A turn-on fluorescent indicator for citrate with micromolar sensitivity

Article abstract of DOI:10.1039/b711139h

A turn-on fluorescent indicator for citric acid (citrate) has been developed, displaying high emission enhancement (+1500%) and low interference by other carboxylates. The sensor is based on the non-emissive copper(ii) complex of a fluorescent amino amide, which, upon addition of citrate decomplexates to yield the emissive ligand. The detection limit estimated for this new chemosensing system is about 0.5 M. This novel approach to the analysis of citrate constitutes an alternative ca. 10²-10³ times more sensitive than the standard method based on the enzyme citrate lyase. The Royal Society of Chemistry.

Full text of DOI:10.1039/b711139h

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www.rsc.org/dalton | Dalton Transactions
A turn-on fluorescent indicator for citrate with micromolar sensitivity†
M. Isabel Burguete, Francisco Galindo,* Santiago V. Luis* and Laura Vigara
Received 20th July 2007, Accepted 2nd August 2007
First published as an Advance Article on the web 21st August 2007
DOI: 10.1039/b711139h
A turn-on fluorescent indicator for citric acid (citrate) has been developed, displaying high emission
enhancement (+1500%) and low interference by other carboxylates. The sensor is based on the
non-emissive copper(II) complex of a fluorescent amino amide, which, upon addition of citrate
decomplexates to yield the emissive ligand. The detection limit estimated for this new chemosensing
system is about 0.5 lM. This novel approach to the analysis of citrate constitutes an alternative ca.
102–103 times more sensitive than the standard method based on the enzyme citrate lyase.
1. Introduction
Citrate is an organic tricarboxylate playing important roles in the
biochemistry of living beings since it is one of the key components
of the Krebs cycle.1 Its concentration has a diagnostic value
as defective levels of citrate have been associated to prostate
cancer.2 Citrate has also been used as a molecular marker for
the diagnosis of urological diseases, since its concentration in
urine can be correlated to some pathological states, like, for
instance, nephrolithiasis (kidney stones),3 or glycogen storage
disease.4 Citrate concentration in this biomedical context is usually
determined by the citrate lyase (CL) spectrophotometric test,5
a well-established methodology.6 This test consists of a series
of enzymatic reactions by citrate lyase, and L-lactate/L-malate
dehydrogenases leading finally to the oxidation of NADH to
NAD+, the extent of which can be correlated to the citrate
concentration. Several variants of this method have been de-
veloped up until now.7 In contrast to other analytical methods
detecting citric acid,8 fluorescent sensors offer advantages in
terms of sensitivity, selectivity and response times.9 Recently some
researchers, pioneered by Anslyn, have focused their attention
to the supramolecular recognition and fluorescent sensing of
citrate, obtaining very remarkable results.10 Among those sensors,
those developed by Wolfbeis and Parker, based on europium
complexes are specially promising for biological imaging.11 In the
biomedical and food sciences the CL method is still the reference
procedure to analyze citrate,2,7,12 and consequently, any new optical
methodology to detect this analyte should be compared with it.
Here we present a new strategy to sense citrate in aqueous medium,
which is between 100 and 1000 times more sensitive than the CL
method.
Chart 1 Structures of 1 and its copper(II) complex 2.
complex 2 leads to a significant emission increase (more than
15-fold) with citrate but it is not so sensitive to the presence of
other carboxylates. This positive change takes place within the
micromolar range of citrate concentration, whereas the enzymatic
CL method is sensitive at millimolar concentrations of this anion.
Hence, we believe that 2, and other related complexes to be
developed in the future, could be a competitive alternative for
the analysis of citrate (especially for fluorescent microscopy and
flow cytometry where high sensitivity is needed).
2. Results and discussion
2.1. Synthesis and characterization
The synthetic route to obtain ligand 1 was adapted from similar
syntheses carried out in our group,14 and is outlined in Chart
2. The N-Cbz protected amino acid L-phenylalanine 3 was
activated with N-hydroxysuccinimide to yield the activated ester 4.
