetric flasks to the
analytical probes for Al sensing reached the lowest detection mark with acetonitrile. The titration solutions were prepared
and quantification limits in both absorption and fluorescence by dilution of stock solutions at a final concentration of
techniques, compared to other metals. The interaction con- 1 × 10− M in acetonitrile. Titrations of the ligands were con-
stants of the complexes all showed very close values, except for ducted by the addition of microliters of standardized metal
4
2
+
3
+
5
2+
3
–Hg , which presented considerably higher values attributed solutions in acetonitrile. The absorption and emission spectra
2+
to the soft–soft attraction between Hg and S-cysteine, reinforcing of these solutions were recorded after additions (λexc = 428 nm
the Pearson’s acid–base concept. This result suggests the impor- and λem = 546 nm for 2 and 3). The stock solutions of the met-
tance of the sulfur atom in the amino acid residue to modulate allic ions were prepared by dissolving an appropriate amount
the metal–ligand interaction constants for soft metal ions. As the of the salts in 10 mL volumetric flasks to the mark with water
presence of water suppresses the sensing ability of the probes, (ca. 0.05 M). The titration solutions of metals were obtained by
these are very promising structures to be applied in the selective the dilution of the stock solution to the concentration of 1.5 ×
−
3
detection of the studied cations in biological aprotic environ- 10 M in acetonitrile. The solid-state emission spectra of 2
ments, such as cell membranes or micellar systems. These and 3 were recorded on a Horiba-Scientific Fluoromax-4 spec-
further studies are under investigation in our group.
trofluorometer using an external fiber-optic device. The fluo-
rescence quantum yield of compounds 1, 2 and 3 was
measured using a solution of acridine yellow in absolute
ethanol as the standard (ϕ = 0.47) and was corrected for
different refraction indexes of solvents. The detection (LOD)
and quantification (LOQ) limits for the metal ions were deter-
4
9
Experimental section
Chemicals and starting materials
The fluorescein (free acid) was purchased from Fluka, Boc-Cys- mined, having in mind their use for real metal ion detection
(
4-MeBzl)-OH and metallic salts (Hg(ClO ) ·3H O, Al(NO ) · and for analytical applications. For these measurements, ten
4 2 2 3 3
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Paper
different analyses for the selected receptor were performed in eluent. The pure product was obtained as an orange solid in
order to obtain the LOD and LOQ.
The LOD and LOQ were obtained by applying the
equations:
86% yield.
RF in EtOAc/hexane 2 : 1 = 0.50. Melting point: 68 °C.
Elemental analysis (found: C, 65.1; H, 6.7; N, 2.2% CHNS
requires: C, 65.3; H, 6.6; N, 2.1) 1H NMR (200 MHz, CDCl ) δ
3
LOD ¼ yblank þ 3 std
LOQ ¼ yblank þ 10 std;
ð1Þ
ð2Þ
8
1
1
0
6
1
1
1
1
6
.36–8.20 (m, 1H), 7.72 (td, J = 6.7, 1.6 Hz, 2H), 7.42–7.28 (m,
H), 7.08–6.80 (m, 3H), 6.64–6.40 (m, 2H), 5.41 (d, J = 8.7 Hz,
H), 4.62 (d, J = 8.7 Hz, 1H), 4.33–3.83 (m, 4H), 1.48 (s, 9H),
where yblank = signal detection limit and std = standard
.98 (t, J = 7.1 Hz, 3H), 0.90 (d, J = 0.8 Hz, 9H), 0.16–0.02 (m,
deviation.
13
3
H). C NMR (50 MHz, CDCl ) δ 186.19, 169.19, 165.41,
58.74, 155.65, 154.09, 152.90, 149.16, 134.07, 132.94, 131.60,
30.99, 130.90, 130.68, 130.57, 130.07, 128.91, 119.95, 119.42,
18.22, 110.23, 106.46, 80.61, 63.95, 61.69, 56.18, 28.52, 25.98,
8.45, 13.78, 0.20, −5.28. MALDI-TOF-MS [calcd (found)]:
Theoretical calculations
All calculations were performed with Gaussian09 (19) using
Density Functional Theory with the B3LYP functional and the
-31G** (C, H, N, O, S, Al) and LanL2DZ (Hg) basis set, as
implemented in Gaussian09. Default optimization parameters
were used throughout the calculations.
