A fluorescent molecular ruler as a selective probe for ω-aminoacids

Article abstract of DOI:10.1039/c0cc05454b

A fluorescent probe for ω-aminoacids behaves as a molecular ruler, changing the yellow fluorescent emission into blue as a function of the distance between the terminal ammonium and the carboxylate groups, permitting the quantitative detection of ω-aminoa

Full text of DOI:10.1039/c0cc05454b

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A fluorescent molecular ruler as a selective probe for x-aminoacidsw
´ ´
´
Daniel Moreno, Jose V. Cuevas, Gabriel Garcıa-Herbosa and Tomas Torroba*
Received 8th December 2010, Accepted 17th January 2011
DOI: 10.1039/c0cc05454b
A fluorescent probe for x-aminoacids behaves as a molecular
ruler, changing the yellow fluorescent emission into blue as a
function of the distance between the terminal ammonium and the
carboxylate groups, permitting the quantitative detection of
x-aminoacids, their metabolites and related drugs, such as
pregabalin or gabapentin, from pharmaceutical formulations.
The concept of a molecular ruler has been very fruitful in the
biological sciences for measuring distances between groups of
biomolecules in dynamic processes,1 and in materials chemistry
for investigating distance-dependent emission behaviours
of fluorophores2 or super-resolution microscopy.3 Molecular
rulers of low molecular weight have been much less studied; in a
relevant example, a molecular ruler was used to unambiguously
assign two stereoisomers of bis-porphyrins.4 This simple con-
cept may have useful applications for the detection of natural
aminoacids but it was never applied before. Except for the
reactive cysteine and related thiol-containing aminoacids,5
fluorescent probes for selective recognition of unprotected
aminoacids are uncommon,6 as well as probes detecting protected
aminoacids,6a,7 and are mostly oriented to the selective
recognition of natural aminoacids and their unnatural enantio-
mers. We have applied the molecular ruler concept to the study
of the interactions of proteinogenic as well as non-proteinogenic
aminoacids and a classic bis-urea receptor tagged with two units
of a new highly solvatochromic fluorescent indicator. In this
communication we report our findings in the selective fluores-
cent discrimination between different classes of o-aminoacids
and their zwitterionic metabolites.
Scheme 1 Synthesis of fluorescent probes 6 and 8.
permitted its insertion in classic receptors. Reaction of 4 with
bis-isocyanate 5 in chloroform for 2 days at room temperature
afforded bisurea 6 in 75% yield. Monourea 8 was obtained in
70% yield by the reaction of 4 with isocyanate 7 in similar
conditions. Compounds 3–4, 6 and 8 were fully characterized by
the usual analytical and spectroscopic techniques (see ESIw). As
compound 4, bisurea 6 is a pale yellow compound with a bright
yellow fluorescence. Bisurea 6 did not undergo any physical
change in the presence of common cations in MeCN or DMSO
but its behaviour in the presence of common anions depended
on the solvent. In pure MeCN a 10À4 M solution of 6 increased
the light yellow colour as the fluorescence was quenched in the
presence of FÀ, BzOÀ, H2PO4À, AcOÀ and CNÀ, a common
behaviour of all coloured aromatic ureas.8 Unexpectedly, the
changes were the opposite in DMSO. In pure DMSO a 10À4 M
solution of 6 decreased its intense yellow colour as the fluoreÀs-
The synthesis of fluorescent probes is depicted in Scheme 1.
Thus, the Suzuki reaction of aryl boronate 1 and 5-bromo-
indanone 2 catalyzed by Pd(PPh3)4 in toluene/butanol/water
in the presence of CsCO3 gave aminoketone 3 in 91% yield.
The Knoevenagel reaction of 3 and malononitrile in the presence
cence was shifted to blue in the presence of FÀ, BzOÀ, H2PO4
,
AcOÀ and CNÀ as Bu4N+ salts. The changes were simÀilar in
DMSO/water 9 : 1, although the sensitivity to H2PO4 was
lost. Fluorescent titration and fitting afforded binding con-
stants for 1 : 1 complexes, confirmed from Job’s plot analysis,
that decreased slightly for FÀ and CNÀ from DMSO to
DMSO/H2O 9 : 1 but increased or were unchanged for BzOÀ
and AcOÀ from DMSO to DMSO/H2O 9 : 1 (see ESIw),
indicating that 6 is a good probe for carboxylates in mixed
solvents. To exploit the carboxylate sensitivity in zwitterions,
we tested 10À4 M solutions of 6 with aqueous solutions of 16
common a-aminoacids. As aminoacids were added, the yellow
fluorescence was clearly shifted to blue in the presence of two or
more equivalents of glutamine, lysine, asparagine and arginine,
all having in common a nitrogen-including functionality at
some distance of the carboxylate function.
of DABCO in toluene at reflux for 2 h gave aminoindane 4 in
abs
95% yield. Compound 4 is a pale yellow (l
= 398 nm,
max
e = 34 500 MÀ1 cmÀ1, in CH2Cl2), highly solvatochromic
fluorescent indicator (lemmax = 537 nm, lmexacx = 366 nm, F = 0.6
in CH2Cl2) (see ESIw), with a reactive amino group that
´
Departamento de Quımica, Facultad de Ciencias,
Universidad de Burgos, 09001 Burgos, Spain.
E-mail: ttorroba@ubu.es; Fax: +34 947258831; Tel: +34 947258088
w Electronic supplementary information (ESI) available: Experimental
procedures, physical and spectral data of new products, fluorimetric
studies, NMR titrations and calculations. See DOI: 10.1039/c0cc05454b
c
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Chem. Commun., 2011, 47, 3183–3185 3183