Coupling with 1-aminoanthracene afforded compound 5, which
In the course of our research on fluorescent peptidomimetic
molecules,13 we found that the copper(II) complex of amino amide
1 derived from L-phenylalanine (complex depicted in Chart 1 as
2), is stable in aqueous-methanolic solutions (pH 7.5–8.0) but
is very sensitive to traces of citrate. Thus, the non-fluorescent
Departamento de Qu´ımica Inorga´nica y Orga´nica, Unidad Asociada de Ma-
teriales Orga´nicos Avanzados (UAMOA), Escuela Superior de Tecnolog´ıa
y Ciencias Experimentales, Universitat Jaume I-CSIC, Avda. Sos Baynat,
s/n, E-12071, Castello´n, Spain. E-mail: francisco.galindo@uji.es (F.G.),
luiss@uji.es (S.V.L.); Fax: +34 964 728214; Tel: +34 964 728751
† Electronic supplementary information (ESI) available: Fig. S1–S5. See
DOI: 10.1039/b711139h
Chart 2 Synthetic route to prepare complex 2.
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was deprotected to yield ligand 1 as the bromohydrate salt.
Marchelli and co-workers have reported that a-amino amides
derived from natural amino acids similar to 1 form stable 2 : 1
(ligand : metal) complexes with Cu(II), yielding in many cases
crystals suitable for X-ray analysis.15 Those structures show a
head-to-tail configuration of the ligands around the Cu(II) center
with a N4 coordination sphere. Moreover, Burrows,16 Polt17 and
Fenton18 have described the formation of metallic complexes with
other organic ligands containing amino amide functionalities. In
all the cases, a basic medium for the deprotonation of the amides
and complex formation was reported to yield neutral complexes.
Hence, 2 was obtained by reaction of two equivalents of 1 and one
equivalent of Cu(II) chloride in basic methanol affording a green
brilliant solid, in 51% yield. The structure postulated in Chart 1
is presented as tentative according to the analogous complexes
described in the literature15–18 and to the following experimental
evidence.
Upon complexation the carbonyl frequency (FT-IR) shifted
from 1663 cm−1 in 1 to 1591 cm−1 in 2 (see ESI†), indicating the
participation of the deprotonated amide group in the coordination
to the metallic cation and in agreement with related systems.16,17,18
The absorption spectrum of 1 (Fig. 1) displays the typically
resolved anthracenic band between 350 and 400 nm, with a
maximum at 366 nm (6247 M−1 cm−1). The absorption spectra
of 1-aminoanthracene consists of a broad non-structured band
between 340 and 440 nm.19 Thus, the pair of electrons at the
nitrogen attached to the fluorescent group are not delocalized
in the anthracene moiety but in the amide group. This fact is
supported by the fluorescence spectrum of 1 (Fig. 1) which displays
a maximum at 418 nm, whereas that of 1-aminoanthracene occurs
at 470 nm.19 The fluorescence quantum yield of 1 was recorded,
with excitation at 356 nm, yielding a value of φ = 0.20 0.02, both
under air and nitrogen atmospheres (using anthracene in degassed
ethanol [φ = 0.27] as standard20).
Fig. 2 Determination of the complex stoichiometry by means of a
fluorescence Job Plot, where f indicates the molar fraction of ligand in
a mixture of ligand and metal, with a total constant concentration of
species of 4 × 10−5 M; water : methanol, 3 : 7, v : v; pH 7.9, NaCl 0.15 M.
k exc = 370 nm.
of 0.7 which indicates the existence of a complex with a 2 : 1
ligand to metal ratio. On the other hand the absorption Job-
Plot (Fig. 3) also showed an inflexion point at 0.4. This fact
could be associated to the presence in aqueous solution of a
minor amount of 1 : 1 complex (theoretical f = 0.5). Thus, it
is reasonable to assume that complex 2 in aqueous solution is in
equilibrium with other species. As reported by Dallavalle,22 who
studied several a-amino amides in the presence of Cu(II), a variety
of species can exist in solution. At pH 7.5–8.0, for instance, the
prolinamide (ligand L) + Cu(II) system is comprised of CuL2H−2
+
as the major species in equilibrium with CuL2H−1+, CuLH−1
,
CuL2 and CuL 2+ as minor components.
