Recognition of free tryptophan in water by synthetic pseudopeptides: Fluorescence and thermodynamic studies

Article abstract of DOI:10.1002/chem.201304851

Pseudopeptidic receptors containing an acridine unit have been prepared and their fluorescence response to a series of amino acids was measured in water. Free amino acids, not protected either at the C or the N terminus, were used for this purpose. The pr

Full text of DOI:10.1002/chem.201304851

DOI: 10.1002/chem.201304851
Full Paper
&
Supramolecular Chemistry
Recognition of Free Tryptophan in Water by Synthetic
Pseudopeptides: Fluorescence and Thermodynamic Studies
Vicente Martꢀ-Centelles, M. Angeles Izquierdo, M. Isabel Burguete, Francisco Galindo,* and
Santiago V. Luis*[a]
Abstract: Pseudopeptidic receptors containing an acridine
unit have been prepared and their fluorescence response to
a series of amino acids was measured in water. Free amino
acids, not protected either at the C or the N terminus, were
used for this purpose. The prepared receptors display a selec-
tive response to tryptophan (Trp) versus the other assayed
amino acids under acidic conditions. The macrocyclic nature
of the receptor is important as the fluorescence quenching
is higher for the macrocyclic compound than for the related
open-chain receptor. Notably, under the experimental acidic
conditions used, both the receptor and guest are fully pro-
tonated and positively charged; thus, the experimental re-
sults suggest the formation of supramolecular species that
contain two positively charged organic molecular compo-
nents in proximity stabilized through aromatic–aromatic in-
teractions and a complex set of cation-anion-cation interac-
tions. The selectivity towards Trp seems to be based on the
existence of a strong association between the indole ring of
the monocharged amino acid and the acridinium fragment
of the triprotonated form of the receptor, which is estab-
lished to be assisted by the interaction of the cationic moiet-
ies with hydrogen sulfate anions.
Introduction
perienced by the bioanalytical sciences in the use of fluores-
cence-based techniques, a current challenge is still the lack of
knowledge about many elementary interactions that occur at
the level of a single amino acid under the considered experi-
mental conditions. An illustrative example was provided re-
cently by Webb and co-workers, who described how free tryp-
tophan can dramatically quench the emission of the popular
probe Alexa 488.[34] Interestingly, half of the quenching was
shown to arise from a ground-state association of the probe
and the amino acid.
Amino acids are essential building blocks for living organisms
as they are the structural basis of proteins and peptides. In this
regard, supramolecular interactions that involve amino acids,
peptides, and proteins are of paramount importance as, for in-
stance, the protein–protein interaction is an important process
in cell recognition.[1–3] On the other hand, the aggregation of
peptides and proteins is a well-known phenomenon, which is
sometimes linked to disease states.[4] Hence, the understanding
of such weak association process within the framework of
supramolecular chemistry, under natural and non-natural con-
ditions, is of fundamental importance.[2,5–10] In this context, the
recognition of amino acids and amino acid-related compounds
has attracted the attention of numerous researchers from
a basic perspective, but also from an applied point of
view.[11–14] A growing number of reports that deal with the bio-
analysis of amino acid mixtures have been published in recent
years.[15–30] Some diagnostic assays are based on photoinduced
electron-transfer processes that involve one or more target
amino acids.[31–33] However, despite the great development ex-
The development of methods for the recognition and sens-
ing of amino acids is vital because of their critical biological
relevance,[18,23,34–37] which is particularly true for the sensing of
tryptophan.[20,30,38–41] The presence of tryptophan is directly re-
lated to the immune system, in which enzymes break down
this amino acid through the kynurenine metabolic path-
way.[42,43] Tryptophan is required for protein synthesis and
other important metabolic functions; in addition, this amino
acid is the only source for the production of several important
molecules in the nervous system, such as serotonin, whereas
tryptophan is used for the synthesis of melatonin in the pineal
gland.[42,44]
Taking into account the importance of basic studies that
have tackled the interaction between amino acids and small
fluorescent molecules and our previous work on pseudopepti-
dic supramolecular systems,[45] we report herein supramolec-
ular studies on a series of acridine-based pseudopeptidic mole-
cules (Figure 1) with different unprotected amino acids by
means of NMR, UV/Vis, and fluorescence (steady-state and
time-resolved) spectroscopy and computational calculations.
The complexation of such receptors with free amino acids has
[a] Dr. V. Martꢀ-Centelles, Dr. M. A. Izquierdo, Dr. M. I. Burguete, Dr. F. Galindo,
Dr. S. V. Luis
Departamento de Quꢀmica Inorgꢁnica y Orgꢁnica
Universitat Jaume I, Av. Sos Baynat, s/n
E-12071 Castellꢂn (Spain)
Fax: (+34)964728214
E-mail: francisco.galindo@uji.es
luiss@uji.es
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201304851.
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Figure 1. The pseudopeptidic receptors containing acridine fragments con-
sidered herein.
Scheme 1. Synthesis of pseudopeptidic receptors 1 and 2. i) DME; ii) HBr/
AcOH 33%, RT then aq. NaOH; iii) TBABr, DIPEA, CH3CN, temperature gradi-
ent=50–818C.
been studied. These pseudopeptidic receptors selectively rec-
ognize free tryptophan (Trp) over alanine (Ala), histidine (His),
methionine (Met), phenylalanine (Phe), tyrosine (Tyr), and
valine (Val). Even at a very low pH values, receptors 1 and 2
and Trp can associate to an appreciable extent, most likely
with the participation of aromatic interactions, which is re-
markable considering that both the receptor and the host are
positively charged.[46]
the presence of tetrabutylammonium bromide (TBABr) and
N,N-diisopropylethylamine (DIPEA) with a temperature gradient
of 50–818C. The crude product was purified by column chro-
matography to afford the pure macrocycle in 63% yield, which
is a remarkable yield taking into account that high dilution
conditions were not employed.[53,54] These relatively high yields
obtained for the macrocyclization reaction suggest a high
degree of preorganization of the open-chain intermediate and
the corresponding transition state, which has been demon-
strated in other cases that used this methodology.[55–60]
Results and Discussion
Recognition studies were carried out with four pseudopeptidic
synthetic receptors based on Val and Phe 1a,b and 2a,b, re-
spectively, and seven free amino acids (i.e., Ala, His, Met, Phe,
Tyr, Trp, and Val) as guests.
Synthesis of the receptors
Characterization of compounds
The design of the acridine pseudopeptides considered herein
is based on our previous studies that revealed how this type
of molecule represents an interesting option for anion recogni-
tion and fluorescent sensing, in particular in acidic media.[47]
The presence of aromatic 1a and 2a and nonaromatic 1b and
2b side chains was considered to be an interesting design
vector because these side chains can participate in different
types of interaction. The synthesis of receptors 1a and 2a and
Compounds 1b and 2b were characterized by spectroscopic
and analytical techniques. Interestingly, in solution with CDCl3,
1
the H NMR signals of the CH2 groups at positions 4 and 5 of
the acridine moiety are remarkably different for the open-
chain derivatives 1a,b and macrocyclic derivatives 2a,b. For
the open-chain compound 1a, these protons appear as one
doublet, whereas in 1b, 2a, and 2b they appear as two
double doublets. This finding points to the presence of an ap-
preciable coupling constant with the amide hydrogen atom in
1b, 2a, and 2b (see Figure 2 and Figure S20 in the Supporting
Information). This effect is higher for the macrocyclic com-
pounds, which is expected for compounds with higher confor-
mational restrictions. However, it is remarkable that an analo-
gous pattern can also be observed for the open-chain com-
pound 2a. Thus, the conformational freedom of this Val-de-
rived compound is notably restricted relative to the Phe-de-
rived analogue 1a (compare spectra (a) and (c) in Figure 2).
