Synthetic tripodal squaramido-based receptors for the complexation of antineoplastic folates in water

Article abstract of DOI:10.1002/ejoc.201100855

Three suitably oriented squaramido ammonium groups form the basis of the first abiotic receptor that employs the noncovalent ammonium-carboxylate electrostatic interaction and a hydrogen bond to achieve high-affinity molecular recognition in water. These

Full text of DOI:10.1002/ejoc.201100855

FULL PAPER
DOI: 10.1002/ejoc.201100855
Synthetic Tripodal Squaramido-Based Receptors for the Complexation of
Antineoplastic Folates in Water
David Quiñonero,[a] Kenia A. López,[a] Pere M. Deyà,[a] M. Nieves Piña,[a] and
Jeroni Morey*[a]
Keywords: Anions / Molecular recognition / Supramolecular chemistry / Noncovalent interactions / Medicinal chemistry /
Antineoplastic drugs
Three suitably oriented squaramido ammonium groups form
and MS. Also, we have proposed a model for coordination
the basis of the first abiotic receptor that employs the nonco- from DFT calculations and NMR spectroscopy studies. Fi-
valent ammonium–carboxylate electrostatic interaction and a
hydrogen bond to achieve high-affinity molecular recogni-
tion in water. These interacctions have been studied experi-
mentally by isothermal calorimetry (ITC), NMR spectroscopy,
nally, a fluorimetric ensemble of the receptor with 5-carboxy-
fluorescein was useful for the quantitative fluorimetric deter-
mination of folate and methotrexate in commercial samples
using competition assays.
(guanidinium and amidinium groups), neutral binding units
(urea, thiourea, amide, and pyrrole moieties), and natural
amino acids (biomimetic receptors). In fact, in nature re-
combinant human dihydrofolate reductase (DHFR) is
known to bind the glutamate moiety of folate through an
arginine residue[10] (i.e., a guanidinium group) in the solid
state. However, in the bacterial proteins R67 DHFR[11] and
Introduction
The recognition and sensing of anionic substrates by pos-
itively charged or neutral synthetic receptors has attracted
considerable attention during the last decade because of the
fundamental role that anions play in many chemical and
biological processes.[1] For instance, anions are present in
biochemical processes such as the activity of enzymes, DNA
regulation, protein biosynthesis, and the transport of hor-
mones.[2] Furthermore, in recent publications, large hydro-
carbon anions, namely, C6H– and C8H–, have been detected
in interstellar media.[3] Generally, the study of real analyti-
cal problems – complex mixtures of bioanalytes[4] (urine,
blood, saliva) environmental analytes (industrial wastes),[5]
cellular imaging, and quality control – concerning anionic
species are carried out in water media and at a specific pH
value, for example, phosphate[6] only exists in a particular
pH range. One important part of anion coordination chem-
istry is the selective complexation of carboxylate anions by
synthetic hosts.[7] For example, sensing and selective detec-
tion of carboxylate anions, such as citrate, malate, and tar-
trate, have been the subject of several studies in food sci-
ence.[8]
Folic acid is a hematopoietic water-soluble B vitamin
that has received a lot of attention because of its important
role in the pathogenesis of cardiovascular diseases, neural
tube defects, and certain kinds of cancer.[9] Carboxylate
anion recognition is usually based on charged binding units
[a] Department of Chemistry, Universitat de les Illes Balears,
Cra. Valldemossa, Km 7.5, 07122 Palma de Mallorca, Illes
Balears, Spain
Fax: +34-971-173-426
E-mail: jeroni.morey@uib.es
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201100855.
Figure 1. Structure of receptors 1a–2b and guests 3a–7.
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thymidylate synthase[12] the glutamate tail of folate and me- in cold (0.0016 mgmLϪ1 at 25 °C) and boiling water (1%),
thotrexate, respectively, is bound to a lysine residue (ammo- but is soluble in very acidic, pH 1–3, and alkaline, pH Ͼ
nium group).
8, aqueous solutions and insoluble in usual apolar organic
Herein, we describe simple abiotic, tripodal, squaramido- solvents. These facts make 3a a very demanding guest for
based receptors capable of tight binding of folate anions sensing.[17] The design of the trisubstituted receptors 1a and
through electrostatic interactions between tetraalkylammo- 1b is based on 1,3,5-tris(aminomethyl)-2,4,6-trimethylbenz-
nium groups and the cooperative action of squaramide ene[18]
units through hydrogen bonding (Figure 1).
and
1,3,5-tris(aminomethyl)-2,4,6-triethylbenz-
ene,[15,19] respectively.
Molecule 1b represents the first folate-binding receptor
composed of an abiotic squaramide that is capable of
achieving high enough affinity to sense folate-like guests,
namely, folic acid (3a), methotrexate (3b), alimta (4, peme-
trexed), and raltitrexed (5) in water.
Nowadays, compounds 3b, 4 and 5 are used as antifolates
drugs,[13] and represent one of the most extensively investi-
gated classes of antineoplastic agents, along with ami-
nopterin, which initially demonstrating clinical activity
more than 50 years ago.[14]
Synthesis of the Receptors
The synthetic route to obtain the receptors 1a–b and 2a–
b was adapted from similar syntheses carried out in our
group.[15] Receptor 1a (Scheme 1) in an acceptable yield is
obtained by coupling an ammonium squaramide iodide salt
(8)[20] with a spacer 1,3,5-tris(aminomethyl)-2,4,6-trimeth-
ylbenzene (9) in ethanol for 8 h at room temperature. Like-
wise, the preparation of receptor 1b is carried out by con-
densing the key intermediate 8 with the spacer 1,3,5-tris-
(aminomethyl)-2,4,6-triethylbenzene (11), but the receptor
is obtained in a very low yield. Better results are obtained
Results and Discussions
In a previous paper,[15] we reported a tripodal receptor (see Scheme 1) in a two-step process, first by obtaining the
capable of recognizing several tricarboxylate salts and intermediate 12, by coupling the squaramide 10[20] with
quantifying citrate concentrations. Since this receptor was spacer 11, followed by complete methylation with methyl
partially soluble in water, it was impossible to use it for iodide.
sensing slightly soluble guests in this solvent. All receptors
Receptors 2a and 2b, based on the more flexible tris(2-
presented herein, 1a–b and 2a–b (Figure 1), have one extra aminoethyl)amine (13),[21] (see Scheme 2), are obtained by
carbon atom in the side chains and all show excellent solu- coupling 13 with the intermediate squaramide 8 or 14,
bility in water, making it possible to sense slightly soluble respectively. At this point, it should be noted that receptor
guests in this solvent. Alkyl ammonium salts are strongly 2a can be prepared on a multigram scale.
solvated in water,[16] showing a solvation shell that extends
The purification of the receptors is simple (chromatog-
to the carbon in the alpha position to the ammonium raphy is not required), since they all precipitate in ethanol
group. Accordingly, we propose that in these new receptors, or methanol and then are exhaustively washed with diethyl
1a–b and 2a–b, the solvation shell is not sterically hindered ether or pentane.