2+
Fig. 1 Comparison between absorption spectra of (a) complex 2 and (b)
ligand 1; and between fluorescence spectra of (c) complex 2 and (d) ligand
1 (k exc = 370 nm); water : methanol, 3 : 7, v : v; pH 7.9, NaCl 0.15 M.
Fig. 3 Determination of the complex stoichiometry by means of an
absorption Job Plot, where f indicates the molar fraction of ligand in
a mixture of ligand and metal, with a total constant concentration of
species of 4 × 10−5 M; water : methanol, 3 : 7, v : v; pH 7.9, NaCl 0.15 M.
The stoichiometry of the complex in solution has been deter-
mined to be 2 : 1 (ligand : metal), using the method of continuous
variations21 (Job-Plot) with the absorption and emission spectra,
in agreement with structures of related complexes as reported
by Marchelli.15 From the fluorescence Job-Plot (Fig. 2), a sharp
variation of emission was recorded at a ligand molar fraction (f )
Mass spectrometric analysis of 2 using the Fast Atom Bom-
bardment (FAB) technique allowed to record a peak with m/z =
741.2, which corresponds to the expected mass [M]+ of the complex
(see ESI†). Additional mass spectra were recorded (Electrospray
Ionization, ESI) in order to gain further evidence on the structure
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of the complex 2. Thus, in negative mode and in the presence
of KCl, the peak m/z = 776.2, corresponding to [M + Cl]− was
recorded. In positive mode, the peaks m/z = 742.4 [M + H]+ and
780.4 [M + K]+ were detected. No evidence for the 1 : 1 complex
formation could be obtained by either of the MS techniques.
In order to further confirm the tetracoordination of the metal
center in complex 2 by 1, with the amides in the deprotonated
form, the reactants 1·HBr and Cu(II) chloride were mixed (0.4 mM
and 0.2 mM respectively in MeOH) in a spectrophotometric cell.
Aliquots of base (KOH in MeOH) were added stepwise. For the
formation of 2, a double deprotonation of 1·HBr is needed (one
for the HBr and the other one for the amide group). Since there are
two ligands in complex 2, then four equivalents of base, relative
to Cu(II), would be required to form the final complex. Fig. 4
and 5 show how the reaction of 2 equivalents of 1·HBr with
1 equivalent of Cu(II) is complete after the addition of exactly
four equivalents of KOH. The same result was obtained using
fluorescence spectroscopy (see ESI†).
2.2. Analytical determinations
Titrations of 2 with citrate were carried out by addition of small
aliquots of concentrated analyte (trisodium citrate) to a solution
of complex 2 (2 × 10−5 M; water : methanol, 3 : 7, v : v; pH 7.9;
NaCl 0.15 M).23 The non fluorescent solution of 2 became highly
emissive as shown in Fig. 6 upon addition of citrate anion. The
presence of citrate was detectable even to the naked eye under
UV illumination (365 nm) as can be seen in the inset of Fig. 6.
This visual feature is especially important for a probe to be useful
for microscopy, for which no satisfactory fluorescent probe is
commercially available to date. The sensitivity of the complex was
evaluated by means of the corresponding titration with citrate over
a wide range of concentrations (from 0.1 lM to 100 lM) as can
be seen in Fig. 7. For comparison, the CL method was also tested
according to the standard commercial protocol6 (from 0.1 lM to
10 mM) and the results are also plotted in Fig. 7. As is clearly
shown, the method using complex 2 is sensitive to concentrations
of citrate almost three orders of magnitude lower than the CL
methodology.