This behavior can be associated to the higher rigidity provided
by the Val side chain (isopropyl) relative to the Phe side chain
(benzyl) according to the conformational-flexibility scale for
amino acids proposed by Huang and Nau,[61] in which Val is
only surpassed in rigidity by isoleucine (Ile) and especially by
proline (Pro).
ꢀ
their actuation as selective fluorescent sensors for H2PO4 ions
has been described previously.[47] An analogous procedure was
followed for the synthesis of 1b and 2b (Scheme 1). The syn-
thesis of the pseudopeptidic compound 1b-Cbz was carried
out by reaction of 4,5-bis(aminomethyl)acridine with the corre-
sponding protected amino acid activated as its hydroxysuccini-
mide ester with dimethoxyethane (DME) as the solvent. 4,5-Bis-
(aminomethyl)acridine was prepared according to previously
reported procedures.[48–51] The carbobenzlyoxy (Cbz) protecting
group was removed by using an HBr/AcOH mixture followed
by neutralization with NaOH to yield the open-chain receptor
1b. The most difficult step in the synthetic scheme is the mac-
rocyclization reaction to obtain macrocycle 2b. In this case,
careful control of the temperature, with the use of a tempera-
ture gradient[47,52] and under conditions similar to those used
for related pseudopeptidic macrocycles, provided significantly
better yields than by performing the reaction at reflux. Thus,
macrocycle 2b was prepared from the reaction of bis(amino
amide) 1b with 1,3-bis(bromomethyl)benzene in acetonitrile in
The electronic absorption spectra and the steady-state fluo-
rescence emission spectra of 1b and 2b were also recorded in
CHCl3. As expected,[62] 1b and 2b have similar properties, thus
showing absorption maxima at l=357 nm and an emission
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Figure 2. 1H NMR spectra (20 mm, CDCl3) of compounds: a) 1a, b) 1b, c) 2a,
and d) 2b.
maximum at l=420 nm when excited at l=357 nm (Figure 3;
see ref. [47] for the related compounds 1a and 2a).
The fluorescence quantum yields for 1b and 2b at acidic
pH values were similar (pH 0.37 and 0.36, respectively) to those
reported for 1a and 2a (pH 0.43 and 0.38, respectively),[47] and
all of them slightly lower than those measured for the parent
acridine (pH 0.65).[62] The quantum yield significantly decreases
upon increase of the pH value, which is expected from the
conversion of a highly emissive fluorophore such as acridinium
(Figure 4) into the less emissive acridine.[63–67] In addition, time-
correlated single-photon counting (TCSPC) was used to charac-
Figure 4. Normalized absorption (left axis) and emission (right axis) spectra
for 1b and 2b in H2O at pH 1.00. lexc =357 nm. Probe concentra-
tion=20 mm.
terize the singlet excited states of 1b and 2b. Values for the
different fluorescence lifetimes tF were obtained by the fitting
the data to the equation I=I0ꢀaiti (see Figure S10 in the Sup-
porting Information). From the excitation and emission curves,
the energy of the singlet excited state ES was also calculated
for each compound (Table 1).
The behavior of pseudopeptidic compounds 1 and 2 in an
acidic medium is comparable to that displayed by acridine,
that is, the emitting species at long wavelengths is clearly the
protonated acridine species (acridinium cation), which displays
fluorescence properties (ES =ca. 62–66 kcalmolꢀ1 and t1 =
ca. 24–32 ns) clearly different to the acridine moiety (ES =
ca. 72 kcalmolꢀ1 and t1 = <10 ns).[47,62,64]
Determination of pKa values
The pKa values for the protonation at the acridine moiety of
these compounds were determined by means of fluorescence
titrations. The variation in the intensity of the fluorescence
emission of the acridinium moiety (excitation wavelength at
isosbestic points: l=363 and 365 nm for 1b and 2b, respec-
tively) at different pH regions was used to determine the
pKa values for the protonation of the acridine moiety in the re-
ceptors by fitting the corresponding fluorescence intensity
versus the pH value for each titration (Figure 5).
The on/off fluorescence switching process at very low
pH values corresponds to the protonation of the nitrogen
atom of the acridine ring because the aliphatic amines should
Figure 3. Normalized absorption (left axis) and emission (right axis) spectra
for 1b and 2b in CHCl3. lex =357 nm. Probe concentration=10 mm.
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Recognition of amino acids
Table 1. Fluorescence properties for 1 and 2 in water (measurements made with a concentra-
tion of 20 mm in water using H2SO4 and NaOH to adjust the pH.)
1H NMR experiments
pH
labs lexc lem
ES1
FF[b] t1
a1 t2
a2 c2 pKa
To analyze the ability of these pseudopep-
tidic compounds to interact with amino
[nm] [nm] [nm][a] [kcalmolꢀ1
]
[ns][c] [%] [ns][c] [%]
Acridine 0.92 354, 354[d] 477
398
65.9
72.7
62.7
0.65 31.6[e]
0.24 9.5[h]
0.38 23.9[e]
–
–
–
–
–
–
–
–
–
1.03 5.42[f]
1.22
1
acids, different H NMR spectroscopic ex-
periments were carried out. It is worth
mentioning that previous work by our
group has focused on the study of N-pro-
tected amino acids and dipeptide recogni-
tion by related pseudopeptidic recep-
tors.[23,59,69] In this case, however, the
study was based on the recognition of
amino acids not derivatized as esters or
protected with carbamate groups, which
is more relevant from a biomedical point
of view, in particular for the development
of sensing methodologies of practical in-
terest. The free amino acids considered
here (i.e., Ala, His, Met, Phe, Trp, Tyr, and
Val; Figure 6) are expected to exist as
zwitterions at neutrality[15] or as cationic
species at the experimental acidic pH
values required for the protonation of the
acridine fragment.
12.65 355 354[g] 427
2a[i,j]
1a[j]
0.55 359, 359[d] 502
397
1.15 1.67ꢁ0.02
0.68 358, 359[d] 501
398
4.16 357 356[k] 432
62.6
71.8
62.2
71.4
62.8
70.4
0.43 26.6[e]
–
–
–
1.15 2.74ꢁ0.01
0.05 1.3[h] 95.4 12.2[h] 4.6 0.96
2b
1b
0.24 358, 357[d] 504
420
4.05 356 356[l] 437
0.36 20.8[e]
–
–
–
0.92 2.47ꢁ0.01
0.03 1.1[h] 78.4 12.4[h] 21.6 0.83
0.30 356, 355[d] 501
416
4.13 356 355[l] 428
0.37 22.4[e]
–
–
–
1.02 2.63ꢁ0.02
0.03 1.1[h] 58.4 12.5[h] 41.6 0.88
[a] lexc =366 nm. [b] Quinine sulphate standard (aqueous H2SO4 0.1m, air); FF =0.53 (taken
from ref. [67]). [c] lexc =372 nm. [d] lem =540 nm. [e] lem =475 nm. [f] Taken from ref. [63].
[g] lem =470 nm. [h] lem =426 nm. [i] It was not possible to measure this sample at pH 4 due to
precipitation of the product. [j] Taken from ref. [47]. [k] lem =450 nm. [l] lem =490 nm.