6
62.28 (662.28) [M + H]. UV-vis [acetonitrile; λmax, nm (εmax
,
3
−1
−1
dm mol cm )]: 337 (10 727), 428 (16 019), 444 (15 835).
Fluorescence emission in acetonitrile (λ = 428 nm, λem
=
exc
1
5
1
46 nm). IR (BaF
2
windows, cm− ): 2954, 2933, 2859, 1774,
Synthesis of organic compounds
717, 1645, 1598, 1523, 1367, 1259, 1153, 1106, 838, 777.
Synthesis of 2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoate
Synthesis of (R)-ethyl 2-(6-((2-((tert-butoxycarbonyl)amino)-
(
1). In a 500 mL round bottomed flask, concentrated sulfuric 3-((4-methylbenzyl)thio)propanoyl)oxy)-3-oxo-3H-xanthen-9-yl)
acid (15 mL) was added dropwise to a solution of fluorescein benzoate (3). The same procedure was performed as for obtain-
30.09 mmol, 10 g) in ethanol (200 mL) at room temperature. ing 2, replacing Boc-Ser(TBDMS)-OH by Boc-Cys(4methyl
(
After stirring and refluxing for 18 hours, the solvent was -benzyl)-OH. The pure product was obtained as an orange
removed in a rotatory evaporator and the resulting mixture was solid in 90% yield. RF in EtOAc/hexane 2 : 1 = 0.54. Melting
diluted in chloroform and transferred to a 1000 mL beaker. point: 66 °C. Elemental analysis (found: C, 68.1; H, 5.7; N, 2.1;
1
Solid sodium bicarbonate was added to the mixture until evol- S, 4.7% CHNS requires: C, 68.4; H, 5.6; N, 2.1; S, 4.8). H NMR
3
ution of carbon dioxide ceased. The heterogeneous mixture (200 MHz, CDCl ) δ 8.43–8.17 (m, 3H), 7.92–7.55 (m, 6H), 7.04
was filtered through a Buchner funnel and the organic phase (ddd, J = 19.0, 15.8, 12.5 Hz, 28H), 6.69–6.41 (m, 4H), 5.33 (d,
was evaporated. The crude product was dissolved in 400 mL of J = 7.6 Hz, 3H), 4.74 (d, J = 7.6 Hz, 4H), 4.06 (q, J = 7.1 Hz, 7H),
ethanol (96%) at boiling temperature. The volume was reduced 3.77 (s, 5H), 2.98 (d, J = 5.4 Hz, 5H), 2.33 (s, 9H), 1.47 (s, 30H),
1
3
to 100 mL by boiling and the resulting solution was allowed to 0.99 (t, J = 7.1 Hz, 8H). C NMR (50 MHz, CDCl3) δ 186.01,
stand for 12 hours at −20 °C, yielding 8.90 g of pure product 172.95, 169.19, 165.09, 158.64, 157.50, 155.13, 153.74, 152.64,
as brown crystals, with a greenish glow. Yield 90%. Melting 149.54, 137.09, 134.15, 133.75, 132.70, 131.35, 130.59, 130.47,
1
point: 242 °C. H NMR (200 MHz, DMSO) δ 8.18 (d, J = 7.4 Hz, 130.38, 129.89, 129.74, 129.36, 129.14, 128.79, 121.87, 119.63,
1
H), 8.00–7.61 (m, 2H), 7.50 (d, J = 7.0 Hz, 1H), 6.80 (d, J = 9.6 119.26, 118.10, 115.21, 110.06, 106.08, 103.81, 80.59, 61.45,
Hz, 2H), 6.56 (d, J = 6.6 Hz, 4H), 3.96 (q, J = 7.0 Hz, 2H), 0.86 53.49, 36.39, 33.11, 28.24, 21.06, 13.57. MALDI-TOF-MS [calcd
1
3
(
1
1
t, J = 7.1 Hz, 3H). C NMR (50 MHz, DMSO) δ 173.93, 165.02, (found)]: 668.23 (668.23) [M + H]. UV-vis [acetonitrile; λmax, nm
3
−1
−1
56.13, 150.49, 133.66, 132.91, 130.61, 130.06, 129.87, 121.75, (εmax, dm mol cm )]: 336 (9360), 428 (14 425), 445 (14 350).