(the alpha carbon is accessible by the solvent) and can be
Receptors 1a–b are more rigid and with a higher degree
formed freely due to the length of the side chain, suggesting of preorganization than the trisubstituted receptors 2a–b.
that this structural feature is key for to the correct solubility The three alkylamino groups present in the hosts are the
in water. It should be mentioned that 3a is sparingly soluble responsible for their excellent solubility in water. In fact, in
Scheme 1. Synthetic route to prepare receptors 1a–b.
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Synthetic Tripodal Squaramido-Based Receptors
ues confirm well-established, strong complexation in water.
To the best of our knowledge, this is the first example in
the literature to address a complexation study between an
abiotic receptor and folate-related guests. The low binding
association constant obtained for receptor 1b and the glut-
amate anion 7 (6.0ϫ102 m–1) suggests that the a priori main
binding force, that is, the ammonium–carboxylate electro-
static interaction, is not the only one responsible for the
high association constants obtained with bigger guests. This
fact is in accordance with the results found for complex
formation between a dianionic guest and a tricationic host:
the association constant obtained for receptor 1b (tri-
cationic host) interacting with folate derivative 3b (dianionic
guest) is surprisingly high (4.5ϫ104 m–1).
Scheme 2. Synthetic route to prepare receptors 2a–b.
In other words, another positive cooperative binding
force is needed to explain these high association constants.
Nevertheless, in water this cooperativity effect has been ra-
rely observed for synthetic hosts.[24b] In our case, this posi-
tive cooperative binding force is most likely to be due to
the existence of a well-established hydrogen bond between
the OH group in the 4-position of the pterin ring of the 3a,
or the NH2 group of the 3b, and one carbonyl group.
ESI-MS supports the formation of the 1b·3a complex
with 1:1 stoichiometry (m/z 1274; see the Supporting Infor-
mation). Adducts 1b·3b (m/z 1539) and 1b·4 (m/z 1260) were
also observed by ESI-MS, which confirmed the formation
of the proposed 1:1 complexes. Complexes of higher stoichi-
ometry were not found in the ESI mass spectra.
their ammonium form, the coulombic force becomes the
dominant interaction between the receptor and the gluta-
mate moiety of folates.
In Table 1 we show the binding constants and thermo-
dynamic experimental results obtained by isothermal calo-
rimetry (ITC; for details see the Supporting Information)
for complexation between receptors 1a–b and 2a–b and di-
carboxylate folate-related guests 3a, 3b, 4, 5, pteroic acid
(6), and the glutamate anion (7). All host–guest complexes
were studied in water at 293 K.
Table 1. Binding constants (Kass, m–1), association Gibbs free ener-
gies (ΔGass kJ/mol), association enthalpies (ΔHass, kJ/mol), and as-
sociation entropies (TΔSass, kJ/mol) for guests 3a–7 and receptors
To gain more insight into the interaction pattern of our
1a–2b in H2O at pH 9.0 in a 0.25 m Tris-HCl buffer system, at complexes, we carried out geometry optimizations of com-
293 K.
plexes 1b·3a and 1b·3b by means of DFT calculations at the
BP86/TZVP level of theory. We optimized the geometry of
all compounds using restricted Kohn–Sham DFT.[25] In
these calculations we used the exchange-correlation BP86
functional, which combined the Becke88 (B) exchange func-
tional,[26] in conjunction with a combination of the Slater–
Dirac exchange and Vosko, Wilk, and Nusair 1980,[27] and
Perdew’s 1986[28] correlation functionals. In these calcula-
tions, the Ahlrichs triple-ζ basis for all atoms (def-TZVP
basis set, TZ hereafter)[29] was used.
Host·guesKt ass
ΔGass
ΔHass
TΔSass
[1a·3a] (3.9Ϯ0.1) ϫ103
[1b·3a] (1.8Ϯ0.1) ϫ104
[2a·3a] (1.20Ϯ0.01) ϫ103
[2b·3a] (5.8Ϯ0.4) ϫ102
–20.3
–23.9
–17.4
–15.5
–15.6
–26.1
–21.7
–25.0
–21.3
–46.0
–31.3
–22.3
–2.2
–2.0
–27.6
–6.4
–25.7
–7.4
–4.9
13.3
13.6
–1.5
15.3
6.7
[1b·7]
(6.0Ϯ0.9) ϫ102
[1b·3b] (4.5Ϯ0.2) ϫ104
[1b·4]
[1b·5]
[1b·6]
(7.20Ϯ0.05) ϫ103
(2.9Ϯ0.4) ϫ104
(6.2Ϯ0.4) ϫ103
–18.3
–25.4
–4.1
Our DFT calculations were carried out using the resolu-
To regulate the pH, a 0.25 m Tris-HCl buffer system was tion of the identity (RI) BP86. Because of the time-consum-
used, which provided the pH of 9.0 required to ensure the ing nature of the calculations, we used the parallel RI-
existence of the anionic form of the assayed guests. In all DFT[30,31] methodology, which uses an auxiliary fitting ba-
examples, the stoichiometry n factor for the complexes was sis set to avoid treating the complete set of two-electron
between 0.7 and 1. The ammonium squarate–carboxylate repulsion integrals, thus speeding up calculations by a fac-
association, in all cases, was an enthalpically driven process tor of 10. Fast evaluation of the Coulomb potential for elec-
and was clearly exothermic.[22] The significant effect of tron densities by using a multipole-accelerated RI approxi-
“preorganization” in folate binding is evident from the data mation linear scaling [O(N)] for large molecules was also
shown in Table 1: the folate affinities of tridentate receptors used.[32] It significantly reduced calculation times for mole-
1a (with a central trimethyl-substituted benzene), 2a (with cules with more than 1000–2000 basis functions.
13 as a spacer) and 2b (receptor similar to 2a, without am-
Moreover, for all compounds, we carried out optimiza-
monium salts) are lower than that of 1b, which has a central tion calculations by simulating water as the solvent within
triethyl-substituted benzene ring.[23] The alternating ar- the conductor-like screening model (COSMO)[33] at the RI-
rangement of groups around the benzene ring provides a BP86/TZ level of theory. COSMO is a continuum solvation
rigid template for a convergent cavity binding site. The model in which the solute molecule forms a cavity within
binding association constants obtained for receptor 1b with the dielectric continuum of permitivity, ε (78.39 for water),
3a, 3b, and 5 are in the order of 104 m–1.[24] These high val- that represents the solvent.