The most interesting results were ob-
served at acidic pH values. Titrations of 1a and 2a with the dif-
ferent amino acids were performed in D2O (2m H2SO4) with
tert-butanol as an internal standard (Figures 7 and 8). Studies
Figure 5. Fluorescence pH titrations of 1b and 2b that monitor the emission
at l=495 and at 500 nm for 1b and 2b, respectively. Probe concentra-
tion=10 mm in aqueous solution (1% DMSO). lex =363 and 365 nm and
pKa =2.63ꢁ0.02 and 2.47ꢁ0.01 for 1b and 2b, respectively.
Figure 6. The l-amino acids considered herein.
carried out with l-Trp were the most significant. For both re-
1
protonate first due to their primary and secondary character
(i.e., 1b and 2b, respectively). This behavior is comparable
with examples reported, for instance, for polyaza pyridino-
phanes able to admit up to six protons, including the protona-
tion of the nitrogen atom at the pyridine moiety.[68]
ceptors, the H NMR signal of the acridine H9 atom displayed
a large upfield shift upon the addition of l-Trp (Ddmax
=
ꢀ0.999 ppm). This shift suggests the presence of an important
interaction between l-Trp and 1a and 2a, which could take
place by p–p stacking between the electron-poor acridinium
cationic fragment and the electron-rich indole ring. In sharp
contrast, 1H NMR titration experiments with l-Met showed
a very small downfield shift (Dd=0.015 ppm for the last point
of the titration ([l-Met]=0.125m)) for the same signal. Experi-
ments with l-Tyr also showed a small upfield change. Finally,
experiments with Ala, Phe, His, and Val did not show any varia-
By comparing these pKa values for compounds derived from
Val 1b and 2b and from Phe 1a and 2a, some differences can
be observed, especially for macrocycles 2a and 2b (pKa =1.67
vs 2.47). Most likely, this difference reflects the different hydro-
phobicity imparted by the side chains, as previously studied
for analogous compounds.[59,60]
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Figure 8. 1H NMR titration of 2a (5 mm) with l-Phe (from 0 to 0.25m) in 2m
H2SO4 in D2O. The acridine H9 signal, which is upfield shifted with the addi-
tion of Trp, is marked with a black dot a) 0, b) 24, c) 45, d) 65, e) 83, f) 115,
g) 143, h) 188, i) 222, j) 250 mm Phe.
Figure 7. 1H NMR titration of 2a (5 mm) with l-Trp (from 0 to 0.25m) in 2m
H2SO4 in D2O. The acridine H9 signal, which is upfield shifted with the addi-
tion of Trp, is marked with a black dot: a) 0, b) 24, c) 45, d) 65, e) 83, f) 115,
g) 143, h) 188, i) 222, j) 250 mm Trp.
1
tion in the H NMR chemical shifts, thus indicating that no no-
where t0 is the fluorescence lifetime in the absence of
a quencher, t is the fluorescence lifetime in the presence of
a quencher, I0 is the fluorescence emission intensity in the ab-
sence of a quencher, I is the fluorescence emission intensity in
the presence of a quencher, KD is the dynamic quenching con-
stant, KS is the static quenching constant, and kq is the dynam-
ic rate quenching constant.
ticeable association between the host and guest takes place,
which can be observed in Figure 8 for the titration of macrocy-
cle 2a with l-Phe.
Fluorescence quenching studies
The fluorescence emission intensity of the receptor was
measured in the presence of increasing concentrations of the
different amino acids in acidified water (1m H2SO4; see Fig-
ure S18 in the Supporting Information). Under such acidic con-
ditions, the receptors are triprotonated[47] and the amino acids
are monoprotonated.[73,74] Amino acids that contain an aliphat-
ic side chain (l-Ala, l-Val), a benzene ring (l-Phe), or a protonat-
ed imidazole ring (l-His) did not disturb the fluorescence emis-
sion of the receptors. On the contrary, those containing elec-
tron-rich side chains (l-Trp, l-Tyr, l-Met) quenched the fluores-
cence intensity of the receptors, although with remarkable dif-
ferences, with Trp displaying the most marked effect. The
quenching was nonlinear as the Stern–Volmer plot curved up-
wards. This deviation from linearity indicates a combination of
both dynamic and static quenching processes, that is, the exis-
tence of an effective complexation in the ground state as sug-
gested by NMR spectroscopic experiments. Titrations were car-
ried out for both enantiomers of each amino acid (see Fig-
ure S18 in the Supporting Information), but no appreciable
enantiodifferentiation was detected.
To gain further insight into the association process detected
for Trp, fluorescence and UV/Vis titration experiments were
performed under related experimental conditions (1m H2SO4).
Both the UV/Vis and fluorescence spectra displayed apprecia-
ble changes with the addition of Trp that which were more
significant than those observed with other amino acids. Strong
quenching of the fluorescence emission of 1 and 2 was only
detected in the case of Trp (Figure 9), only slightly quenched
by Tyr and Met, and not quenched at all by Ala, Val, Phe, or
His.
Stern–Volmer analysis [Eqs (1) and (2)] of the fluorescence
quenching allows its nature to be determined, hence disclos-
ing dynamic (collisional) and static (ground-state association)
effects.[70,71] Theoretically, pure dynamic quenching provides
a linear Stern–Volmer plot from the quenching experiments
from steady-state emission measurements [Eq. (1)], whereas
a deviation from linearity indicates an interaction between the
host and the guest in the ground state and Equation (2) ap-
plies.[72] The dynamic quenching constant is related to the ex-
cited-state lifetime according to Equation (3). Plot deviation
from the linearity in the plot obtained:
Additionally, time-resolved fluorescence experiments for the
titration of the hosts 1a, 2a, 1b, and 2b with Trp were also
performed to confirm the coexistence of static and dynamic
quenching. In this type of experiment, only the dynamic
quenching of the excited state is observed, thus allowing the
calculation of the corresponding dynamic quenching constant
KD. Therefore, the combination of time-resolved and steady-
state fluorescence experiments allowed determination of the
t0=t ¼ I0=I ¼ 1 þ KD½Qꢂ
I0=I ¼ ð1 þ KD½QꢂÞð1 þ KS½QꢂÞ
KD ¼ kqt
ð1Þ
ð2Þ
ð3Þ
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Figure 11. Stern–Volmer plot of the titration of 2a (20 mm) with l-Trp (from
0 to 36 mm) in aqueous H2SO4 (1m). Solid lines correspond to fitting to
Equations (1) and (2). lex =366 nm.
viously calculated KD constant from the time-resolved fluores-
cence fitting to Eq. (1)], thus allowing the calculation of the KS
constant. The latter process must be associated to the forma-
tion of a nonfluorescent complex in the ground state. By using
the calculated KS value, it is possible to obtain the associated
Gibbs free energy DG,[75] which is approximately ꢀ1.0 kcal
molꢀ1 in the case of l-Trp. The recognition process is slightly
more favorable for the l enantiomer, although the enantiodis-
crimination is low.
Figure 9. Variation of the fluorescence and absorbance in the titration of 2a
(20 mm) with l-Trp (from 0 to 36 mm) in H2SO4 (1m).