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Fig. 1 Effect of the addition of 3 equiv. of aminoacids in water to
10À4 M solutions of 6 in DMSO: L (Leu), M (Met), S (Ser), T (Thr),
C (Cys), P (Pro), N (Asn), Q (Gln), F (Phe), Y (Tyr), W (Trp), K (Lys),
R (Arg), H (His), D (Asp), E (Glu) (lexc = 366 nm).
Fig. 3 Plot of log K1–2 for 2 : 1 complexes of o-aminoacids and 6.
There was no change in the presence of even 3 equivalents of
common basic a-aminoacids such as proline, hystidine or
tryptophan (Fig. 1). Fluorescent titrations of 6 (10À4 M in DMSO)
and aqueous aminoacid solutions showed decreasing of the
intensity of emission as aminoacids were added. Titration
profiles fitted satisfactorily 1 : 1 binding models, from which
binding constants were calculated (log Ka1rginine = 3.93 Æ 0.05,
log Ka1sparagine = 3.93 Æ 0.04, log Kg1lutamine = 4.17 Æ 0.04,
log Kl1ysine = 3.66 Æ 0.04). Job’s plot analyses of fluorescence
titration with lysine or asparagine revealed a maximum at a
50% mole fraction, in accord with the proposed 1 : 1 binding
stoichiometry (see ESIw). In contrast, the less fluorescent
monourea 8 was also sensitive to the same anions but lacked
any kind of selectivity in the presence of aminoacids.
increases from b-alanine to 6-aminohexanoic acid and then
decreases slightly for 7-aminoheptanoic acid. This fact indicates
that 6 behaves as a molecular ruler for carboxylates having an
ammonium group at increasing distances from the carboxylate
function. Typical titration curves for 6 and GABA are given in
Fig. 4. Job’s plot analysis of fluorescence titrations with
6-aminohexanoic acid revealed a maximum at a 66% mole
fraction, in accord with the proposed 2 : 1 binding stoichiometry
(Fig. 4). GABA is an important metabolite, some significant
drugs such as pregabalin9 and gabapentin10 are based on GABA.
Because its similarity to GABA, pregabalin and gabapentin can
be selectively detected from their pharmaceutical formulations
and quantitatively analyzed from their binding constants with 6
(Table 2), measured from fluorescent titrations of their aqueous
solutions and 6.
We then extended the study of the changes in fluorescence of
6 in the presence of metabolic o-aminoacids and related
metabolites or drugs. Thus, while consecutive addition of
3 equivalents of alanine in water did not promote any change,
addition of 3 equivalents of aqueous solutions of b-alanine,
g-aminobutyric acid (GABA), 5-aminovaleric acid, 6-amino-
hexanoic acid or 7-aminoheptanoic acid resulted in dramatic
changes of fluorescence from yellow to blue (Fig. 2) after the
second equivalent.
Quantitative fluorescent titration of 10À4 M solutions of 6 in
DMSO and o-aminoacids (lexc = 390 nm) showed decreasing
of the intensity of emission centred at 557 nm as aminoacids
were added. Titration profiles best fitted 2 : 1 binding models,
from which association constants were calculated (Table 1).
The graphical representation of the values of the log K1À2
gives the tendencies for the homologous series of the o-aminoacids
(Fig. 3). Thus, the value of the first constant decreases from
b-alanine to 6-aminohexanoic acid and then increases for
7-aminoheptanoic acid, but the value of the second constant
Fig. 4 (top) Fluorescence titration curves and titration profile of a
10À4 M solution of 6 in DMSO with an aqueous solution of GABA.
(bottom) Job’s plot of 6 (10À4 M, DMSO) and 6-aminohexanoic acid.
Table 2 Binding constants for complexes from fluorescence titrations
of 10À4 M solutions of 6 in DMSO and drugs or metabolites in water
Fig. 2 Effect of the addition of 3 equiv. of aminoacids or aminoacid
metabolites in water to 10À4 M solutions of 6 in DMSO: A (Ala), B
(b-alanine), G (g-aminobutyric acid, GABA), 7 (5-aminovaleric acid),
8 (6-aminohexanoic acid), 9 (7-aminoheptanoic acid), 10 (sarcosine),
11 (creatinine), 12 (creatine), 13 (L-carnitine) (lexc = 366 nm).
6
Gabapentin
Pregabalin
Carnitine
Creatine
log K1
log K2
3.91 Æ 0.29
3.01 Æ 0.26
2.90 Æ 0.03
3.06 Æ 0.09
4.01 Æ 0.13
3.80 Æ 0.06
3.33 Æ 0.02
—
Table 1 Binding constants for 2 : 1 complexes from fluorescence titrations of 10À4 M solutions of 6 in DMSO and o-aminoacids in water