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Geometries of all structures were optimized with the an- vation shell of the anion. The chemistry of life mainly takes
alytical gradient method without symmetry constraints. place in water; therefore, the design and synthesis of recep-
All of the theoretical calculations described herein were tors that are competitive in aqueous media are of special
carried out by using TURBOMOLE version 5.10.[34]
importance, particularly for folate-type guests. For this
The resulting complex structure for 1b·3a is shown in reason, the low association constant of 7 can be explained
Figure 2. The substrate lies atop in an asymmetric composi- by the fact that the guest cannot establish the additional
tion. One carboxylate group of the 3a is bound to one of hydrogen-bonding interaction with receptor 1b.
the ammonium arms by ion-pair formation, (average
However, despite being a monocarboxylate anion, pter-
N+R4···O– distance of 3.24 Å) and the other carboxylate oic acid (6) is structurally adequate to establish this sort
moiety interacts with two ammonium cation arms. In ad- of hydrogen bond, giving rise to an acceptable association
dition, hydrogen-bonding interactions are observed be- constant of 6.2ϫ103 m–1. This unexpectedly large associa-
tween the N–H groups of squaramide and the oxygen atoms tion constant can also be due to the existence of a cation–
of carboxylate groups. In fact, the carboxylate group that π interaction between an aminomethyl ammonium moiety
is interacting with two ammonium units is hydrogen of the host and the phenyl ring of 6. In fact, this interaction
bonded to one squaramide and the carboxylate bound to is observed in the theoretical calculations for complex
one ammonium unit is hydrogen bonded to two squaramide [1b·3b] with a calculated distance of 3.7 Å between the am-
moieties. Furthermore, there is an additional hydrogen monium moiety and the phenyl ring of 3b.[37]
bond between the O–H group of the pterin ring of 3a and
The overall binding scheme proposed herein is in good
the carbonyl group of the squaramide unit (with an agreement with the experimental signals and shift changes
1
HO···O=C distance of 2.78 Å).
observed in the H NMR spectra. When one equivalent of
Therefore, the asymmetric binding mode observed, which 3a is added to an NMR sample of receptor 1b in D2O,[37]
explains the high stability of these folate complexes, is a at basic pH, the signals for the e and f methylene protons
consequence of three factors: first, the existence of electro- of the squaramide receptor arms and the c protons of the
static forces due to the binding of ammonium residues to methylene group of the central benzene ring of 1b are split
carboxylate anions; second, the hydrogen-bonding donor into three new narrow groups of signals (see the Supporting
capability of the squaramide unit, which is superior to urea Information). Moreover, the F and G proton signals of the
and thiourea,[35] is one important reason for this increase glutamate moiety of 3a are also split in the same way.[37]
of the aromatic character of the squaramide ring upon com- The new appearance of proton signals in the 1H NMR spec-
plexation of the anion;[36] and third, the formation of one tra confirms the formation of a strong complex in an asym-
1
additional hydrogen bond between the guest and a carbonyl metric binding mode in D2O. In addition, the H NMR
group of the squaramide ring.
spectra show clear differentiation of proton signals. Our
Positive cooperativity among the above-mentioned non- DFT calculations showed that two ammonium units were
bonded interactions enlarges the binding phenomena in hydrogen bonded to carboxylates, but in the 1H NMR spec-
water and, consequently, our hosts efficiently compete with trum there was no significant chemical shift or splitting of
water molecules that are initially tightly bonded in the sol- the signal of the d protons. These two results are not con-
Figure 2. Calculated structure of the 1b·3a complex. (C gray; O red; N blue). Distances marked in angstroms.
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Synthetic Tripodal Squaramido-Based Receptors
tradictory because the same experimental behavior is often
observed in the recognition of anions with squaramide am-
monium salts.[38]
Encouraged by the results obtained with receptor 1b, we
planned to obtain a sensing system to be employed for prac-
tical uses. Our aim was to check the potential applicability
of our approach with a commercial sample of folic acid and
extend this approach to related folate samples, such as 3b.
We chose to employ the competitive indicator method [Indi-
cator-Displacement Assay (IDA)][39] that has been widely
used in the literature for determining association constants,
for instance, in the determination of inorganic anions and
carboxylates. This methodology is relatively simple and pre-
cise and allows synthetic receptors to work as sensors with-
out introducing additional changes in covalent architecture.
In our approach, we used a fluorescence indicator, such as
5-carboxyfluorescein.
Figure 4. Fluorescence titration of 1b (2ϫ10–5 m; 0.25 m Tris-HCl
buffer) with 3a. λexc = 525 nm. Upper curve corresponds to a final
concentration of 3a of 2.1ϫ105 m. Inset: (a) final and (b) initial
solutions (λ = 365 nm excitation).
First, we determined the binding constants of 1b with 5-
carboxyfluorescein by evaluating the decrease in the fluo-
rescence intensity of 5-carboxyfluorescein in the presence of
increasing amounts of receptor 1b in water with 0.25 m Tris-
HCl buffer (pH 9), at room temperature (23 °C). The bind-
ing constant determined by this method was found to be
Kass = 4.2ϫ103 m–1 with a 1:1 binding algorithm. This
binding constant is 4.2-fold lower than that obtained for 3a
by ITC experiments under the same conditions, which allow
the use of competition assays for the determination of 3a
and 3b in water. We have determined a calibration plot at a
maximum fluorescence emission band, λ = 525 nm, for the
than 7% and the interval of determination was between 1
and 50 ppm of 3a or 3b.
This method for the determination of 3a and 3b is
cheaper than existing ones,[43] the results are obtained
quickly (within hours), and does not require extensive
knowledge of specific techniques for carrying it out, for in-
stance, enzymatic methods. In certain circumstances, it
could be an excellent method for the routine process of de-
termining quantities of 3a and 3b in solids or liquids.