Following a similar methodology, fitting to the Stern–Volmer
equations was performed for the different titrations (the results
are summarized in Table 2). The most effective quencher was
clearly Trp with a KD constant of approximately 83–93mꢀ1 for
1a and 2a, whereas the KD constant was approximately 60–
75mꢀ1 for 1b and 2b. Therefore, the isopropyl side chain de-
rived from Val in 1b and 2b seems to decrease the dynamic
quenching constant relative to Phe-derived compounds. The
same effect of the amino acid side chain in receptors 1 and 2
was also observed for Tyr and Met. Regarding the ground-state
complexes, the KS binding constants with Trp and Tyr are in
the ranges 20–60 and 2–4mꢀ1, respectively, and are negligible
with Met. The observation of a moderate association constant
with Trp in the ground state by means of fluorescence spec-
troscopy is in agreement with the chemical shifts recorded by
KD and KS quenching constants. As an example, Figure 10
shows the fluorescence-decay curves for the titration of 2a
with l-Trp.
Both types of measurements can be combined in the same
graph as for 2a with l-Trp (Figure 11). It can be observed that
the Stern–Volmer plot for the time-resolved fluorescence meas-
urements fits to a straight line. On the other hand, the repre-
sentation of the steady-state measurements shows an upward
curvature that can be fitted to Equation (2) [(by using the pre-
1
using H NMR spectroscopy, hence confirming the existence of
a supramolecular complex with Trp. This finding is remarkable
considering that quite extreme conditions were employed (i.e.,
1m H2SO4) and highlights the tendency of amino acids (in this
case, Trp) to form supramolecular complexes, even in the most
harsh environment. Remarkably, the association constants for
the macrocyclic receptors are approximately 50% higher than
for the open-chain compounds, which could be ascribed to
the well-known macrocyclic effect (preorganization of hosts).[75]
From the calculated KD constants, it is possible to obtain the
corresponding dynamic quenching rate constants kq by using
Equation (3). Table 3 summarizes the obtained kq constants for
the fluorescence quenching of the studied acridine-based re-
ceptors, which are in the range (2.0–3.9)ꢂ109 mꢀ1
cal small molecule in water, the diffusion rate is 7.4ꢂ
s
ꢀ1. For a typi-
Figure 10. Fluorescence-decay curves for the titration of 2a (20 mm) with l-
Trp (from 0 to 36 mm) in aqueous H2SO4 (1m). lex =372, lem =475 nm.
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known process of photo-induced electron transfer (PET). The
acridinium cation is an excellent oxidizing agent in the first ex-
cited state and, accordingly, the emission of acridinium and 9-
methylacridinium cations has been described to be strongly
quenched by electron-rich donors through PET with kq =(3.5–
Table 2. Parameters obtained from fitting to Stern–Volmer plots from the
titration of 1a, 2a, 1b, and 2b (20 mm) with a series of l- and d-amino
acids (from 0 to 36 mm) in aqueous H2SO4 (1m). lexc =366 nm.[a]
Aaa
[Mꢀ1
]
1a
2a
1b
2b
[b]
4.7)ꢂ109 mꢀ1 sꢀ1 [77]
In our case, we calculated the feasibility of
.
l-Trp
KD
86.5ꢁ1.7
32.5ꢁ0.3
92.8ꢁ1.7
23.9ꢁ0.1
61.5ꢁ0.8
58.4ꢁ0.6
80.5ꢁ1.2
85.7ꢁ1.4
72.3ꢁ1.0
2.68ꢁ0.13
77.4ꢁ1.1
2.44ꢁ0.13
83.7ꢁ1.2
52.0ꢁ0.8
83.5ꢁ1.0
45.4ꢁ0.4
54.3ꢁ0.4
49.5ꢁ0.6
81.4ꢁ1.8
87.5ꢁ2.1
70.2ꢁ1.1
3.67ꢁ0.18
75.3ꢁ1.1
3.75ꢁ0.20
74.0ꢁ0.2
32.1ꢁ0.4
70.8ꢁ0.5
30.7ꢁ0.3
51.0ꢁ0.4
49.2ꢁ0.3
65.2ꢁ1.3
68.5ꢁ1.5
59.1ꢁ0.4
2.60ꢁ0.19
60.0ꢁ0.6
3.06ꢁ0.17
59.1ꢁ1.2
59.2ꢁ0.5
74.1ꢁ0.7
40.7ꢁ0.2
43.6ꢁ0.5
43.0ꢁ0.3
69.2ꢁ1.5
63.8ꢁ1.5
59.3ꢁ1.0
3.11ꢁ0.15
54.7ꢁ0.8
3.34ꢁ0.20
[c]
KS
KD
KS
KD
KD
KD
KD
KD
KS
KD
KS
PET for Trp, Tyr, and Met to the pseudopeptidic receptors by
using the Rehm–Weller formulation[78] (see the Supporting In-
formation). In all the cases, the resulting DG values associated
with the PET process are negative (DG=ꢀ20, ꢀ15, and
ꢀ7 kcalmolꢀ1 for Trp, Tyr, and Met, respectively), which sup-
ports employing the PET mechanism to explain the emission
quenching.
[b]
d-Trp
[c]
[d]
[d]
[e]
[e]
[f]
l-Met
d-Met
l-Tyr
d-Tyr
l-Tyr
[g]
[f]
d-Tyr
[g]
[a] Steady-state fluorescence experiments were measured at 500 nm for
compounds 1a, 2a, and 2b; and at 495 nm for compound 1b. Studies
with l-Ala, l-His, l-Val, and l-Phe indicated that these amino acids do not
quench the fluorescence of 1a, 1b, 2a, and 2b under the used experi-
mental conditions. [b] KD obtained from fluorescence lifetime measure-
ments. [c] KS obtained from steady-state fluorescence measurements
using the KD constant. [d] KD obtained from steady-state fluorescence
measurements. [e] KD obtained from steady-state fluorescence measure-
ments to the first points; a small variation from the linearity of the fits
was observed associated to a small binding constant. [f] KD obtained from
steady-state fluorescence measurements to the first points. [g] KS ob-
tained from steady-state fluorescence measurements using the KD con-
stant.
Thermodynamic studies
To understand the association process better, that is, whether
it is entropy or enthalpy controlled, the association constants
for 1a and 2a with l-Trp were determined by using UV/Vis ti-
trations at different temperatures. The use of UV/Vis spectros-
copy afforded values of KS analogous to those obtained by
fluorescence, but the absorption data were more robust for
thermodynamic studies than the emission data because emis-
sion is more sensitive to temperature changes than absorption.
In this regard, Figure 9 shows the marked change in the ab-
sorption spectra of the receptor with the addition of Trp,
which can be associated with the formation of the
Table 3. Dynamic quenching constants kq (Mꢀ1 sꢀ1) obtained from fittings to Stern–
Volmer plots of the titration of 1a, 2a, 1b, and 2b (20 mm) with a series of l- and d-
amino acids (from 0 to 36 mm) in aqueous H2SO4 (1m).
supramolecular complex. By assuming that the static
quenching is due the formation of a 1:1 complex be-
tween Trp and the acridine-based host molecule (A),
the corresponding equilibrium is defined by Equa-
tion (4) and the equilibrium constant by Equation (5).