Fig. 6 Fluorescence titration of 2 (2 × 10−5M; water : methanol, 3 : 7,
Fig. 4 Change of the absorption spectra of a mixture of 1·HBr (0.4 mM)
and CuCl2 (0.2 mM) upon addition of KOH, in MeOH.
v : v; pH 7.9; NaCl 0.15 M) with citrate. k
= 370 nm. Upper curve
exc
corresponds to a final citrate concentration of 4 × 10−5M. Inset: (a) initial
and (b) final solutions (365 nm exc.).
Fig. 5 Change of the absorbance at 349, 366, 400, and 425 nm of a
mixture of 1·HBr (0.4 mM) and CuCl2 (0.2 mM) upon addition of KOH,
in MeOH.
Fig. 7 Comparative sensitivity towards citrate of: CL method (dashed
line, absorption measurements) and complex 2 (solid line, fluorescence
measurements).
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A representation of the lower concentration range revealed
linearity up to 10 lM of citrate, with a R = 0.99764 (Fig. 8).24 This
calibration allowed the determination of several concentrations of
citrate not available directly to the CL test (known concentration
of prepared solutions are given in brackets): 3.2 (3.6), 4.3 (4.1), 6.6
(6.1), 9.0 (8.2) lM.
It was found that the intensity reached about 40% of that recorded
with citrate.
From a mechanistic point of view the results can be explained
invoking the decomplexation of non emissive 2 to yield free
fluorescent 1 and copper citrate species (a complex mixture as
reported previously26). In fact, the fluorescence quantum yield
of the final solution, after titration of 2 with citrate, is φ =
0.22
0.02, which matches, under the experimental error, the
value of 1 measured independently (φ = 0.20 0.02). The absence
of emission in 2 is in agreement with the internal fluorescence
quenching reported for many emissive ligands when complexed
with copper(II) (chelation enhanced quenching (CHEQ) effect).27
In order to confirm the metal stripping from complex 2,
EDTA (ethylenediaminetetraacetic acid), a stronger chelator than
citrate,28 was added to a solution of 2. As expected, the non
fluorescent solution of 2 became highly emissive upon addition
of 40 lM of EDTA. Attempts to analyze mathematically the
titration curves of 2 vs carboxylates resulted in poor fittings,
probably as a result of the complex equilibria of species taking
place in aqueous solution. A potentiometric study is probably
needed in order to shed some light into the thermodynamics of
the complexation–decomplexation process and this is expected to
be carried out in future studies. The literature reports an analogous
decomplexation-induced fluorescence enhancement, reported by
Reymond, to monitor protease activities,29 in which the stripping
of Cu(II) is the basis of the signalling mechanism.
Fig. 8 Calibration curve for 2 (2 × 10−5M; water : methanol, 3 : 7, v :
v; pH 7.9; NaCl 0.15 M). Fluorescence intensity at 418 nm vs citrate
concentration (R = 0.99764). k exc = 370 nm.
The selectivity of 2 for citrate determination was evaluated by
the corresponding fluorescence titrations with other carboxylates.
No significant fluorescence increase took place with succinate,
acetate, lactate, glutarate and fumarate (up to 40 lM). With
mandelate, tartrate and malate, a small fluorescence enhancement
of ca. 10–15% occurred (Fig. 9). The selectivity for citrate over
malate is specially important for biomedical and food analysis
since both species are commonly found in many real samples.
This causes mutual interferences in analytical protocols, and to
avoid this problem new methodologies are continuously under
development.25 The amino acid L-glutamate was also tested
provided that it is a potential tri-coordinating species (two
carboxylates and one amino group) as citrate (three carboxylates).