5-carboxyfluorescein ensemble upon addition of 3a (see Conclusions
Figure 3) and 3b.[37] The increase in the intensity of the
The effective sensing of antineoplastic folate anions has
emission band upon incremental additions of 3a is shown
in Figure 4. This progressive increase is due to displacement
of 5-carboxyfluorescein from the receptor by the 3a guest,
restoring the original fluorescence and, therefore, signaling
the presence of 3a.
been successfully achieved by using simple tripodal sensors
in water. We have demonstrated the potential utility of the
squaramide ammonium binding unit for the molecular re-
cognition of dicarboxylate anions, such as 3a and antineo-
plastic folate-like guests, in water. In addition, we have
shown that a sensing ensemble of 1b and 5-carboxyfluores-
cein is capable of quantitatively determining the amount of
3a and 3b anions of a commercial preparation. Finally, this
study proposes a mechanism for how these new tritopic re-
ceptors are coordinated with the folate guests, emphasizing
the important role of the hydrogen bond between the squar-
ate and pteridine rings.
Experimental Section
General: Reactions were carried out in oven-dried glassware under
an atmosphere of argon, unless otherwise indicated. All commer-
cially available reagents: glutaric acid, 3a, Pteroic acid, 13, 1,3,5-
triethylbenzene, 2,4,6-tris-(bromomethyl)mesitylene, 3,4-diethoxy-
3-cyclobutene-1,2-dione, and N,N-dimethyl-1,3-propanediamine
were supplied by Sigma Aldrich. Compound 3b was supplied by
Fluka. Compound 4 was supplied by Sequoia Research Products.
Compound 5 was purchased from AK Scientific and used without
further purification, unless otherwise stated. Diethyl ether and eth-
anol used for synthesis were supplied by Scharlau Chemie and were
distilled from calcium hydride immediately before use. All other
Figure 3. Calibration curve at λ = 525 nm for the ensemble of 1b
and 5-carboxyfluorescein upon addition of 3a in H2O, at pH 9.0 in
0.25 m Tris-HCl (R = 0.9995).
The viability and potential applicability[39] of our fluores-
cence ensemble was checked for the fluorimetric quantita-
tive determination of 3a and 3b[40] in commercially available
pills of folic acid (acfol®)[41] and 3b (Metoject®),[42] respec-
tively. The relative error of these measurements was lower reagents and solvents were purchased from Scharlau Chemie or
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Sigma Aldrich and used without further purification. NMR sol-
vents were purchased from Euriso-top. For the ITC solvent, doubly
distilled deionized water was obtained from a Millipore-Q system
with 18 MΩcm resistivity.
(CH3)2NCH2 chain], 2.11 [s, 18 H, N(CH3)2], 1.63 [quintuplet,
J(H,H) = 5.6, 7.1 Hz, 6 H, CH2 chain], 1.10 [t, J(H,H) = 7.3 Hz,
9 H, CH3 ethyl ArH] ppm. 13C NMR (75.4 MHz [D6]DMSO): δ =
182.8, 182.7, 168.2, 167.2, 144.1, 132.8, 56.4, 45.5, 42.0, 41.7, 29.2,
23.1, 16.8 ppm. FTIR (KBr): ν = 3169 (s), 2934 (s), 2779 (m), 1797
˜
Instrumentation: 1H and 13C NMR spectra were recorded on a
Bruker Avance spectrometer at 300 and 75 MHz at 23 °C. Chemical
shifts are reported as part per million (δ, ppm) referenced to the
residual protium signal of deuterated solvents. Spectral features are
tabulated in the following order: chemical shift (δ, ppm), multi-
plicity (s = singlet, d = doublet, t = triplet, q = quartet, m = com-
plex multiplet), number of protons, coupling constants (J, Hz), as-
signment. Electrospray mass spectra (ESMS) were recorded with a
Micromass, Autospec3000 spectrometer equipped with an electro-
spray module. The ITC experiments were carried out on a Micro-
cal, ultramicrocalorimeter MCS-ITC instrument. The ITC instru-
ment was periodically calibrated by using an internal electric heater,
following the procedures recommended by the manufacturer. The
fluorescence titrations were registered with an Aminco-Browman
series2 spectrophotomers. IR spectra were obtained on a Nicolet
Impact 400 instrument in the solid state. Spectral features are tabu-
lated as follows: wavenumber (cm–1), intensity (s = strong, m =
medium, w = weak). Melting points were measured on a Tottoli
apparatus.
(m), 1653 (s), 1582 (s), 1341 (s), 1096 (m), 747 (w) cm–1. HRMS
(ESI, H2O/EtOH): calcd. for C42H64N9O6 [M + H]+ 790.4901,
C42H63N9O6Na [M + Na]+ 812,4799; found 790.5446, 812.5301.
Compound 1b: Methyl iodide (79 μL, 1.27 mmol) was added, using
a syringe, to a solution of 12 (0.22 g, 0.28 mmol) in acetone
(50 mL) and DMF (25 mL). The mixture was heated under reflux
in an atmosphere of argon for 12 h, after which time it was cooled
to room temperature. The resulting white solid was isolated by cen-
trifugation, after decanting the supernatant, and purified by wash-
ing with cold acetone (3ϫ10 mL) then it was dried to give 1b
1
(0.46 g, 39%); m.p. 209 °C. H NMR (300 MHz, [D6]DMSO): δ =
7.35 (s, 3 H, NH), 7.28 (s, 3 H, NH), 4.84 (s, 6 H, NCH2 ArH),
3.54 [q, J(H,H) = 5.6 Hz, 6 H, NCH2 chain], 3.31 [t, J(H,H) =
5.6 Hz, 6 H, (CH3)3N+CH2 chain], 3.05 [s, 27 H, +N(CH3)3], 2.72
[q, J(H,H) = 7.1 Hz, 6 H, CH2 ethyl ArH], 1.94 (m, 6 H, CH2
chain), 1.10 [t, J(H,H) = 7.3 Hz, 9 H, CH3 ethyl ArH] ppm. 13C
NMR (75.4 MHz [D6]DMSO): δ = 183.1, 182.7, 168.2, 167.4,
144.1, 132.6, 63.3, 54.6, 53.0, 42.0, 41.7, 26.4, 25.0, 23.1, 16.8 ppm.
FTIR (KBr): ν = 3451 (s), 3222 (s), 2962 (m), 1798 (m), 1662 (m),
˜
Determination of Thermodynamic Parameters by ITC
1595 (s), 1536 (s), 1447 (m), 1340 (w), 1256 (w) cm–1. HRMS (ESI,
Titration Conditions: 40–45 injections of an 8–10 mm solution of
the folates (6 μL each) in H2O with a 0.25 m Tris-HCl buffer system
(pH 9.0) were introduced into a sample cell at 293 K containing a
solution of guest (1.5 mL) in H2O with a 0.25 m Tris-HCl buffer
system. The heat of dilution was subtracted prior to data analysis
by Origin MicroCal software. In all cases the c parameter, defined
as c = Kass[G]tn, was kept between 10 and 1000. Errors were calcu-
lated at a confidence level of 95%. Kass, ΔH, and ΔS were obtained
at 293 K by curve fitting by using Origin 5.0 software, as im-
H2O/EtOH): calcd. for C45H72N9O6I2 [M
C45H72N9O6I3Na [M
1238.3047.