Aaa
1a kq [Mꢀ1 sꢀ1
]
2a kq [Mꢀ1 sꢀ1
]
1b kq [Mꢀ1 sꢀ1
]
2b kq [Mꢀ1 sꢀ1
]
l-Trp
d-Trp
l-Met
d-Met
l-Tyr
(3.25ꢁ0.06)ꢂ109
(3.49ꢁ0.06)ꢂ109
(2.31ꢁ0.03)ꢂ109
(2.20ꢁ0.02)ꢂ109
(2.72ꢁ0.04)ꢂ109
(2.91ꢁ0.04)ꢂ109
(2.74ꢁ0.02)ꢂ108
(3.50ꢁ0.05)ꢂ109
(3.49ꢁ0.04)ꢂ109
(2.27ꢁ0.02)ꢂ109
(2.07ꢁ0.03)ꢂ109
(2.94ꢁ0.05)ꢂ109
(3.15ꢁ0.05)ꢂ109
(2.52ꢁ0.04)ꢂ108
(3.30ꢁ0.01)ꢂ109
(3.16ꢁ0.02)ꢂ109
(2.28ꢁ0.02)ꢂ109
(2.20ꢁ0.01)ꢂ109
(2.64ꢁ0.02)ꢂ109
(2.68ꢁ0.03)ꢂ109
(7.19ꢁ0.01)ꢂ108
(2.84ꢁ0.06)ꢂ109
(3.56ꢁ0.03)ꢂ109
(2.10ꢁ0.02)ꢂ109
(2.07ꢁ0.01)ꢂ109
(2.85ꢁ0.05)ꢂ109
(2.63ꢁ0.04)ꢂ109
(3.50ꢁ0.01)ꢂ108
A þ Trp Ð A ꢃ Trp
ð4Þ
ð5Þ
KS ¼ ½A ꢃ Trpꢂ=ð½Aꢂ½TrpꢂÞ
d-Tyr
NaCl
The representation of lnK versus 1/T (van’t Hoff
plot) allowed us to obtain the respective enthalpic
and entropic parameters DH8 and DS8 involved in
the binding process according to the expression of the Gibbs
free energy of binding defined in Equation (6).[75]
109 mꢀ1 sꢀ1 [62]
which is in agreement with the obtained results;
,
therefore, these rates are about one half of the diffusion-con-
trolled limit.[76] Sauer and co-workers reported bimolecular
fluorescence quenching constants in the range (2.0–5.0)ꢂ
109 mꢀ1 sꢀ1 for several bioprobes that interact with Trp, which
is in close agreement with our results. Additionally, a moderate
association in the ground state has been described for those
dyes with association constants in the range 96–206mꢀ1.[38]
More recently, Webb and co-workers described the fluores-
cence quenching of the bioprobe ALEXA 488 by Trp with a dy-
s
namic rate of kq =3.5ꢂ109 mꢀ1 ꢀ1; however, in that case, the
association of the dye and the amino acid was weaker
(15.1mꢀ1) than in the present situation.[34]
Regarding the physical mechanism that results in the
quenching of the fluorescence emission of the receptors by
Trp, it is reasonable to consider the participation of the well-
DG ¼ DHꢀTDS ¼ ꢀRTlnKS
ð6Þ
The variation of the absorbance in the titration of 1a and
2a with l-Trp at 5–358C in the UV/Vis titrations is displayed in
Figure 12. It can be observed that the maximum variation of
the absorbance decreases with the temperature and the curva-
ture also follows the same trend, thus indicating that the com-
plex formation is disfavored at higher temperatures. Moreover,
both the maximum variation of the absorbance and the curva-
ture are less marked in the open-chain compound 1a relative
to the macrocyclic compound 2a, which clearly suggests that
binding constants for macrocycle 2a are larger than for 1a,
thus confirming the existence of a macrocyclic effect.[79,80]
Chem. Eur. J. 2014, 20, 7465 – 7478
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Table 4. Parameters obtained from fitting to 1:1 Host–Guest model for
the titration of 1a and 2a (60 mm) with l-Trp (from 0 to 83 mm) in aque-
ous H2SO4 (1m).
T [8C]
KS [Mꢀ1] 1a
KS [Mꢀ1] 2a
5
23.2ꢁ0.5
17.4ꢁ0.5
13.6ꢁ1.1
10.7ꢁ0.7
51ꢁ3
15
25
35
44ꢁ4
37ꢁ4
31.1ꢁ1.6
Figure 13. Variation of the lnK with 1/T from fitting to the 1:1 host–guest
model for the titration of 1a and 2a with l-Trp (from 0 to 83 mm) in aque-
ous H2SO4 (1m). Probe concentration=60 mm.
shows excellent linearity, and therefore it was possible to
obtain the entropic and enthalpic contributions to the binding
process (Table 5; note the small errors associated with each
calculated thermodynamic parameter). In both compounds,
the DS and DH values are negative. Despite the remarkable
fact that the host and guest are positively charged these re-
sults confirm the presence of a significant enthalpic contribu-
tion that brings both molecules together. The unfavorable neg-
ative DS value can be rationalized in terms of the desolvation/
solvation process during complex formation, thus leading to
a host–guest complex more organized than the free molecules
Figure 12. Variation of the Abs for the titration of: a) 1a and b) 2a (60 mm)
with l-Trp (from 0 to 83 mm) in aqueous H2SO4 (1m) at different tempera-
tures (from 5 to 358C). Solid lines correspond to fitting to the 1:1 binding
model.
A fitting for each titration to a 1:1 binding model was per- in solution. The comparison of the macrocyclic versus the
formed by using Equation (7),[81] where DAbs is the increment open-chain structure is also interesting. On the one hand, the
of the absorbance, DAbsmax is the maximum increment of the enthalpy contribution is more favorable (more negative) for
absorbance, c is the concentration of guest, c0 is the concen- the open-chain compound 1a relative to the macrocyclic com-
tration of the host, and KS is the association constant
(Figure 12). From the results obtained from the fitting at each
Table 5. Parameters obtained from the fitting to a 1:1 Host–Guest model
temperature, the corresponding binding constants KS were ob-
for the titration of 1a and 2a (60 mm) with l-Trp (from 0 to 83 mm) in
aqueous H2SO4 (1m).
tained (Table 4).
[kalmolꢀ1
]
1a
2a
DAbs ¼ DAbsmax=2ðc=c0 þ 1 þ 1=ðKSc0Þ
DS
ꢀ9.5ꢁ0.2
ꢀ4.4ꢁ0.1
ꢀ2.8ꢁ0.1
ꢀ1.5ꢁ0.1
ꢀ2.3ꢁ0.5
ꢀ2.8ꢁ0.1
ꢀ0.7ꢁ0.1
ꢀ2.1ꢁ0.3
ð7Þ
2
1=2
ꢀf½c=c0 þ 1 þ 1=ðKSc0Þꢂ ꢀ4c=c0g
Þ
DH
T DS [a]
[a]
DG
From the obtained binding constants, the corresponding
[a] Calculated at 298 K.
van’t Hoff representations were plotted (Figure 13). The data
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pound 2a. This outcome is reasonable because the macrocy-
clic compound locates the three positive charges closer than
in the open-chain compound 1a, and the electrostatic repul-
sion is more difficult to avoid. But, on the other hand, the en-
tropic contribution is more favorable (less negative) for macro-
cyclic compound 2a as long as the macrocyclic compound is
more preorganized and its conformation does not change
upon complexation as much as the open-chain derivative 1a.
Overall, as a result of the combination of the former elements
at 258C, the binding is more exergonic for the macrocyclic
compound (ꢀ2.1 kcalmolꢀ1) than for the open-chain analo-
gous derivative (ꢀ2.1 versus ꢀ1.5 kcalmolꢀ1, respectively).