2.3. Combinatorial experiments
Encouraged by the results obtained with the copper(II) complex
of amino amide 1, we decided to test, in a combinatorial fashion,
the same amino amide with a series of metals and carboxylates.
The purpose was to find, at least qualitatively, some other metallic
complexes capable to give fluorogenic sensing of citrate (or other
anion). Solutions of the following metallic cations were placed in
the B–H rows of a 96-well plate (as chloride salts): Cu(II), Ni(II),
Zn(II), Cd(II), Co(II), Cu(I) and Mg(II). The ligand 1 was added
to each of the 96 wells. The most appropriate medium for this
combinatorial-qualitative experiment was found to be a 2 : 1 (v :
v) water : methanol mixture, at pH 7.4 (HEPES 6 mM, NaCl
6 × 10−2 M).30 Finally the following carboxylates were added to
columns 2–12 of the plate : acetate, glutarate, malonate, succinate,
tartrate, citrate, lactate, benzoate, ftalate, fumarate and maleate.
As can be seen in Fig. 10, the unique metallic cations capable
to induce quenching of 1, at pH 7.4, were Cu(II) and Cu(I) (see
column 1 in Fig. 10). Moreover, the unique carboxylate capable of
restoring the fluorescence (visually) was citrate. If Cu(II) and Cu(I)
were compared, the emission quenching was much more effective
with the divalent cation.
The apparent inability of other known quenchers of fluores-
cence, like nickel(II) for instance, to form a complex with 1 must
be attributed to the mild pH conditions (pH 7.4) selected to carry
out the experiment. Recording the fluorescence spectra of 1 at
different pH and in the presence of Cu(II), Ni(II) or Zn(II) revealed
that complexation of 1 by Ni(II) in fact can occur, but at a pH more
basic than in the case of Cu(II), in agreement with other amino
amide metallic complexes.31 On the other hand, Zn(II) is not able
to quench at any pH the emission of 1, which is fluorescent over the
whole range of pH (see ESI†). We are currently investigating the
Fig. 9 Fluorescence response of 2 (2 × 10−5M; water : methanol, 3 :
7, v : v; pH 7.9; NaCl 0.15 M) for citrate and other carboxylates (final
concentration: 40 lM).
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Micromass Instruments). Infrared spectra were recorded in a
Perkin-Elmer 2000 FT-IR spectrometer. UV-vis absorption spec-
tra were recorded in a Hewlett-Packard 8453 apparatus. Steady-
state fluorescence spectra were acquired in a Spex Fluorolog 3–
11 equipped with a 450 W xenon lamp. Emission spectra were
obtained from air equilibrated samples (otherwise stated) exciting
at 370 nm, in right angle mode and using 1 × 1 cm (3 ml) quartz
cells. The curves were processed with the appropriate correction
files. Excitation spectra were also recorded in order to assure
that no impurities were responsible for the recorded emissions.
All measurements were performed at 295 K. Emission quantum
yields were determined using anthracene in degassed ethanol as
standard.20 All the absorptions were maintained below Abs = 0.10.
The samples and the reference were excited at the same wavelength
of 356 nm (iso-absorptive) and the emission spectra were corrected
to take into account the refractive indices of each medium.
Fig. 10 Schematic representation of the combinatorial experiment carried
out with 1. Final concentrations: ligand 1: 6 × 10−5 M. Metallic salts: 3 ×
10−5 M. Carboxylates: 1.2 × 10−4 M. Medium: water : methanol, 2 : 1
(v : v) pH 7.4 (HEPES 6 mM, NaCl 6 × 10−2 M). Volume in each well:
300 lL. Color code: blue indicates strong fluorescence upon illumination
(365 nm), grey indicates complete quenching, and cyan indicates partial
quenching. The yellow arrow points to the maximum change of emission
from non-emissive complex to released ligand upon addition of citrate.
