Selective recognition of tryptophan through inhibition of intramolecular charge-transfer interactions in an aqueous medium

Article abstract of DOI:10.1021/ol062636m

A novel donor-acceptor conjugate 1 was synthesized, and its interactions with various amino acids have been investigated as compared to the model system 2. The conjugate 1 unusually forms an intramolecular charge-transfer complex in the aqueous medium and undergoes selective binding interactions with tryptophan. The uniqueness of this system is that it selectively recognizes tryptophan among all other amino acids and involves synergistic effects of π-stacking, electrostatic, and donor-acceptor interactions.

Full text of DOI:10.1021/ol062636m

ORGANIC
LETTERS
2
007
Vol. 9, No. 3
17-420
Selective Recognition of Tryptophan
through Inhibition of Intramolecular
Charge-Transfer Interactions in an
Aqueous Medium
4
Mahesh Hariharan, Suneesh C. Karunakaran, and Danaboyina Ramaiah*
Photosciences and Photonics, Chemical Sciences and Technology DiVision,
Regional Research Laboratory (CSIR), TriVandrum 695019, India
rama@csrrltrd.ren.nic.in; d_ramaiah@rediffmail.com
Received October 27, 2006
ABSTRACT
A novel donor−acceptor conjugate 1 was synthesized, and its interactions with various amino acids have been investigated as compared to
the model system 2. The conjugate 1 unusually forms an intramolecular charge-transfer complex in the aqueous medium and undergoes
selective binding interactions with tryptophan. The uniqueness of this system is that it selectively recognizes tryptophan among all other
amino acids and involves synergistic effects of
π-stacking, electrostatic, and donor−acceptor interactions.
Selective recognition of amino acids has great significance
as they assemble together to yield proteins, enzymes,
structural elements, and many macromolecules of biological
Most known receptors for amino acids employ the comple-
mentary hydrogen bonding for their recognition. However,
7
such recognition in an aqueous medium would be limited
due to the competitive hydrogen bonding of the solvent.8
The selective recognition involving electrostatic interactions
would also be limited due to the dipolar nature of all the
1
-3
function.
Of all the amino acids, the recognition of
tryptophan (Trp) is vital as it forms the precursor for the
4
secretion of the sleep inducing hormone, melatonin, and has
9
also been implicated as a possible cause of Schizophrenia
natural amino acids. There are a few reports in the
5
,6
10
due to its improper metabolism under in vivo conditions.
literature, which employ autofluorescence for the detection
and quantification of Trp. However, such a method would
be limited because other aromatic amino acids such as
tyrosine (Tyr) and phenylalanine (Phe), which exhibit similar
(
1) (a) Folmer-Anderson, J. F.; Kitamura, M.; Anslyn, E. V. J. Am. Chem.
Soc. 2006, 128, 5652. (b) Shinoda, S.; Okazaki, T.; Player, T. N.; Misaki,
H.; Hori, K.; Tsukubu, H. J. Org. Chem. 2005, 70, 1835. (c) Comeau, A.
B.; Seto, C. T. Org. Lett. 2004, 6, 83. (d) Hossain, M. A.; Schneider, H.-J.
J. Am. Chem. Soc. 1998, 120, 11208. (e) Sirish, M.; Schneider, H.-J. Chem.
Commun. 1999, 907.
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(
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H.; Yeung, K. T.; Cheng, Y. K.; Yang, R. H. Org. Lett. 2005, 7, 5825.
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Soc. 2003, 125, 13819. (b) Huang, X.; Fujioka, N.; Pescitelli, G.; Koehn,
F. E.; Williamson, R. T.; Nakanishi, K.; Berova, N. J. Am. Chem. Soc.
2002, 124, 10320.
1
(
2
1
0.1021/ol062636m CCC: $37.00
© 2007 American Chemical Society
Published on Web 01/10/2007