As the supramolecular species considered involve the inter-
action of two positively charged entities, it is reasonable to
assume that the counteranions present can have a significant
effect and participate in the supramolecular species. Most of
the checked anions provided some interference in the fluores-
cence experiments, but their effect on the binding constants
could be studied by means of additional UV titration experi-
ments of 2a (60 mm) with l-Trp (0–83 mm) in different aqueous
solutions of H3PO4, HCl, and HBr (1m) at 58C. From fitting to
a 1:1 host–guest model, the following binding constants were
obtained: KS =41ꢁ3, 37.0ꢁ1.0, and 21.5ꢁ1.1mꢀ1 for HCl, HBr,
and H3PO4, respectively. These binding constants confirm the
involvement of the anions in the binding of both charged spe-
cies and indicates that H2SO4 is best suited for the generation
of the acidic media required for fluorescence sensing.
Figure 14. The pseudopeptidic structures used for the computational calcu-
lations.
Computational models
Different computational calculations were carried out to postu-
late a tentative binding model. According to the experimental
ꢀ
data, the HSO4 counterion must play an important role, most
likely as a stabilizing element for the host–guest complex.
Therefore, the computational calculations were carried out for
the ternary complex formed between the receptor, Trp, and
HSO4ꢀ. To make the calculations computationally accessible,
the starting structures of the pseudopeptidic receptors were
simplified and the pseudopeptidic structures 1c and 2c de-
rived from alanine were used for the calculations (Figure 14).
Initially, different Monte Carlo conformational searches were
performed with the Spartan ‘08 software[82] by using the
MMFFaq force field. For each case, the most stable conformer
obtained was fully optimized by using the Gaussian 09 soft-
ware at the PM6 level of theory with water as the solvent.[83] In
these simulations, the amine groups of the pseudopeptides
were fully protonated to simulate the acidic pH values at
which the experiments were carried out. In the absence of hy-
drogen sulfate counteranions, it was not possible to obtain
a minimized supramolecular complex due to the electrostatic
repulsion between the two cationic species. In contrast, when
four hydrogen sulfate counteranions were included in the sim-
ulation to counterbalance the positive charge of the protonat-
ed host and guest, it was possible to optimize the structure of
the supramolecular complex, thus obtaining the geometry of
the supramolecular complexes displayed in Figure 15. In such
complexes, the distances between the center of the indole
Figure 15. Optimized geometries (PM6, solvent=water) for the complexes
with l-Trp in the presence of 4 HSO4 counteranions and receptors 1c (a)
and 2c (b).
ꢀ
ring of Trp and the center of the acridine ring of the pseudo-
peptide are 4.46 and 4.48 ꢃ (MMFFaq and PM6, respectively;
solvent=water) for 1c and 4.17 and 4.20 ꢃ (MMFFaq and PM6,
respectively; solvent=water) for 2c. Although this model can
be considered to be very simplistic, the obtained structures for
the complexes are in good agreement with the experimental
observations and show how aromatic interactions between the
acridinium and indole rings are possible and can be responsi-
ble for the strong fluorescence quenching observed in the
presence of Trp and for the selectivity observed for this amino
acid.
Alternatively, optimizations in the presence of explicit water
molecules allowed us to minimize the supramolecular complex
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in the presence and absence of any counteranion (see the Sup-
porting Information). The calculated structures show that in
the absence of the corresponding counteranions formation of
the supramolecular complex forces the existence of stronger
p–p interactions. Very interestingly, there is a good agreement
between the structures calculated by both methodologies
when the four couteranions are present. Thus, the distances
between the center of the indole ring of Trp and the center of
the acridine ring of the pseudopeptide in the presence of
ꢀ
4HSO4 counteranions are 4.65 and 4.10 ꢃ for 1c and 2c, re-
spectively; however, in the absence of counteranions, those
distances are 3.84 and 3.87 ꢃ, respectively.
Additional evidence for the participation of this type of
supramolecular species, according to the former model, was
provided by DOSY NMR spectroscopic experiments. The diffu-
sion coefficients for 2a in the absence and presence of l-Trp
were measured. The measurements were carried out in 5m
H2SO4 in D2O with tetraethylammonium (5 mm) as the internal
standard. The concentrations of the guest and host were 10
and 50 mm for 2a and l-Trp, respectively, to ensure that about
60% of the complex is formed (according to the calculated
constants). From the obtained diffusion coefficients (i.e.,
D(2a+l-Trp)=(1.10ꢁ0.03)·10ꢀ10
and
D(2a)=(1.28ꢁ
0.05)·10ꢀ10 m2 sꢀ1 as the average of four different experiments),
the hydrodynamic radius was obtained by using the Stokes–
Einstein equation R=kT/(6phD): R(2a+l-Trp)=(8.25ꢁ0.37)
and R(2a)=(7.36ꢁ0.30) ꢃ. The agreement of these data with
the computational models can be considered to be good. For
the structures computed by using a continuous solvation
model, the calculated radii of the spheres with origins in the
center of mass that include all atoms (Figure 16; i.e.,
Figure 16. Rmax values for the optimized geometries (MMFFaq): a) H32a3+ in
the presence of 3 HSO4ꢀ counteranions (Rmax(2a)=7.67 ꢃ); b) the complex of
R
max(2a+l-Trp)=8.88 and Rmax(2a)=7.67 ꢃ) are only slightly
+
H32a3+with l-Trp–H2 in the presence of 4 HSO4ꢀ counteranions
(Rmax =8.88 ꢃ); c) H32a3+ in the presence of 3 HSO4ꢀ counteranions with ex-
plicit water molecules (Rmax =7.99 ꢃ); d) the complex of H32a3+with l-Trp–
larger than those obtained experimentally. This outcome is to
be expected taking into consideration that only approximately
60% of the complex is formed under the experimental condi-
tions used. In fact, the computed ratio of Rmax(2a+l-Trp)/
Rmax(2a) is 1:1.16, whereas the experimentally obtained ratio of
R(2a+l-Trp)/R(2a) is 1:1.120ꢁ0.011.[84] For the models calculat-
ed in the presence of explicit water molecules, some interest-
ing data can be obtained. When the presence of the counter-
anions is also considered explicitly, the results show an excel-
lent agreement with those obtained by using the continuum
model and those obtained from DOSY experiments, with
Rmax(2a+l-Trp)=9.33 and Rmax(2a)=7.99 ꢃ. Some overestima-
tion of the Rmax values is observed, which, in the case of the
complex, could be assignable to partial complex formation,
but the value obtained for the Rmax ratio (1:1.17) is again very
close to the experimental value, as mentioned above. In the
+
H2 in the presence of 4 HSO4ꢀ counteranions with explicit water molecules
(Rmax =9.33 ꢃ); e) H32a3+ with explicit water molecules (Rmax =6.13 ꢃ); f) the
+
complex of H32a3+with l-Trp-H2 with explicit water molecules
(Rmax =7.93 ꢃ).
sation theory of attraction between like charges assisted by
counterions.[85–87]
Conclusion
Two new pseudopeptidic receptors derived from valine (open-
chain and macrocyclic) and containing an acridine spacer have
been designed, synthesized, and characterized. Both receptors
and the previously prepared phenylalanine-derived com-
pounds can associate with free tryptophan in 100% aqueous
medium under very acidic conditions. Other studied amino
acids show low (Tyr) or negligible (Met, His, Phe, Val, Ala) com-
plexation. Macrocyclic compounds bound more efficiently to
Trp than the related open-chain compounds, most likely be-
cause of their more favorable preorganization. The recognition
process was studied using 1H NMR, UV/Vis, and fluorescence
spectroscopic analysis and computational calculations, and
ꢀ
absence of HSO4 ions, however, the calculated values are
Rmax(2a+l-Trp)=7.93 and Rmax(2a)=6.13 ꢃ, and the Rmax ratio
is 1:1.29, which are much less consistent with the experimental
values. These experiments, along with the observed influence
of the nature of the anion present, strongly suggest the in-
volvement of the counteranions in the supramolecular species.