4.2. Synthesis
The synthesis of the N-hydroxysuccinimide ester 4 starting from
commercially available 3 was made as described previously,14 with
a yield of 86%. Compound 4 (5.0 g, 12.6 mmol) was dissolved in
anhydrous THF (40 ml) and 1-aminoanthracene (2.7 g, 12.6 mmol)
dissolved in dry THF (10 ml) was added slowly with stirring. The
reaction mixture was refluxed for 8 h under an inert atmosphere.
The solid formed was filtered off and washed with basic water,
neutral water and hot 2-propanol. The product 5 (dark green) was
dried under reduced pressure (60–70 ◦C) for 24 h. Yield: 4.2 g,
70%. mp 236–238 ◦C. 1H NMR (500 MHz, DMSO-d6) d 3.04 (dd,
1H, J = 12.7, 9.7 Hz), 3.22 (dd, 1H, J = 13.4, 5.5 Hz), 4.75 (m,
1H), 5.06 (s, 2H), 7.25 (m, 1H), 7.33 (m, 1H), 7.43 (d, 6H, J =
7.1 Hz), 7.50 (t, 2H, J = 7.8 Hz), 7.54 (dd, 2H, J = 6.4, 2.9 Hz),
7.61 (d, 1H, J = 7.0 Hz), 7.80 (d, 1H, J = 8.0 Hz), 7.95 (d, 1H,
J = 8.5 Hz), 8.00 (m, 1H), 8.10 (m, 1H), 8.53 (s, 1H), 8.60 (s, 1H),
10.20 (s, 1H); 13C NMR (125 MHz, DMSO-d6) d 37.6, 56.7, 65.3,
120.6, 121.3, 124.9, 125.6, 125.7, 126.2, 126.3, 127.5, 127.6, 127.7,
128.0, 128.2, 129.3, 130.7, 131.0, 131.7, 133.0, 136.9, 137.7, 156.0,
171.2. FTIR (KBr) 3269, 1684, 1653, 1531 cm−1. Anal. calcd. for
C31H26N2O3: C, 78.5; H, 5.5; N, 5.9; found. C, 78.1; H, 5.8; N, 5.9.
ESI-MS m/z = 497.2 [M + H]+, 513.3 [M + Na]+.
analytical applicability of new combinations of fluorescent ligands
and metallic quenchers.
3. Conclusion
In summary, a new strategy for citrate sensing in aqueous medium
with micromolar sensitivity has been described. This methodology
for sensing citrate, based on a “decomplexation-induced fluores-
cence enhancement” process (or fluorescence restoration of a latent
fluorescent ligand), can constitute a competitive alternative to the
citrate lyase based methodologies.32 The concept is not exclusive
to Cu(II) amino amidates as described in this paper but could
be expanded in the future to other complexes and analytes, just
by choosing the appropriate combination of ligand, metal and
working pH.
Synthesis of
1
[(S)-2-amino-N-(anthracen-1-yl)-3-phenyl-
4. Experimental
propanamide]: compound 5 (3.0 g, 6.3 mmol) was added to
HBr/AcOH (33%) (14 ml) and the mixture was stirred at rt until
CO2 evolution ceased. At this point, diethyl ether was added to
the solution, which led to the deposition of a green precipitate.
This was filtered off and washed with additional ether. Dark green
4.1. Materials and methods
All commercially available reagents (Aldrich or Fluka) were
used without further purification. Solvents for reactions were
distilled over an adequate drying agent. Water for fluorescence
◦
1
solid (1·HBr). Yield (2.6 g, 97%); mp (dec.) 210 C; H NMR
(500 MHz, DMSO-d6) d 3.28 (m, 1H), 3.40 (bs, 1H), 4.55 (s, 1H),
7.33 (m, 1H), 7.40 (m, 4H), 7.51 (t, 1H, J = 7.8 Hz), 7.57 (m,
2H), 7.61 (d, 1H, J = 7.1 Hz), 7.98 (d, 1H, J = 8.5 Hz), 8.00 (d,
1H, J = 8.0 Hz), 8.10 (d, 1H, J = 8.0 Hz), 8.27 (s, 1H), 8.50 (bs,
3H), 8.61 (s, 1H), 10.45 (s, 1H); 13C NMR (125 MHz, DMSO-d6)
d 37.3, 54.1, 120.7, 121.1, 124.9, 126.0, 126.1, 126.2, 126.5, 127.3,
127.9, 128.1, 128.6, 129.7, 130.8, 131.1, 131.6, 131.9, 134.9, 167.5.