–
I]+ 1088.3684,
+
Na]+ 1238.2632; found 1088.3682,
Compound 2a: A solution of 13 (6.82 g, 0.047 mol) in methanol
(50 mL) was added dropwise to stirred solution (51.5 g,
a
0.140 mol) of 8 in methanol (100 mL). The reaction mixture was
stirred overnight at room temperature (20 °C) in an atmosphere of
argon. After this time, the white precipitate was collected from the
reaction mixture by filtration, washed with cold diethyl ether
(70 mL) and methanol (120 mL), and dried under high vacuum to
give 2a (34 g, 65%); m.p. 152 °C 1H NMR (300 MHz, [D6]DMSO):
δ = 7.61 (s, 3 H, NH), 7.38 (s, 3 H, NH), 3.58 (m, 12 H), 3.32 (m,
6 H), 3.10 [s, 27 H, +N(CH3)3], 2.72 [q, J(H,H) = 7.1 Hz, 6 H],
1.98 (m, 6 H, CH2 chain), ppm. 13C NMR (75.4 MHz [D6]DMSO):
δ = 183.1, 182.6, 168.2, 63.4, 55.0, 52.8, 42.1, 41.0, 24.9 ppm. FTIR
plemented by MicroCalTM
.
Compound 1a: A solution of 9 (207 mg, 1 mmol) in ethanol (20 mL)
was added dropwise to a stirred solution (1.10 g, 3.0 mmol) of 8 in
ethanol (10 mL). The reaction mixture was stirred overnight at
room temperature (20 °C) in an atmosphere of argon. After this
time, the white precipitate was collected from the reaction mixture
by centrifugation, washed with cold ethanol (3ϫ10 mL) and Et2O
(2ϫ10 mL), and dried under high vacuum to give host 1a (0.65 g,
(KBr): ν = 3445 (s), 3220 (m), 2953 (w), 1799 (w), 1653 (m), 1592
˜
(s), 1544 (s), 1447 (m), 1345 (w) cm–1. HRMS (ESI, H2O/EtOH):
1
56%); m.p. 254 °C. H NMR (300 MHz, [D6]DMSO): δ = 7.92 (s,
+
calcd. for C36H63N10O6I2 [M – I]+ 985.3022; found m/z 985.2855.
1 H, NH), 7.35 (s, 5 H, NH), 4.89 (s, 6 H, NCH2 ArH), 3.56 [q,
J(H,H) = 5.6 Hz, 6 H, NCH2 chain], 3.31 [t, J(H,H) = 5.6 Hz, 6
H, (CH3)3N+CH2 chain], 3.07 [s, 27 H, +N(CH3)3], 2.50 (9 H, CH3
methyl ArH), 1.97 (m, 6 H, CH2 chain), ppm. 13C NMR
(75.4 MHz [D6]DMSO): δ = 183.1, 182.7, 168.2, 167.4, 144.1,
132.3, 63.3, 54.6, 53.0, 42.0, 41.7, 35.1, 27.2, 25.0, 16.1 ppm. FTIR
Compound 2b: A solution of 13 (50 mg, 0.34 mmol) in ethanol
(15 mL) was added dropwise to
a stirred solution (0.27 g,
1.2 mmol) of 10 in ethanol (20 mL). The reaction mixture was
stirred overnight at room temperature (20 °C) in an atmosphere of
argon. After this time, the white precipitate was collected from the
reaction mixture by centrifugation, washed with cold ethanol
(3ϫ10 mL), and dried under high vacuum to give 2b (0.182 g,
(KBr): ν = 3445 (s), 3222 (w), 1799 (w), 1656 (m), 1597 (s), 1544
˜
(s), 1477 (m), 1342 (w) cm–1. HRMS (ESI, H2O/EtOH): calcd. for
C42H66N9O6I2 ([M – I]+ 1046.3220; found 1046.3226.
1
78%); m.p. 241 °C, H NMR (300 MHz, [D6]DMSO): δ = 7.45 (s,
3 H, NH), 7.32 (s, 3 H, NH), 3.58 (m, 12 H), 2.65 (t, 6 H), 2.21 (t,
6 H), 3.10 [s, 18 H, N(CH3)2], 1.63 (m, 6 H), ppm. 13C NMR
(75.4 MHz [D6]DMSO): δ = 182.9, 182.7, 168.6, 168.0, 56.5, 55.2,
Compound 12: A solution of 11 (0.21 g, 0.84 mmol) in ethanol
(20 mL) was added dropwise to
a stirred solution (0.67 g,
2.9 mmol) of 10 in ethanol (20 mL). The reaction mixture was
stirred overnight at room temperature (20 °C) in an atmosphere of
argon. After this time, the white precipitate was collected from the
reaction mixture by centrifugation, washed with cold ethanol
(3ϫ10 mL), and dried under high vacuum to give 12 (0.43 g, 66%);
m.p. Ͼ 300 °C. 1H NMR (300 MHz, [D6]DMSO): δ = 7.25 (s, 3 H,
NH), 7.18 (s, 3 H, NH), 4.84 [d, J(H,H) = 4.5 Hz, 6 H, NCH2
ArH], 3.84 [q, J(H,H) = 5.6 Hz, 6 H, NCH2 chain], 2.75 [q, J(H,H)
= 7.1 Hz, 6 H, CH2 ethyl ArH], 2.20 [t, J(H,H) = 5.6 Hz, 6 H,
45.6, 42.1, 29.1 ppm. FTIR (KBr): ν = 3445 (s), 3220 (m), 2952
˜
(w), 1799 (w), 1648 (m), 1594 (s), 1546 (s), 1435 (m), 1345 (w) cm–1.
HRMS
(ESI,
H2O/EtOH):
calcd.
for
C33H55N10O6
[M + H]+ 687.4306; found 687.4304.
Supporting Information (see footnote on the first page of this arti-
cle): ITC experiments. 1H and 13C NMR spectra, MS (ESI) charac-
terization, and fluorescence titration data including fitting curves;
details of the quantum chemical calculations.