absorption and fluorescence emissions, albeit in low yields,11
would interfere with the recognition. Progress in this area
would require new strategies for the recognition of Trp in
the aqueous medium and subsequent signaling of the event
through optical changes.12
In this context, we designed a novel donor-acceptor
conjugate 1, for the selective recognition of Trp by making
use of its beneficial electron-donating, electrostatic, and
π-stacking properties (Figure 1 and Supporting Information
Figure 2. Absorption spectra of 1 (8.5 µM) and the model
compound 2 (8.15 µM) in an aqueous medium. Inset shows the
corresponding emission spectra. Excitation wavelength ) 340 nm.
The picosecond time-resolved fluorescence studies of 1 in
an aqueous medium showed a biexponential decay with
lifetimes of 4.3 (22%) and 121 ns (78%), when monitored
at 400 nm (Figure 3). However, when the emission was
Figure 1. Structures of the viologen-linked pyrene conjugate 1,
the model compound 2, and selected amino acids.
Figures S1-S4). Interestingly, our results indicate that 1
forms a fluorescent intramolecular charge-transfer (ICT)
complex in the aqueous medium and exhibits unusual
selectivity for Trp. Uniquely, this system is devoid of
hydrogen bonding but recognizes Trp selectively through
inhibition of the ICT complex involving π-stacking, elec-
trostatic, and donor-acceptor interactions and signals the
event through changes in steady-state and time-resolved
1
fluorescence, H NMR, and cyclic voltammetric (CV)
techniques.
Figure 2 shows the absorption spectra of the viologen-
linked pyrene conjugate 1 and the model compound 2 in an
aqueous medium. In addition to the absorption corresponding
to the pyrene chromophore at 344 nm, the viologen-linked
pyrene derivative 1 shows a new and diffused band in the
long wavelength region that extends up to 440 nm. Similarly,
the fluorescence spectrum of 1, on the other hand, showed
a broad and red-shifted emission centered at 475 nm, in
addition to the characteristic pyrene chromophore emission
at 400 nm (inset of Figure 2). In contrast, the model
compound 2 exhibited only the absorption and fluorescence
spectra that are characteristic of the pyrene chromophore.
Figure 3. Picosecond time-resolved fluorescence spectra of 1 (13
µM) in an aqueous medium. Emission collected at (a) 400 and (b)
4
75 nm. Excitation wavelength ) 335 nm. Inset shows excitation
spectra of 1 (8.5 µM) in an aqueous medium. Emission monitored
at (a) 400 and (b) 475 nm.
monitored at 475 nm, we observed only monoexponential
decay with a lifetime of 4.3 ns (100%).
(
9) (a) Folmer-Anderson, J. F.; Kitamura, M.; Anslyn, E. V. J. Am. Chem.
Soc. 2005, 127, 7986. (b) Schmuck, C.; Bickert, V. Org. Lett. 2003, 5,
579. (c) Sasaki, S.; Hashizume, A.; Citterio, D.; Fujii, E.; Suzuki, K.
To have a better understanding of the species that exhibited
long wavelength emission at 475 nm in the case of derivative
1, we have recorded the fluorescence excitation spectra,
following the emission at different wavelengths (inset of
Figure 3). When monitored at 400 nm, the fluorescence
excitation spectrum of 1 is found to be characteristic of the
pyrene moiety. In contrast, when monitored at 475 nm, the
excitation spectrum showed a peak around 400 nm that
extended up to 440 nm. This band is different from the
absorption of the pyrene chromophore (344 nm).
4
Tetrahedron Lett. 2002, 43, 7243. (d) Sirish, M.; Chertkov, V. A.; Schneider,
H.-J. Chem.-Eur. J. 2002, 8, 1181.
(
10) Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter,
P. In Molecular Biology of the Cell, 4th ed.; Garland Science: New York,
002.
11) (a) Galian, R. E.; Pastor-Perez, L.; Miranda, M. A.; Perez-Prieto, J.
2
(
Chem.-Eur. J. 2005, 11, 3443. (b) Wang, X.; Nau, W. M. J. Am. Chem.
Soc. 2004, 126, 808.
(
12) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley,
A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997,
7, 1515.
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418
Org. Lett., Vol. 9, No. 3, 2007