The participation of anions in the assembly of two species
with the same charge assisted by anions suggested in our
model is similar to those described by the counterion-conden-
Chem. Eur. J. 2014, 20, 7465 – 7478
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a good agreement between all the data was observed. Very in-
terestingly, the results reported herein clearly highlight the as-
sociation between two positively charged species, in which the
interaction involves aromatic interactions and, most likely, elec-
trostatic and hydrogen-bonding interactions that involve the
counteranions and solvent molecules. This strong interaction
between the indole ring of the tryptophan and the acridinium
ring of the host seems to be responsible for the selectivity ob-
served for this amino acid. A direct extrapolation of our results
to the biological realm is not possible due to the non-natural
very acidic conditions required for our experiments. However,
the results obtained illustrate the potential to establish supra-
molecular species between two positively charged species, in
particular when assisted by the proper counteranions, which
can be of interest for understanding different association pro-
cesses of biological relevance. Thus, the formation of a supra-
molecular complex needs to involve strong favorable interac-
tions to compensate for this initial electrostatic repulsion.
On the other hand, as the selective recognition process of
tryptophan is associated with strong quenching of the fluores-
cence of the acridinium subunit at the receptor, the present re-
sults open new approaches for the development of fluorescent
selective sensors for an important biological target, such as the
amino acid tryptophan.
Fluorescence measurements
Titrations with amino acids in water: The compounds were dis-
solved in 1m H2SO4 to obtain 1 mm stock solutions from which
20 mm solutions in 1m H2SO4 were prepared. These solutions were
titrated by adding increasing volumes of stock solutions of 0–
36 mm of the corresponding amino acids in 1m H2SO4.
pH titrations in water: The different compounds were dissolved in
dimethyl sulfoxide (DMSO) to obtain 1 mm stock solutions. Those
solutions were diluted to 10 mm with water containing a mixture of
several buffers to facilitate titrations between pH 9 and 1 (sodium
acetate (40 mm), sodium phosphate (40 mm), sodium borate
(40 mm), and sodium carbonate (40 mm)). A small final percentage
of DMSO (1–2%) was required to avoid the precipitation of the
compounds due to their low solubility in water. Slight variations in
the pH values of the solutions were achieved by adding the mini-
mum volumes of 0.1–1.0m NaOH or 0.1–1.0m HCl (typically 10 mL
added to 10 mL), in such a way that dilution effects were negligi-
ble. The pKa values were obtained from the nonlinear curve-fitting
of the data with Origin 6.1.
Fluorescence-lifetime determination: Compounds were dissolved
in water and H2SO4 to obtain stock solutions (1 mm). These solu-
tions were diluted to 20 mm with water. Variations in the pH value
of the solutions were achieved by adding H2SO4 or NaOH. The
samples were purged with nitrogen.
Quantum-yield determination: Solutions of the compounds were
prepared as described for the fluorescence-lifetime determination.
Fluorescence quantum yields of 1, 2, and acridine are reported rel-
ative to quinine sulphate (aqueous solution in 0.1m H2SO4, air)
with FF =0.53.[67] The experiments were carried out in optically
matching solutions. Emission spectra were recorded upon excita-
tion at lexc =366 nm. The quantum yield was calculated by using
Equation (8). In this expression, it is assumed that the sample and
the reference are excited at the same wavelength so that it is not
necessary to correct for the different excitation intensities at differ-
ent wavelengths.
Experimental Section
Materials and methods
All the commercially available reagents and solvents were used as
received, without further purification. Acridine was recrystallized
from ethanol/water as described previously.[66]
2
Ff ¼ Fr ꢄ ðArFs=AsFrÞ ꢄ ðns =nr2Þ
ð8Þ
Steady-state fluorescence spectroscopy: For fluorescence titra-
tions in water, steady-state fluorescence spectra were recorded on
a Spex Fluorolog 3–11 fluorimeter equipped with a 450 W xenon
lamp. Fluorescence spectra were recorded in the front-face mode.
where Ff is the quantum yield, F is the integrated intensity, A is
the optical density, and n is the refractive index. The subscript r
refers to the reference fluorophore of known quantum yield and
subscript s to the sample. The spectra were appropriately correct-
ed.
Time-resolved fluorescence spectroscopy: Time-resolved fluores-
cence measurements were carried out by using time-correlated
single-photon counting (TCSPC) on an IBM-5000J apparatus. Sam-
ples were excited with a NanoLED at l=372 nm with a full width
at half maximum (FWHM) value of l=1.3 ns and a repetition rate
of 100 kHz. Data were fitted to the appropriate exponential model
after deconvolution of the instrument response function by using
an iterative deconvolution technique with the IBH DAS6 fluores-
cence-decay-analysis software, in which reduced c2 and weighted
residuals serve as parameters for the goodness of fit.
NMR diffusion studies
All DOSY experiments were conducted in quadruplicate on a Varian
NMR System 500 MHz spectrometer at 300 K equipped with PER-
FORMA IV pulse-field gradients. Data were collected with the bipo-
lar pulse-pair stimulated-echo sequence. The diffusion parameters
were optimized to obtain a 90–95% on signal-intensity decay. Typi-
cal values are diffusion times (D) of 100 ms, with encoding gradi-
ent pulses of total a duration d of 2 ms. An array of 30 values of
gradient strength was acquired with 14 scans per value. Diffusion
coefficients were calculated by using the values of the intensity of
the observed signal from the Stejskal–Tanner equation[88] with
Origin 6.1 software.
NMR spectroscopy: 1H and 13C NMR spectra were recorded on
1
a 500 MHz spectrometer (500 and 125 MHz for H and 13C, respec-
1
tively) or a 300 MHz spectrometer (300 and 75 MHz for H and 13C,
respectively).
IR spectroscopy: Attenuated total reflectance (ATR) FTIR spectra
were acquired on a JASCO 6200 equipment with a MIRacle single-
reflection ATR diamond/ZnSe accessory. The raw IR data were pro-
cessed with the JASCO spectral manager software.
Computational studies
UV/Vis spectroscopy: UV/Vis absorption measurements were re-
corded on a Hewlett–Packard 8453 spectrophotometer, equipped
with a temperature-control accessory Peltier Agilent 89090A.
Monte Carlo conformational searches with the MMFFaq force field
were carried out with the Spartan ‘08 software for triprotonated
model compounds 1c and 2c[82] and their supramolecular com-
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plexes with monoprotonated tryptophan in the presence and ab-
sence of hydrogen sulfate counteranions. The most stable con-
formers were optimized at the PM6 level of theory with water as
the solvent in the Gaussian 09 software.[83] All the geometries were
fully optimized and were checked to be true minima by analysis of
the vibration normal modes. The 2a model employed for the cal-
culation of the Rmax value was obtained from the 2c MMFFaq most
stable conformers by adding the phenyl substituents. Explicit
water molecules were added by using the solvate plugin (TIP3BOX
solvent model, the box salvation model, and a box size of 5) imple-
mented in the UCSF Chimera package and the resulting solvated
structure was minimized with the Spartan ‘08 software. Chimera
was developed by the Resource for Biocomputing, Visualization,
and Informatics at the University of California (San Francisco, USA;
supported by NIGMS P41-GM103311).[89]
174.3, 146.8, 136.7, 136.4, 129.5, 127.7, 126.6, 125.7, 60.4, 40.6, 30.9,
19.7, 16.1 ppm; IR (ATR): n˜ =1430, 1638, 3066, 3277 cmꢀ1; HRMS
(ESI-TOF): m/z calcd for C41H39N5O2: 436.2713 [M+H+]; found
436.2717; elemental analysis calcd (%) for C41H39N5O2: C 68.94, H
7.64, N 16.08; found: C 68.82, H 7.62 N, 15.82.
