Squaraines as Fluoro-Chromogenic Probes for Thiol-Containing Compounds and Their Application to the Detection of Biorelevant Thiols

Article abstract of DOI:10.1021/ja031987i

A highly selective colorimetric chemodosimeter for thiol-containing compounds in aqueous solutions is reported. The design protocol makes use of a highly specific reaction between thiols and the electrophilic four-membered ring of highly colored, fluoresc

Full text of DOI:10.1021/ja031987i

Published on Web 03/10/2004
Squaraines as Fluoro-Chromogenic Probes for Thiol-Containing Compounds
and Their Application to the Detection of Biorelevant Thiols
Jose V. Ros-Lis,† Beatriz Garc´ıa,† Diego Jime´nez,† Ramo´n Mart´ınez-Ma´n˜ez,*,† Fe´lix Sanceno´n,†
Juan Soto,† Fernando Gonzalvo,‡ and M. Carmen Valldecabres‡
GDDS, Departamento de Qu´ımica, UniVersidad Polite´cnica de Valencia, Camino de Vera s/n,
E-46071 Valencia, Spain, and Hospital de la Ribera, Ctra de Corbera, Km 1, E-46600 Valencia, Spain
Received December 29, 2003; E-mail: rmaez@qim.upv.es
Scheme 1. Molecular Structure of the Squaraine Derivatives L1
Amino acids are very well-known compounds with an angular
importance in biological systems as they join together to yield
and L2
proteins, enzymes, structural elements, and many other molecules
of biological activity. This role of building blocks in living systems,
along with the discovery that a number of disorders are associated
with changes in concentration of particular amino acids, has resulted
in a rising interest in their detection in fields such as chemistry,
biochemistry, and clinical chemistry.1
Natural R-amino acids show similar properties due to the special
arrangement of their carboxyl and amino groups. Therefore, che-
mosensors relying on the coordination with these common moieties
are not as selective as wanted although additional coordination with
its potential application to the detection of biorelevant thiols. Here
other groups in the amino acid’s side chains has sometimes resulted
the target concept is specific reactivity instead of selective
in some preferential coordination. This is the case of two fluorogenic
coordination.3 Related with that “chemodosimeter” approach several
chemosensors for R-amino acids, one containing a Zn2+ complex
thiol-reactive probes are also known and used for the determination
of thiol-containing compounds. These are common alkylating agents
such as iodoacetamides, maleimides, benzylic halides, etc. However,
these and other reported thiol reagents show certain limitations,
as binding site for carboxylates2a (preferential sensing of amino
acids bearing aromatic residues due to π-staking interaction with
an anthracene group) and the other a heteroditopic system containing
an aza-crown ether for ammonium binding and guanidinium units
typically comprising reactivity with other nucleophiles such as
for carboxylate coordination2b (preferential response with lysine and
amines including amino acids with amine residues (histidine or
glycine). Both contain anthracene as signaling unit and follow the
tyrosine), instability in light, hydrolysis of the linkage to the
general “binding site-signaling unit” approach. On the other hand,
fluorophore, etc. Additionally, many of these reagents show signals
near the UV zone, and relatively few (usually rather complex and
high in cost) are designed to display transduction near the IR regions
of the spectra.
a terpyridine-Zn2+ complex also bearing guanidinium groups2c has
been described that shows certain selectivity for aspartate via
metal-ligand coordination and hydrogen bonding involving the
side-chain carboxylate and guanidinium groups. This case follows
We have focused our attention to water-soluble squaraine deriv-
a “displacement” approach in which color change was observed as
atives such as the compounds shown in Scheme 1, L1 and L2.
the aspartate binding induces the displacement of the pyrocatechol
Squaraines are electrophiles because of their formally electron-
violet dye previously bound to the receptor. As an attractive
deficient central four-membered ring. For instance, they have been
alternative to these preferential coordination systems, a higher
proved to react with the nucleophilic anion cyanide.4 Another
selective amino acid signaling has been achieved when the inter-
interesting aspect with relation to squaraines is that they show a
action between the receptor and the amino acid does not rely
very intense band at long wavelengths and are usually highly
primarily on the coordination of the carboxylate-ammonium groups
but rather on the interaction with the side-chain residues of certain
amino acids. Following this approximation, three recent examples
have been described. The first one follows a “displacement”
approach and is based on the interaction of the imidazolate residue
of the histidine with a dimetallic Cu2+ complex of a bisdien
macrocycle.2d The other two are so-called “chemodosimeters”. One
displays a highly selective fluorescence signal due to coordination
of cysteine and cysteine derivatives (such as methionine, homocys-
teine, and glutathione) to a Pt(II) center, in a Ru(II)-Pt(II) trinuclear
complex2e and the liberation of the highly fluorescent cis-[Ru(phen)-
(CN)2]. Another recent example displays visual detection of cysteine
and homocysteine via thiazolidine formation with a xanthene dye.2f
We are interested in further exploring the high potential of the
“chemodosimeter” approach toward thiol-selective recognition and
fluorescent even in aqueous environments. Thus, both L1 and L2
show a band at ca. 640 nm (ꢀ ca. 1 × 105 mol cm-1) and have a
quantum yield in acetonitrile/water, 20:80 v/v, of ca. 0.1.