The nature of the unusual species observed in the case of
the viologen-linked pyrene conjugate 1 was further inves-
tigated through the effects of concentration, solvent, and
temperature (Supporting Information Figures S5-S8). With
the increase in concentration of the conjugate 1 in the
aqueous medium, we observed an increase in emission
intensity at 475 nm as well as at 400 nm corresponding to
the pyrene chromophore. However, the relative change in
fluorescence intensity (I475/I400) with an increase in concen-
tration of 1 showed a sigmoidal nature. On the other hand,
the fluorescence spectra of 1 in methanol and phosphate
buffer (pH 7.4; 10 mM) showed only the emission that is
characteristic of the pyrene chromophore. The broad and
structureless emission at 475 nm was not observed under
these conditions, indicating its sensitivity to the nature of
13
the medium. As the temperature increases, the emission at
75 nm showed a decrease in intensity with a concomitant
Figure 4. Change in the fluorescence spectrum of 1 (13 µM) in
4
an aqueous medium with an increase in the addition of Trp. [Trp]
increase in the intensity of the pyrene chromophore at 400
nm. However, the emission at 475 nm could be observed
even at 75 °C, indicating the stability of this species at these
-4
(
a) 2.9 and (k) 27.0 × 10 M. Inset shows the Benesi-Hildebrand
analysis. Excitation wavelength ) 340 nm.
1
4
temperatures.
along with a concomitant increase in intensity at 400 nm,
corresponding to the ICT complex and the pyrene chro-
mophore, respectively. The Benesi-Hildebrand analysis of
the fluorescence changes observed at 475 nm gave a 1:1
stoichiometry for the complex formed between the conjugate
The new broad absorption band observed (370-440 nm)
in the case of the conjugate 1 could be attributed to the
formation of the fluorescent intramolecular charge-transfer
(
ICT) complex between the pyrene chromophore and the
viologen moiety. This assignment is based on the intermo-
lecular fluorescence quenching of the pyrene chromophore
by methylviologen, characterization of the pyrene radical
1
M
and Trp with an association constant of Kass ) 1300 ( 29
-
1
-1
and the change in free energy of -17.7 kJ mol . This
value is in good agreement with the association constant (Kass
cation and reduced viologen by laser flash photolysis
-1
studies,15 and the theoretically calculated favorable change
) 1261 ( 33 M ) calculated using the fluorescence changes
observed but excited at 400 nm (Supporting Information
Figure S9). When we titrated the conjugate 1 with Phe and
Tyr, we obtained significantly lower association constants
16
in free energy (∆G ) -1.6 eV) for such an electron transfer
between pyrene and viologen moieties. Further evidence for
the involvement of a fluorescent ICT complex is obtained
from the negligible results with the model compound 2,
which lacks the viologen unit. In addition, we observed both
folded-sandwich and extended conformers through AM1
calculations, wherein the former one is expected to undergo
effective ICT interactions.
-
1
of 73 and 52 M , respectively, whereas other amino acids
such as Gly, Cys, Thr, Leu, and His under similar conditions
exhibited negligible changes in both the absorption and the
fluorescence properties of 1 (Supporting Information Figure
S10). These results indicate that the conjugate 1 interacts
selectively with Trp through altering the ICT complex
formation, whereas all other amino acids exhibit negligible
interactions (Supporting Information Figure S11). Interest-
ingly, the selectivity of the conjugate 1 toward Trp was
observed even in the presence of equimolar amounts of other
amino acids.
Our next objective was to evaluate and employ the
beneficial properties of the viologen-linked pyrene 1 for the
recognition of amino acids because this conjugate forms a
novel fluorescent intramolecular charge-transfer (ICT) com-
plex in the aqueous medium. We selected a few naturally
occurring important aromatic and, for comparison, aliphatic
L-amino acids (Figure 1) and have investigated their interac-
The complex formation between the conjugate 1 and Trp
was further demonstrated using picosecond time-resolved
1
tions with the conjugate 1 through photophysical, H NMR,
1
fluorescence, CV, and H NMR techniques. In CV, conjugate
and CV techniques. Figure 4 shows the changes in the
fluorescence spectra of the conjugate 1 with an increase in
concentration of Trp in the aqueous medium. We observed
a regular decrease in the fluorescence intensity at 475 nm
1
exhibited two reversible one-electron reduction processes
centered at -0.46 and -0.74 V, characteristic of the viologen
moiety (Figure 5). However, in the presence of Trp, we
observed a significant decrease in current intensity of 7.2
µA (32%) and 13.5 µA (33%), indicating the formation of a
(13) (a) Schneider, H.-J.; Kramer, R.; Simova, S.; Schneider, U. J. Am.
Chem. Soc. 1988, 110, 6442. (b) Schneider, H.-J. Angew. Chem., Int. Ed.
Engl. 1991, 30, 1417.
1
7
strong complex between 1 and Trp. The time-resolved
decay analysis indicated that 1 alone exhibits a monoexpo-
nential decay with a lifetime of 4.3 ns when monitored at
(
14) Birks, J. B. Photophysics of Aromatic Molecules; Wiley-Inter-
science: New York, 1970; Chapter 9.
15) (a) Hariharan, M.; Joseph, J.; Ramaiah, D. J. Phys. Chem. B 2006,
(
1
9
1
10, 24678. (b) Joseph, J.; Eldho, N. V.; Ramaiah, D. Chem.-Eur. J. 2003,
, 5926. (c) Joseph, J.; Eldho, N. V.; Ramaiah, D. J. Phys. Chem. B 2003,
07, 4444.
(17) (a) Neelakandan, P. P.; Hariharan, M.; Ramaiah, D. Org. Lett. 2005,
7, 5765. (b) Neelakandan, P. P.; Hariharan, M.; Ramaiah, D. J. Am. Chem.
Soc. 2006, 128, 11334. (c) Jisha, V. S.; Arun, K. T.; Hariharan, M.; Ramaiah,
D. J. Am. Chem. Soc. 2006, 128, 6204.
(
16) Murov, S. L.; Carmichael, I.; Hug, G. L. In Handbook of
Photochemistry, 2nd ed.; Marcel Dekker, Inc.: New York, 1993.
Org. Lett., Vol. 9, No. 3, 2007
419