6192
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Eur. J. Org. Chem. 2011, 6187–6194

Synthetic Tripodal Squaramido-Based Receptors
Am. Chem. Soc. 2005, 127, 184–200; b) S. K. Kim, B.-G. Kang,
H. S. Koh, Y. J. Yoon, S. J. Jung, B. Jeong, K.-D. Lee, J. Yoon,
Org. Lett. 2004, 6, 4655–4658 (dicarboxylates); c) C. Schmuck,
Chem. Eur. J. 2000, 6, 709–718; d) C. Schmuck, J. Am. Chem.
Soc. 2005, 127, 3373–3379; e) A. Metzger, E. V. Anslyn, Angew.
Chem. 1998, 110, 682; Angew. Chem. Int. Ed. 1998, 37, 649–
652; f) A. Metzger, V. M. Lynch, E. V. Anslyn, Angew. Chem.
1997, 109, 911; Angew. Chem. Int. Ed. Engl. 1997, 36, 862–865;
g) C. Bazzicalupi, A. Bencini, A. Bianchi, L. Borsari, C.
Giorgi, B. Valtancoli, J. Org. Chem. 2005, 70, 4257–4266; h) C.
Bazzicalupu, A. Bencini, A. Bianchi, C. Borri, A. Danesi, E.
García-España, C. Giorgi, B. Valtancoli, J. Org. Chem. 2008,
73, 8286–8295; i) C. Gomy, A. R. Schmitzer, J. Org. Chem.
2006, 71, 3121–3125.
a) A. Metzger, E. V. Anslyn, Angew. Chem. 1998, 110, 682; An-
gew. Chem. Int. Ed. 1998, 37, 649–652; b) M. I. Burguete, F.
Galindo, S. V. Luis, L. Vigara, Dalton Trans. 2007, 4027–4033;
c) C. Schmuck, M. Schwegmann, Org. Biomol. Chem. 2006, 4,
836–838; d) L. Fabbrizzi, F. Foti, A. Taglietti, Org. Lett. 2005,
7, 2603–2606; e) J. L. Lavigne, E. V. Anslyn, Angew. Chem.
1999, 111, 3903; Angew. Chem. Int. Ed. 1999, 38, 3666–3669.
a) H. I. Morrinson, D. Schaubel, M. Desmeules, D. T. Wigle,
JAMA J. Am. Med. Assoc. 1996, 275, 1893–1896; b) O. Nygard,
S. M. Vollset, H. Refsum, I. Stensvold, A. Tverdal, J. E. Nord-
rehaug, P. M. Ueland, G. Kvale, JAMA J. Am. Med. Assoc.
1995, 274, 1526–1533; c) E. B. Rimm, W. C. Willet, F. B. Hu,
L. Sampson, G. A. Colditz, J. E. Manson, C. Hennekens, M. J.
Stampfer, JAMA J. Am. Med. Assoc. 1998, 279, 359–364.
J. F. Davies, T. J. Delcamp, N. J. Prendergast, V. A. Ashford,
J. H. Freisheim, J. Kraut, Biochemistry 1990, 29, 9467–9479.
E. E. Howell, Biochemistry 2005, 44, 12420–12433.
W. Welch, C. R. Sage, T. J. Stout, T. Klein, J. Ruppert, R. M.
Acknowledgments
This work was funded by the Ministerio de Educación y Ciencia
(MEC) (grant number CTQ2008-00841/BQU) and the Comunitat
Autònoma de les Illes Balears (CAIB) (grant number FPI09
45692991-H). K. A. L. thanks the DGR+D+I (Govern Balear) for
a predoctoral fellowship. D. Q. thanks the Spanish Ministerio de
Ciencia e Innovación (MICINN) for a Ramón y Cajal Contract.
[1] For comprehensive reviews of anion recognition, see: a) P. A.
Gale, Coord. Chem. Rev. 2001, 213, 79–128; b) P. D. Beer, P. A.
Gale, Angew. Chem. 2001, 113, 502; Angew. Chem. Int. Ed.
2001, 40, 486–516; c) P. A. Gale, Coord. Chem. Rev. 2000, 199,
181–233; d) C. Caltagirone, P. A. Gale, Chem. Soc. Rev. 2009,
38, 520–563; e) C. R. Bondy, S. J. Loeb, Coord. Chem. Rev.
2003, 240, 77–99; f) Supramolecular Chemistry of Anions (Eds.:
A. Bianchi, K. Bowman-James, E. García-España), Wiley-
VCH, Weinheim, Germany, 1997; g) Anion Receptor Chemistry
(Eds.: J. L. Sessler, P. A. Gale, W.-S. Cho), Royal Society of
Chemistry, Cambridge, U.K., 2006; h) Encyclopedia of Supra-
molecular Chemistry (Eds.: J. L. Atwood, J. W. Steed), Marcel
Dekker, New York, 2004; i) E. A. Katayev, Y. A. Ustynyuk,
J. L. Sessler, Coord. Chem. Rev. 2006, 250, 3004–3037; j) M. D.
Best, S. L. Tobey, E. V. Anslyn, Coord. Chem. Rev. 2003, 240,
191–221; k) Z. Xu, S. K. Kim, J. Yoon, Chem. Soc. Rev. 2010,
39, 1457–1466; l) Z. Xu, N. J. Singh, S. K. Kim, D. R. Spring,
K. S. Kim, J. Yoon, Chem. Eur. J. 2011, 17, 1163–1170.
[2] For reviews, see: a) R. Martínez-Máñez, F. Sancenón, Chem.
Rev. 2003, 103, 4419–4476; b) L. Pu, Chem. Rev. 2004, 104,
1687–1716; c) C. Schmuck, Coord. Chem. Rev. 2006, 250, 3053–
3067; d) B. Kuswandi, Nuriman, W. Verboom, D. N. Rein-
houdt, Sensors 2006, 6, 978–1017, and references cited therein.
[3] a) M. C. McCarthy, C. A. Gottlieb, H. Gupta, P. Thaddeus,
ApJ. 2006, 652, L141–L143; b) S. Brünken, H. Gupta, C. A.
Gottlieb, M. C. McCarthy, P. Thaddeus, ApJ. 2007, 664, L43–
L46.
[4] a) S. L. Tobey, E. V. Anslyn, Org. Lett. 2003, 5, 2029–2031; b)
S. Van Arman, A. W. Czarnik, J. Am. Chem. Soc. 1990, 112,
5376–5377; c) S. K. V. Yathavakilla, M. Fricke, P. A. Creed,
D. T. Heitkemper, N. V. Shockey, C. Schwegel, J. Caruso, J. T.
Creed, Anal. Chem. 2008, 80, 775–782; d) A. W. Martínez, S. T.
Phillips, M. J. Butte, G. M. Whitesides, Angew. Chem. 2007,
119, 1340; Angew. Chem. Int. Ed. 2007, 46, 1318–1320; e) Z.