FTIR (KBr) 3425, 3028, 1663 (carbonyl), 1549 cm−1. Anal. calcd.
for C23H21BrN2O: C, 65.6; H, 5.0; N, 6.7; found. C, 65.2; H, 5.4; N,
6.6. ESI-MS m/z = 341.3 [M − Br]+. UV-vis absorption (MeOH),
k (e, M−1 cm−1) 349 nm (4830), 366 nm (6247), 385 nm (5192);
Fluorescence emission (MeOH), kmax = 418 nm (exc. at 370 nm).
R
measurements was Milliporeꢀ quality. MeOH for fluorescence
determinations was spectroscopic grade. The enzymatic analysis
of citrate was performed using the citrate lyase kit commercialized
by Boehringer Mannheim and according to the protocol described
by the manufacturer.
NMR spectra were recorded on a Varian INOVA 500 spec-
trometer (500 MHz for 1H and 125 MHz for 13C). Chemical shifts
are reported in ppm using residual undeuterated solvent peaks
as internal standards. Mass spectra (ESI) were recorded on a
Micromass Quattro LC spectrometer equipped with an electro-
spray ionisation source and a triple-quadrupole analyzer. FAB-
mass spectrum was recorded in a VG Autospec (VG Analytical,
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The synthesis of complex 2 (bis-[(S)-2-amino-N-(anthracen-1-
yl)-3-phenylpropanamidato] copper(II)) was made analogously to
similar complexes described in the literature.15–18 Ligand 1·HBr
(0.22 g, 0.427 mmol) and CuCl2·2H2O (0.04 g, 0.214 mmol) were
dissolved in MeOH (40 ml) (no color change was observed).
Then KOH (0.05 g, 0.854 mmol) was added and the solution
became darker immediately. The mixture was refluxed for 1 h. The
solution was concentrated under reduced pressure and the crude
washed exhaustively with hot water. The complex 2 was dried
under vacuum (65 ◦C, 20 h). Brilliant green crystals were obtained
(not suitable for X-ray diffraction) soluble in DMSO and partially
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◦
soluble in alcohols. Yield (0.081 g, 51%). mp 193–195 C; FTIR
(KBr) 3437, 3047, 1591 (carbonyl), 1455 cm−1; UV-vis absorption
(MeOH), k (e, M−1 cm−1) 351 nm (6900), 371 nm (8850), 393 nm
(9650), 425 nm (2525). FAB-MS (positive) m/z 741.20 [M]+, ESI-
MS (positive) m/z 742.4 [M + H]+, 780.4 [M + K]+; ESI-MS
(negative) m/z 776.2 [M + Cl]−. The global yield for the synthesis
of 2 starting from 3 was 30%.
Acknowledgements
Financial support from the Spanish Ministerio de Edu-
cacio´n y Ciencia (MEC, projects CTQ2006-15672-C05-02/BQU,
CTQ2006-26251-E/BQU [Programa Explora]), Generalitat Va-
lenciana (projects GV/2007/277 and ARVIV/2007/079), and
Fundacio´ Caixa Castello´-UJI (project P1·1B2004–38) is acknowl-
edged. F. G. thanks the financial support from MEC (Ramo´n y
Cajal Program). L. V. thanks the financial support from MEC
(FPU fellowship).
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4032 | Dalton Trans., 2007, 4027–4033
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