Figure 5. Square wave voltammograms of 1 (0.28 mM) with an
increase in the addition of Trp. [Trp] (a) 0, (b) 1.02, (c) 2.0, and
Figure 6. Minimum energy conformer of 1 in the absence and
presence of Trp obtained through AM1 calculations.
(d) 2.95 mM in water and a scan rate of 100 mV/s. Inset shows the
picosecond time-resolved fluorescence spectra of 1 (13 µM) in an
aqueous medium in the absence (a) and presence (b) of Trp (27 ×
-
4
10
M). Emission collected at 475 nm and an excitation wave-
length of 335 nm.
revealed a 1:1 stoichiometry for the complex, wherein the
indole unit of Trp is located in close proximity to the folded
conformer of 1. As a consequence of the complexation, the
folded conformer of 1 transforms to a relatively stable,
expanded, tweezerlike structure. The evidence for such a
transformation is obtained from the time-resolved fluores-
4
75 nm, whereas a significantly decreased lifetime of 3 ns
was observed in the presence of Trp (inset of Figure 5). The
successive additions of Trp to a solution of 1 in D O resulted
2
in broadening and an upfield shift (∆δ 0.1 ppm) of protons
1
cence, CV, and H NMR studies. We observed a significantly
corresponding to the pyrene and viologen moieties in the
reduced lifetime and intensity of the species corresponding
to the ICT complex in the picosecond time-resolved studies.
Further, the decrease in current intensity and broadening and
shielding of protons of the viologen moiety due to the
interactions with the indole moiety confirm the formation
of a stable complex between Trp and the conjugate 1.
In summary, we have developed a novel donor-acceptor
conjugate which can form a fluorescent intramolecular
charge-transfer complex in the aqueous medium. Results
indicate that the formation of the ICT complex is favored
through the cooperativity effect arising from hydrophobic,
π-stacking, and electrostatic interactions of the units present
in the system. Interestingly, this conjugate forms a novel
molecular recognition system that can discriminate Trp from
other natural amino acids involving synergistic effects of both
π-stacking and electrostatic interactions and signals the event
through changes in the fluorescence intensity. Further studies
are in progress to understand the effect of a spacer on the
ICT complex formation and the nature of interactions
involved in the recognition of amino acids.
1
18
H NMR spectra (Supporting Information Figure S12). The
Benesi-Hildebrand analysis of the corresponding changes
in chemical shift values gave an association constant of 1337
-
1
(
32 M . This is in good agreement with the values
obtained from the fluorescence data and the changes in
chemical shift of the protons corresponding to Trp (downfield
shift; ∆δ 0.1 ppm) observed with the increase in addition of
the conjugate 1 (Supporting Information Figure S13, Kass
)
-
1
1
257 ( 19 M ).
The interesting observation is that the conjugate 1 exhibits
unusually high selectivity for Trp among all other amino
acids through inhibition of the ICT complex. This could be
attributed to the enhanced π-electron cloud of the indole moi-
ety of Trp, which promotes better π-stacking interactions
with the pyrene and viologen moieties,19 followed by the
electronic interactions between the carboxyl group of Trp and
the viologen moiety. These interactions lead to the formation
of a stable complex between 1 and Trp. In contrast, other
aromatic amino acids such as Tyr and Phe and aliphatic amino
acids Gly, Cys, Thr, and Leu showed negligible interactions
due to their lower π-electron density and hence are incapable
of forming stable complexes with the conjugate 1.
Acknowledgment. This work was supported by CSIR
and DST, Government of India. This is contribution no. PPD-
235 from RRL, Trivandrum, India.
We further carried out the minimum energy AM1 calcula-
tions to understand the nature of complexation between the
conjugate 1 and Trp. As shown in Figure 6, these calculations
Supporting Information Available: Details of synthesis
and calculations, Figures S1-S13 showing fluorescence, and
1
(
18) NMR titrations of Trp using different concentrations of the conjugate
(3-13 µM) afforded similar association constants ((2%).
19) (a) Bush, M. E.; Bouley, M. D.; Urbach, A. R. J. Am. Chem. Soc.
005, 127, 14511. (b) Werner, F.; Schneider, H.-J. HelV. Chim. Acta 2000,
3, 465. (c) Malta, L. F. B.; Senra, J. D.; Medeiros, M. E.; Antunes, O. A.
H NMR spectra of the conjugate 1 under various conditions.
1
This material is available free of charge via the Internet at
http://pubs.acs.org.
(
2
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C. Supramol. Chem. 2006, 18, 327.
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