Lin, M. Wu, M. Schäferling, O. S. Wolfbeis, Angew. Chem.
2004, 116, 1767; Angew. Chem. Int. Ed. 2004, 43, 1735–1738.
[5] a) B. A. Moyer, J. F. Birdwell, P. V. Bonnesen, L. H. Delmau,
in: Macrocyclic Chemistry: Current Trends and Future Perspec-
tives (Ed.: K. Gloe), Springer, Dordrecht, 2005, pp. 383–405;
b) Analytical Methods in Supramolecular Chemistry (Ed.: C. A.
Schalley), Wiley-VCH, Weinheim, Germany, 2007; c) Core
Concepts in Supramolecular Chemistry and Nanochemistry
(Eds.: J. W. Steed, D. R. Turner, K. J. Wallace), Wiley, Chiches-
ter, U.K., 2007; d) M. A. Palacios, R. Nishiyabu, M. Márquez,
P. Anzenbacher Jr., J. Am. Chem. Soc. 2007, 129, 7538–7544,
and references cited therein.
[8]
[9]
[10]
[11]
[12]
Stroud, A. Jain, J. Mol. Biol. 2001, 313, 813–829.
[13]
a) G.-M. Liand, S. R. Presnell, in: Encyclopedia of Biological
Chemistry (Eds.: W. J. Lennarz, M. D. Lane), Royal Society of
Chemistry, Cambridge, 2004, vol. 1, p. 671; b) R. Messmann,
C. Allegra, Antifolates in Cancer Chemotherapy & Biotherapy
(Eds.: B. Chabner, D. Longo), Lippincott Williams & Wilkins,
Philadelphia, 2001, p. 241; c) N. Kohler, C. Sun, J. Wang, M.
Zhang, Langmuir 2005, 21, 8858–8864; d) Pemetrexed: A.
Hanauske, V. Chen, P. Paoletti, C. Niyikiza, Oncologist 2001,
6, 363–373; e) N. J. Vogelzang, J. J. Rusthoven, J. Symanowski,
J. Clin. Oncol. 2003, 21, 2636–2644; f) L. Paz-Ares, S. Bezares,
J. M. Tabernero, D. Castellanos, H. Cortes-Funes, Cancer
2003, 97, 2056–2063; g) C. Shih, V. J. Chen, L. S. Gossett, Can-
cer Res. 1997, 57, 1116–1123.
S. Farber, L. K. Diamond, R. D. Mercer, R. F. Sylvester, J. A.
Wolff, N. Engl. J. Med. 1948, 238, 787–793.
A. Frontera, J. Morey, A. Oliver, M. N. Piña, D. Quiñonero,
A. Costa, P. Ballester, P. M. Deyà, E. V. Anslyn, J. Org. Chem.
2006, 71, 7185–7195.
M. Rekharsky, Y. Inoue, J. Am. Chem. Soc. 2000, 122, 4418–
4435.
The Merck Index, 11th ed., Merck, New Jersey, 1989, p. 660.
1,3,5-Tris(bromomethyl)-2,4,6-trimethylbenzene: a) A. W.
van der Made, R. H. van der Made, J. Org. Chem. 1993, 58,
1262–1263; b) Z. Hou, T. D. P. Stack, C. J. Sunderland, K. N.
Raymond, Inorg. Chim. Acta 1997, 263, 341–355; c) C. E. Wil-
lans, K. M. Anderson, P. C. Junk, L. J. Barbour, J. W. Steed,
Chem. Commun. 2007, 3634–3636; d) M. G. Sarwar, B.
Dragisic, S. Sagoo, M. S. Taylor, Angew. Chem. Int. Ed. 2010,
49, 1–5.
1,3,5-Tris(bromomethyl)-2,4,6-triethylbenzene: a) K. V. Kilway,
J. S. Siegel, Tetrahedron 2001, 57, 3615–3627; b) K. J. Wallace,
R. Hanes, E. V. Anslyn, J. Morey, K. V. Kilway, J. Siegel, Syn-
thesis 2005, 2080–2083; c) A. Vacca, C. Nativi, M. Cacciarini,
R. Pergoli, S. Roelens, J. Am. Chem. Soc. 2004, 126, 16456–
16465; d) C. Guarise, G. Zaupa, L. J. Prins, P. Scrimin, Eur. J.
Org. Chem. 2008, 3559–3568.
[14]
[15]
[16]
[17]
[18]
[6] a) T. Zhang, E. V. Anslyn, Tetrahedron 2004, 60, 11117–11124;
b) S. L. Tobey, B. D. Jones, E. V. Anslyn, J. Am. Chem. Soc.
2003, 125, 4026–4027; c) R. Prohens, G. Martorell, P. Ballester,
A. Costa, Chem. Commun. 2001, 1456–1457; d) H. N. Lee, Z.
Xu, S. K. Kim, K. M. K. Swamy, Y. Kim, S.-J. Kim, J. Yoon,
J. Am. Chem. Soc. 2007, 129, 3828–3829; e) G. V. Zyryanov,
M. A. Palacios, P. Anzenbacher Jr., Angew. Chem. 2007, 119,
7995; Angew. Chem. Int. Ed. 2007, 46, 7849–7852; f) G. V.
Oshovsky, D. N. Reinhoudt, W. Verboom, Angew. Chem. 2007,
119, 2418; Angew. Chem. Int. Ed. 2007, 46, 2366–2393; g) M.
Boiocchi, M. Bonizzoni, L. Fabbrizzi, G. Piovani, A. Taglietti,
Angew. Chem. Int. Ed. 2004, 43, 3847–3852.
[19]
[7] a) A. B. Descalzo, K. Rurack, H. Weisshoff, R. Martínez-
Mañez, M. D. Marcos, P. Amoros, K. Hoffmann, J. Soto, J.
Eur. J. Org. Chem. 2011, 6187–6194
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6193

D. Quiñonero, K. A. López, P. M. Deyà, M. N. Piña, J. Morey
FULL PAPER
[20] M. N. Piña, M. C. Rotger, B. Soberats, P. Ballester, P. M. Deyà,
A. Costa, Chem. Commun. 2007, 9, 963–965.
[21] a) M. Allevi, M. Bonizzoni, L. Fabbrizzi, Chem. Eur. J. 2007,
13, 3787–3795; b) M. M. Ibrahim, N. Shimomura, K. Ichi-
kawa, M. Shiro, Inorg. Chim. Acta 2001, 313, 125–136; c) M.
[25] W. Kohn, L. J. Sham, Phys. Rev. 1965, 140, A1133–A1138.