The reactivity of L1 and L2 was studied with amino acids. Besides
the presence of the amino group, natural R-amino acids also contain
in the side chain some other potentially reactive nucleophilic groups
such as alcohols (serine, threonine), amines (lysine, histidine), and
thiols (cysteine). In a typical assay acetonitrile/water, 20:80 v/v,
solutions of L1 ([L1] ) 1.21 × 10-5 mol dm-3) were buffered at
pH 6 (MES 0.01 mol dm-3), and 10 equiv of different natural
R-amino acids were added. The results are displayed in Figure 1
that shows a remarkable bleaching in the presence of cysteine. A
concomitant complete fluorescence quenching was also observed.
A very similar decoloration and fluorescence quenching was found
for L2.
These chromo- and fluorogenic effects are most likely due to
the thiol residue of the cysteine amino acid. Additional studies (see
† Universidad Polite´cnica de Valencia.
‡ Hospital de la Ribera.
9
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J. AM. CHEM. SOC. 2004, 126, 4064-4065
10.1021/ja031987i CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Determination of Total Aminothiols in Human Plasma
Using L1
determined total
aminothiols
added cysteine
measured
(mol dm-1 × 10-6
)
recovery
(%)
sample
(mol dm-1 × 10-6
)
(mol dm-1 × 10-6 a
)
1
306
196
390
196
390
494
710
535
730
98.4
102.0
94.2
2
372
95.8
a Known amounts of cysteine added to the plasma.
Figure 1. Photograph showing the color change in acetonitrile/water, 20:
80 v/v, at pH 6 (MES 0.01 mol dm-3) of the squaraine L1 ([L1] ) 1.21 ×
10-5 mol dm-3) in the presence of 10 equiv of certain amino acids. From
left to right and top to bottom: no amino acid, phenylalanine, threonine,
arginine, histidine, asparagine, leucine, alanine, proline, valine, glycine,
lysine, glutamine, methionine, isoleucin, serine, cysteine, tryptophan,
glutamic acid, and aspartic acid.
development of straightforward, low-cost, and undemanding probes
for the determination of biomarkers such as total aminothiols in
blood might be of general interest. Total cysteine and cysteine
derivatives in human plasma have been determined using L1 and a
colorimetric addition standard method with fine results (see Table
1 and Supporting Information).
In summary, L1 and L2 are specific fluoro-chromo chemodo-
simeters for thiol-containing compounds in aqueous environments.
The squaraine derivatives are quite simple, easy to synthesize, and
show favorable optical properties with absorption and emission not
far from the IR region. The squaraine derivatives have been
successfully applied to the determination of low-molecular mass
aminothiols in a complex multicomponent mixture such as human
plasma. The design principle utters the high potential applicability
of the chemodosimeter approach in the search for new or improved
chromogenic selective or specific probes for target biorelevant
molecules.
Scheme 2. Proposed Reaction between Squarine Derivatives and
Thiol-Containing Compounds
Acknowledgment. We thank the Ministerio de Ciencia y
Tecnolog´ıa (MAT2003-08568-C03-02, REN2002-04237-C02-01).
J.V.R.L. thanks the Ministerio de Educacio´n, Cultura y Deporte
for a Doctoral Fellowship. We would like to thank Prof. Angel
Maquieira for helpful discussion in the analytical determinations.
Supporting Information) showed that L1 and L2 gave decoloration
and fluorescence quenching in the presence of other thiol-containing
compounds, whereas alcohols, phenols, and amines (primary,
secondary, and tertiary) or sulfides produced no color changes. At
this neutral pH even known nucleophiles such as cyanide gave no
color variation. The bleaching process was tentatively assigned to
the reaction shown in Scheme 2 (see Supporting Information for
more details).
Additional studies were carried out to demonstrate the use of L1
or L2 as chromo-fluorogenic chemodosimeters. They could surely
be used for the postcolumn HPLC detection of thiol-containing
compounds. However, we took a further step and studied their
potential applicability for the direct determination of biorelevant
thiols (such as cysteine and cysteine derivatives, see below) in a
complex matrix such as human blood.
In plasma, cysteine can be found free or linked to other amino
acids, for instance, forming cysteine-glycine and glutathione. These
together with homocysteine are known as low-molecular mass
aminothiols. In blood, they are in a large percentage bound to
proteins, but after reduction they are found free in the plasma. Thus,
free low-molecular weight aminothiols in human plasma include
cysteine as the main component (ca. 83% of the total aminothiols)
and the cysteine derivatives, glutathione (ca. 3%), cysteine-glycine
(ca. 11%), and homocysteine (ca. 3%).5 L1 and L2 were found to
react with these aminothiols but not with other sulfur-containing
compounds such as sulfides or disulfides. Recently, the ability of
cysteine and cysteine derivatives to act as biomarkers has begun
to be explored, and their determination in biological fluids has
gained importance in clinical chemistry. For instance, the deter-
mination of aminothiols is important for the diagnosis of certain
diseases such as vascular disorders, arteriosclerosis, etc. Their
determination is usually carried out using rather sophisticated and
time-consuming chromatographic techniques.6 In this respect, the
Supporting Information Available: Synthetic procedures, reactiv-
ity studies of L1 and L2, and its use in the determination of total
aminothiols in human plasma. This material is available free of charge
via the Internet at http://pubs.acs.org.
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