6
b-Alanine
GABA
5-Aminovaleric
6-Aminohexanoic
7-Aminoheptanoic
log K1
log K2
4.49 Æ 0.02
3.93 Æ 0.09
4.15 Æ 0.02
4.10 Æ 0.07
3.98 Æ 0.04
5.17 Æ 0.05
3.70 Æ 0.06
5.58 Æ 0.07
4.18 Æ 0.05
5.51 Æ 0.06
c
3184 Chem. Commun., 2011, 47, 3183–3185
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Some important aminoacid metabolites, commonly used as
nutritional supplements, such as L-carnitine11 or creatine,12
have structural features suitable for their detection by 6. There-
fore, we tested 10À4 M solutions of 6 with their aqueous
solutions. As metabolites were added, the yellow fluorescence
was clearly shifted to blue in the presence of two or more
equivalents of L-carnitine or creatine. There was no change in
the presence of even 3 equivalents of sarcosine or creatinine
(Fig. 2). Fluorescent titration of 6 (10À4 M in DMSO) and
aqueous creatine or L-carnitine solutions showed decreasing of
the intensity of emission as metabolites were added. Titration
profiles fitted a 1 : 1 binding model for creatine and a 2 : 1
binding model for L-carnitine, from which binding constants
were calculated (Table 2). Job’s plot analysis of fluorescence
titrations with L-carnitine revealed a maximum at a 66% mole
fraction, in accord with the proposed 2 : 1 binding stoichio-
the distance between the terminal ammonium and the carboxylate
groups. At lexc = 390 nm only decreasing of the yellow emission
was measured but by tuning the lexc to 366 nm the blue
fluorescence was seen (under the TLC UV-light) or measured in
the spectrofluorometer (see ESIw). Moreover, the sensitivity to
the detected metabolites is preserved in a large extension by
performing the experiments in the presence of several equivalents
of the undetected aminoacids (see ESIw), thus making the system
suitable for the quantitative detection of o-aminoacids and their
metabolites from nutritional supplements, and some important
drugs related to them, such as pregabalin or gabapentin, from
their pharmaceutical formulations.
We gratefully acknowledge financial support from the
Ministerio de Ciencia e Innovacio
12631-BQU), Junta de Castilla y Leo
cion y Cultura y Fondo Social Europeo (Projects BU023A09
´
n, Spain (Project CTQ2009-
´
n, Consejerıa de Educa-
´
´
metry (see ESIw). We calculated detection limits of 10À4
M
and GR170).
solutions of 6 in DMSO, calculated in fluorescence emission
by the blank variability method,13 and selected metabolites
in water, that were 1.93 Â 10À7 M for L-asparagine, 3.09 Â
10À6 M for GABA and 1.38 Â 10À6 M for L-carnitine. We also
Notes and references
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1
carried out H NMR titration experiments of a 10À1 M solu-
tion of 6 with b-alanine, both in DMSO-d6. By addition of
increasing amounts of b-alanine, the two urea NH signals at
d 8.8 and 6.7 moved to a lower field and progressively disappeared
as the H-bonds were formed. The aromatic signals became
more complicate but their chemical shifts remained unchanged
(see ESIw). Apparently, formation of the 1 : 1 and 2 : 1 complexes
affected the urea protons and restricted rotation of the
aromatic rings. With this in mind, we optimized the geometries
of 1 : 1 complexes between 6 and zwitterionic GABA or
pregabalin, stabilized by 3 molecules of DMSO, at the ONIOM
(B3LYP/6-31G*:AM1) level by using GAUSSIAN 03 (Fig. 5 for
the 6 : GABAÁ3DMSO complex).14
The presence of at least 3 molecules of DMSO was necessary
in both cases to stabilize the zwitterionic structures of amino-
acids, otherwise additional hydrogen bonds between the
ammonium and the carboxylate groups were produced during
the calculation, with proton transfer from the ammonium to
the carboxylate groups. The structures converged to stationary
points having four hydrogen bonds between the urea NH
protons and the carboxylate groups that agreed with the
1H-NMR titration results and accounted for the interactions
that gave rise to the molecular ruler effect.
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Fig. 5 Optimized geometry of the 6 : GABAÁ3DMSO complex at the
ONIOM (B3LYP/6-31G*:AM1) in GAUSSIAN 03.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 3183–3185 3185