[26] A. D. Becke, Phys. Rev. A 1988, 38, 3098–3100.
[27] a) P. A. M. Dirac, Proc. R. Soc. London Ser. A 1929, 123, 714–
733; b) J. C. Slater, Phys. Rev. 1951, 81, 385–390; c) S. Vosko,
L. Wilk, M. Nusair, Can. J. Phys. 1980, 58, 1200–1211.
Kruppa, B. König, Chem. Rev. 2006, 106, 3520–3560; d) P. S. [28] J. P. Perdew, Phys. Rev. B 1986, 33, 8822–8824.
Lakshminarayanan, I. Ravlkumar, E. Suresh, P. Ghosh, Inorg.
Chem. 2007, 46, 4769–4771.
[29] a) A. Schäfer, C. Huber, R. Ahlrichs, J. Chem. Phys. 1994, 100,
5829–5835; b) K. Eichkorn, F. Weigend, O. Treutler, R.
Ahlrichs, Theor. Chem. Acc. 1997, 97, 119–124.
[30] M. v. Arnim, R. Ahlrichs, J. Comput. Chem. 1998, 19, 1746–
1757.
[31] K. Eichkorn, O. Treutler, H. Öhm, M. Häser, R. Ahlrichs,
Chem. Phys. Lett. 1995, 242, 652–660.
[22] In polar media, especially water or aqueous alcoholic solution,
the binding phenomena can even be endothermic and entropy
driven due to reorganization of the solvent upon complexation:
a) M. Haj-Zaroubi, N. W. Mitzel, F. P. Schmidtchen, Angew.
Chem. 2002, 114, 111; Angew. Chem. Int. Ed. 2002, 41, 104–
107; b) R. Prohens, M. C. Rotger, M. N. Piña, P. M. Deyà, J.
Morey, P. Ballester, A. Costa, Tetrahedron Lett. 2001, 42, 4933–
4936; c) R. Meissner, X. Garcias, S. Mecozzi, J. Rebek Jr., J.
Am. Chem. Soc. 1997, 119, 77–85.
[32] M. Sierka, A. Hogekamp, R. J. Ahlrichs, J. Chem. Phys. 2003,
118, 9136–9148.
[33] A. Klamt, G. Schüürmann, J. Chem. Soc. Perkin Trans. 2 1993,
5, 799–805.
[23] This fact is due to minor steric effects of methyl groups relative
to ethyl groups in the benzene ring between the alternate ar-
rangement of the methyl (ethyl) groups and the aminomethyl-
ene substituents in the trisubstituted benzene derivatives; for
examples, see: a) G. Hennrich, E. V. Anslyn, Chem. Eur. J.
2002, 8, 2218–2224; b) K. V. Kilway, J. S. Siegel, J. Am. Chem.
[34] R. Ahlrichs, R. M. Bär, M. Hacer, H. Horn, C. Kömel, Chem.
Phys. Lett. 1989, 162, 165–169.
[35] a) D. Quiñonero, A. Frontera, G. A. Suñer, J. Morey, A. Costa,
P. Ballester, P. M. Deyà, Chem. Phys. Lett. 2000, 326, 247–254;
b) C. Garau, A. Frontera, P. Ballester, D. Quiñonero, A. Costa,
P. M. Deyà, Eur. J. Org. Chem. 2005, 179–183.
Soc. 1992, 114, 255–261; c) D. J. Iverson, G. Hunter, J. F. [36] D. Quiñonero, R. Prohens, C. Garau, A. Frontera, P. Ballester,
Blount, J. R. Damewood, K. Mislow, J. Am. Chem. Soc. 1981,
103, 6073–6083; d) T. D. P. Stack, Z. Hou, K. N. Raymond, J.
Am. Chem. Soc. 1993, 115, 6466–6467.
A. Costa, P. M. Deyà, Chem. Phys. Lett. 2002, 351, 115–120.
[37] See the Supporting Information: Figure S6.
[38] M. N. Piña, PhD thesis, Universitat de les Illes Balears (Spain),
2005, p. 72.
[39] E. V. Anslyn, J. Org. Chem. 2007, 72, 687–699.
[40] Spanish Patent application P24718ES00 (31 March, 2009).
[41] Acfol, Italfarmaco, Alcobendas, Madrid, Spain.
[42] Metoject, 10 mgmLϪ1, Gebro Pharma, Medac GmbH, Ham-
burg, Germany.
[43] For comprehensive reviews of folate determination, see: a) J.
Arcot, A. Shrestha, Trends Food Sci. Technol. 2005, 16, 253–
266; b) Microbiological method by AOAC [Method 992.05
(2002) and AACC 7469]; c) C. Wang, S. Li, L. Ting, X. Xu, C.
Wang, Microchim. Acta 2006, 152, 233–238; d) D. Hoegger, P.
Morier, C. Vollet, D. Heini, F. Reymond, J. S. Rossier, Anal.
Bioanal. Chem. 2007, 387, 267–275; e) J. D. M. Patring, J. A.
Jastrebova, S. B. Hjortmo, T. A. Andlid, I. M. Jägerstad, J.
Agric. Food Chem. 2005, 53, 2406–2411.
[24] Receptors capable of binding dicarboxylate in water have been
reported; for example, a receptor that employs tris-cationic
units based on the guanidiniumcarbonyl pyrrole group in coop-
eration with an ammonium group binds N-acetylglutamate
with
a 2:1 association model K1 = =
460 m–1 and K2
3.300 m–1 (90% water/10% DMSO), see: a) C. Schmuck, L.
Geiger, J. Am. Chem. Soc. 2005, 127, 10486–10487; on the
other hand, a receptor employing diguanidinium and CuII
groups binds glutarate and succinate with Ka = 1.400 m–1 and
Ka = 5.500 m–1 in water, respectively, see: b) A. D. Hughes, E. V.
Anslyn, Proc. Natl. Acad. Sci. USA 2007, 104, 6538–6543; an-
other receptor with a cholic acid based fluorescent neutral
group binds glutarate, succinate, and l-glutamate with Ka
=
2.93ϫ105 m–1, Ka = 1.73ϫ105 m–1 and Ka = 5.57ϫ106 m–1 in
CH3OH/H2O (1:1), respectively, see c) S.-Y. Liu, L. Fang, Y.-
B. He, W.-H. Chan, K.-T. Yeung, Y.-K. Cheng, R.-H. Yuang,
Org. Lett. 2005, 7, 5825–5828.
Received: June 13, 2011
Published Online: September 19, 2011
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Eur. J. Org. Chem. 2011, 6187–6194