Compound 2b: Dry acetonitrile (165 mL) was added to 1b
(0.500 g, 1.148 mmol), TBABr (0.1850 g, 0.574 mmol), and DIPEA
(1.965 mL, 11.48 mmol) placed in a 250 mL round-bottom flask.
The reaction mixture was stirred at room temperature until com-
plete dissolution of the reagents, and then 1,3-bis(bromomethyl)-
benzene (0.3030 g, 1.148 mmol) dissolved in a small amount of dry
acetonitrile was added. The reaction mixture was heated using
a temperature gradient from 50.0 to 81.68C (to reflux) for 24 h in
a nitrogen atmosphere. After cooling, the solvent was eliminated
by vacuum. Purification of the crude product was carried out by
column chromatography over silica gel (dichloromethane/metha-
nol 100:0!100:5 with a few drops of aqueous ammonia). The
macrocycle was obtained as a white solid (0.3889 g, 0.723 mmol,
63%). M.p. 100–1028C; [a]2D5 =103.98 (c=0.01 in CHCl3); 1H NMR
Synthesis of receptors
Compound 1b-Cbz: Aqueous NaOH (1m, 10 mL) and MeOH (1 mL)
were added to 4,5-bis(aminomethyl)acridine·2HCl (0.200 g,
0.645 mmol) placed in a 50 mL round-bottom flask. After complete
dissolution of the solid, the solution was extracted three times
with dichloromethane (15 mL). The organic layers were collected
and dried over anhydrous MgSO4. The solvent was then vacuum
evaporated to afford 4,5-bis(aminomethyl)acridine (0.142 g,
0.600 mmol, 93%) as a yellowish solid. This solid was dissolved in
3
(500 MHz, CDCl3): d=8.67 (s, 1H), 8.13 (d, J(H,H)=5.7 Hz, 2H), 7.84
(d, 3J(H,H)=8.4 Hz, 2H), 7.73 (d, 3J(H,H)=6.6 Hz, 2H), 7.42 (t,
3J(H,H)=7.5 Hz, 2H), 7.31 (s, 1H), 7.05–7.19 (m, 3H), 5.10 (dd,
3
3J(H,H)=7.9, 14.9 Hz, 2H), 4.78 (dd, J(H,H)=5.0, 14.9 Hz, 2H), 3.96
(d, 3J(H,H)=12.6 Hz, 2H), 3.54 (d, 3J(H,H)=12.6 Hz, 2H), 3.01 (d,
3J(H,H)=4.6 Hz, 2H), 1.98 (dd, 3J(H,H)=6.4, 11.9 Hz, 4H), 0.85 (d,
3J(H,H)=6.9 Hz, 6H), 0.68 ppm (d, 3J(H,H)=6.8 Hz, 6H); 13C NMR
(126 MHz, CDCl3): d=174.0, 146.6, 140.4, 136.7, 136.4, 129.2, 129.1,
128.9, 127.6, 127.6, 126.4, 125.6, 69.8, 54.4, 40.6, 31.5, 19.6,
DME (15 mL) in
a 50 mL round-bottom flask. Cbz-l-Val-OSu
(0.418 g, 1.2 mmol) was dissolved in DME (10 mL) and slowly
added to the reaction flask. A white precipitate was rapidly formed
and the mixture was stirred at room temperature for 20 h and
then at 508C for 6 h. After cooling, a white precipitate was collect-
ed by filtration and washed with cold water (50 mL) and a small
amount of methanol to afford the expected compound 1b-Cbz as
17.9 ppm; IR (ATR): n˜ =3290, 2870, 1646, 1387, 1555, 1513 cmꢀ1
;
HRMS (ESI-TOF): m/z calcd for C33H39N5O2: 538.3181 [M+H+];
found: 538.3180; elemental analysis calcd (%) for C33H39N5O2: C
73.71, H 7.31, N 13.02; found: C 73.47, H 7.37, N 12.79.
a white solid (0.266 g, 0.378 mmol, 63%). M.p. 252–2558C; [a]D25
=
13.948 (c=0.01 in DMSO); 1H NMR (500 MHz, [D6]DMSO): d=9.10
Acknowledgements
3
(s, 1H), 8.50 (t, 2H, J=5.7 Hz), 8.06 (d, J(H,H)=8.4 Hz, 2H), 7.71 (d,
3J(H,H)=6.7 Hz, 2H), 7.47–7.61 (m, 2H), 7.33 (d, 3J(H,H)=4.5 Hz,
12H), 4.72–5.42 (m, 8H), 3.97 (t, 3J(H,H)=7.9 Hz, 2H), 2.02 (dd,
3J(H,H)=6.9, 13.5 Hz, 2H), 0.80–1.01 ppm (m, 12H); 13C NMR
(75 MHz, [D6]DMSO): d=172.2, 156.9, 146.3, 137.8, 137.4, 129.0,
128.4, 128.3, 128.3, 127.9, 126.6, 126.3, 126.2, 66.1, 61.3, 39.4, 30.8,
20.0, 18.9 ppm; IR (ATR) n˜ =1454, 1645, 3064, 3282 cmꢀ1; HRMS
(ESI-TOF): m/z calcd for C41H45N5O6: 704.3448 [M+H+]; found
704.3458; elemental analysis calcd (%) for C41H45N5O6: C 69.97, H
6.44, N 9.95; found: C 69.86, H 6.48, N 10.12.
Financial support from was provided by the Spanish MINECO
(CTQ2012-38543-C03-01) and Generalitat Valenciana (PROME-
TEO 2012/020). V.M.-C acknowledges the Spanish Ministry of
Science (FPU AP2007-02562) and Generalitat Valenciana
(APOSTD/2013/041) for personal support. The technical sup-
port of the SCIC of the UJI for different instrumental tech-
niques is acknowledged.
Compound 1b: A HBr/AcOH solution (33%, 50 mL) was added to
1b-Cbz (2.000 g, 2.84 mmol) placed into a 100 mL round-bottom
flask. After complete dissolution of the solid, a yellow solution was
formed. The reaction was stirred in a nitrogen atmosphere for 1 h.
The resulting colorless solution was poured into a 250 mL beaker
containing diethyl ether (150 mL). A white precipitate was collect-
ed by filtration and washed with diethyl ether. The solid was redis-
solved in distilled water (30 mL), the solution was washed twice
with CHCl3 (30 mL), and was basified with solid NaOH until pH 12–
13. NaCl was added until saturation and the aqueous phase was
extracted three times with CHCl3 (30 mL). The organic phase was
dried over anhydrous MgSO4, and the solvent was eliminated by
vacuum to afford 1 as a white solid (1.014 g, 2.329 mmol, 82%).
M.p. 1268C; [a]2D5 =ꢀ73.608 (c=0.01 in CHCl3); 1H NMR (500 MHz,
Keywords: fluorescence
pseudopeptides · supramolecular chemistry · tryptophan
·
molecular
recognition
·
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