14.39, 103.24, 60.83, 13.29. UV-vis in acetonitrile; λmax, nm Fluorescence emission in acetonitrile (λexc = 428 nm, λem
εmax, dm mol cm )]: 452 (19 953). Fluorescence emission 546 nm). IR (BaF2 windows, cm ): 2974, 2918, 1771, 1715,
=
3
−1
−1
−1
(
in acetonitrile (λexc = 452 nm, λem = 519 nm).
1639, 1598, 1509, 1263, 1158, 860.
Synthesis of (S)-ethyl 2-(6-((2-((tert-butoxycarbonyl)amino)-3-
((tert-butyldimethylsilyl)oxy)propanoyl)oxy)-3-oxo-3H-xanthen-
9
-yl)benzoate (2). In a round bottomed flask of 50 mL under
Acknowledgements
an inert atmosphere, Boc-Ser(TBDMS)-OH (3.0 mmol, 1.05 g);
N,N‘-dicyclohexylcarbodiimide (DCC, 3 mmol, 0.6 mL) and di- E. Oliveira acknowledges the post-doctoral grant from Funda-
chloromethane (20 mL) were added while stirring for ção para a Ciência e a Tecnologia (FCT-MEC) (Portugal) SFRH/
1
5 minutes. Subsequently, 1 was added (1.1 mmol, 0.386 g) BPD/72557/2010. A. Gonçalves and V. Pilla thank CNPq/Brazil
and the reaction was followed until all the fluorescein for the PhD and post-doctoral (202553/2014-0) grant
derivative was consumed. After 18 hours, the reaction medium respectively. C. Lodeiro thanks the CNPq program, Science
was filtered to remove the urea generated by the consumption without borders 2014–2016 grant (Brazil). Financial support
of DCC, and the solvent was removed under reduced pressure from the Scientific PROTEOMASS Association (Portugal), the
in a rotatory evaporator. The product was purified in a chroma- University of São Paulo through the NAP-CatSinQ (Research
tographic column using flash silica as the stationary phase Core in Catalysis and Chemical Synthesis), FAPESP, CAPES and
and an ethyl acetate/hexane mixture in the ratio 2 : 1 as the FAPEMIG (Brazil) (APQ-00360-14) is acknowledged. This work
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was supported by the Unidade de Ciências Biomoleculares
Aplicadas-UCIBIO which is financed by national funds from
V. Rondeau, J. Toxicol. Environ. Health, Part B, 2007, 10
(Suppl 1), 1–269.
FCT/MEC (UID/Multi/04378/2013) and co-financed by the 23 S. Han, J. Lemire, V. P. Appanna, C. Auger, Z. Castonguay
ERDF under the PT2020 Partnership Agreement (POCI-01- and V. D. Appanna, Cell Biol. Toxicol., 2013, 29, 75–84.
145-FEDER-007728). Part of this work was developed within 24 V. Rondeau, D. Commenges, H. Jacqmin-Gadda and
0
the scope of the project CICECO-Aveiro Institute of Materials,
J. F. Dartigues, Am. J. Epidemiol., 2000, 152, 59–66.
POCI-01-0145-FEDER-007679 (FCT Ref. UID/CTM/50011/2013), 25 B. Daly, J. Ling and a. P. de Silva, Chem. Soc. Rev., 2015, 44,
financed by national funds through the FCT/MEC and when 4203–4211.
appropriate co-financed by FEDER under the PT2020 Partner- 26 S. Das, M. Dutta and D. Das, Anal. Methods, 2013, 5, 6262.
ship Agreement. S. M. S. thanks FCT for the FCT Investigator 27 J. Chan, S. C. Dodani and C. J. Chang, Nat. Chem., 2012, 4,
starting grant IF/00973/2014.
973–984.
28 K. P. Carter, A. M. Young and A. E. Palmer, Chem. Rev.,
2014, 114, 4564–4601.
2
3
9 X. Li and J. S. Taylor, Bioorg. Med. Chem., 2004, 12, 545–552.
0 A. Barba-bon, A. M. Costero, S. Gil, M. Parra and J. Soto,
Chem. Commun., 2012, 48, 3000–3002.
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