Design and synthesis of a novel enediynyl pentapeptide with predominantly β-turn structural motif and its potential as a fluorescence-based chemosensor

Article abstract of DOI:10.1016/j.bmc.2005.03.038

A novel enediynyl pentapeptide in the protected form 1 was synthesized and characterized. It exists predominantly in β-turn structural motif as revealed by variable temperature NMR and CD spectroscopy. In the presence of transition metal ions and gold nan

Full text of DOI:10.1016/j.bmc.2005.03.038

Bioorganic & Medicinal Chemistry 13 (2005) 4096–4102
Design and synthesis of a novel enediynyl pentapeptide
with predominantly b-turn structural motif and its potential as
a fluorescence-based chemosensor
Amit Basak,a,* Subhendu Sekhar Baga and Ajoy Basakb
aContribution from the Bioorganic Laboratory, Department of Chemistry Indian Institute of Technology, Kharagpur 721 302, India
bRegional Protein Chemistry Center, Diseases of Aging, Ottawa Health Research Institute, Ottawa University,
725 Parkedale Ave, Ottawa, Canada ON K1Y 4E9
Received 7 January 2005; revised 19 March 2005; accepted 21 March 2005
Available online 25 April 2005
Abstract—A novel enediynyl pentapeptide in the protected form 1 was synthesized and characterized. It exists predominantly in b-
turn structural motif as revealed by variable temperature NMR and CD spectroscopy. In the presence of transition metal ions and
gold nanoparticles, the fluorescence intensity of the peptide got enhanced with remarkable quantum yield with the Z-enediynyl x-
amino acid acting as a fluorophoric reporter. The interesting photophysical behaviors with alkali and alkaline earth metal ions are
also reported.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
of biologically active molecules, such as lipids, oligonu-
cleotides, and peptides. Since fluorescence spectroscopy
is a very sensitive technique, fluorophore-bound gold
nanoparticles are useful probes for biomolecular label-
ing (e.g., as immunoprobes).8 Both small peptide (e.g.,
GHK based ones reported by Leblanc and co-workers9)
and protein based fluorescence sensors (e.g., mimics for
zinc finger motif as developed by Berg and Shi10a,b and
Imperiali and co-workers10c,d) have been designed and
their photophysical properties evaluated. In these de-
signs, the fluorophoric activities of dansyl and anthra-
cene moieties have been exploited. As part of our
efforts to prepare scaffolds for b-turn peptidomimetics11
as well as metal ion specific peptides, we have designed
and synthesized a pentapeptide 1 where a novel enediy-
nyl x-amino acid is acting as fluorophoric reporter. To
our knowledge, this is the first report of application of
the intrinsic fluorophoric property of Z-enediyne in
monitoring the binding process of metal ions and gold
nanoparticles.
Ever since the discovery of naturally occurring enediy-
nes, efforts are ongoing to find analogous rationally de-
signed molecules having less toxicity without sacrificing
the antitumor activity of the parent system.1 Evaluation
of the parameters controlling the Bergman cyclization
(BC) is another important component that will help in
the successful design of such molecules.1 The enediynes,
possessing extended unsaturation, is likely to be per-
turbed by photoirradiation. However, this aspect has
so far been restricted only to the study of BC under
photoirradiation.2 Because of the presence of special
structural features, in which a double bond is flanked
between two acetylenes in a cis fashion, it occurred to
us that the enediynes may serve as a probe for fluores-
cence spectroscopy provided suitable chelating append-
ages are incorporated in the two arms. The
fluorescence sensing, for example, of metal ions, is of im-
mense interest in biomedical research3 and as chemical
logics.4 Efforts have also been directed toward the devel-
opment of biological nanosensors and optoelectronic
nanodevices.5–7 Colloidal gold functionalized with spe-
cific binding groups can be used to label a wide variety
2. Results and discussion
2.1. Synthesis of the pentapeptide
Keywords: Enediyne; Peptide; b-Turn; Fluorescence; Chemosensor.
Corresponding author. Tel.: +91 322283300; fax: +91 322282252;
e-mail: absk@chem.iitkgp.ernet.in
*
The key step in the synthesis of 1 is the preparation of
the enediynyl amino acid in carboxy-protected form 2.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.03.038

A. Basak et al. / Bioorg. Med. Chem. 13 (2005) 4096–4102
4097
COOCHPh2
R
R
d
Br
Br
Br
b
f
a
O
H
N
N
N
H
CO2CHPh2
7, R = N3
CO2CHPh2
CO2CHPh2
c
Fmoc
76%
Me
O
4
3
8
5, R = OH
e
CO2CHPh2
OH
2, R = NH2
g
6, R = OMs
9
Me
H
O
O
Me
O
N
H
N
TsO-H3+N
O
OCHPh2
10
N
H
OCHPh2
Ph
O
2
Ph
H
N
O
RO2C
Me
H
N
12
O
11
N
Fmoc
H
N
BuOtOCHN
OCHPh2
Ph
Me
O
N
H
N
O
13 R = COOCHPh2
14 R= H
Me
O
O
O
11
1
Scheme 1. Reagents and conditions: (a/b) Pd(PPh3)4, Et3N, 9/10, Cu2Br2; (c) MsCl, CH2Cl2; (d) NaN3, DMF; (e) PPh3,THF–H2O; (f) 14, EDC,
HOBT; (g) (i) TFA/anisole/CH2Cl2, (ii) 12, EDC, HOBT, CH2Cl2–DMF.
This was accomplished by sequential Pd(0)-mediated
Sonogashira coupling12 (Scheme 1). The two arms were
then extended using the partially protected dipeptides
Ala-Phe 12 and Ala-Pro 14 via standard EDC mediated
coupling protocol to furnish the target N,C-diprotected
pentapeptide 1 [Fmoc-Pro-Ala-Eda-Ala-Phe-COOCHPh2,
where Eda = enediyne amino acid] as a pale brown
solid. The reason for choosing such a sequence of amino
acids in 1 is its similarity with linear gramicidin S ana-
logues as prepared by Sneider and Kelly,13 where a
dibenzofuran based amino acid is flanked between two
hydrophobic amino acids. In our system, the enediyne
amino acid is linked between two hydrophobic alanine
residues. Mass and extensive 2D NMR experiments con-
firmed the structure of 1.
Figure 1. Overlayed deconvulated CD spectra of peptide 1 (333 mM)
in MeOH in the absence and presence of various amounts of CaCl2.
2.2. Conformational studies by CD and VT-NMR
The secondary structure of peptide 1 was estimated by
recording its CD spectrum in methanol, which showed
a strong maximum at ꢀ198 nm followed by several
broad minima at ꢀ205, 212, and 222 nm, indicating that
the peptide predominantly adopts a b-turn like structure
(Fig. 1) at least in the solvent used for the study. The
peptide secondary structure estimation using CD estima
program shows a 60% turn like structure, the existence
of which implies the possible presence of intramolecular
H-bond between the peptide strands on the two arms of
the enediyne framework. This could be assessed by
determining the variation of chemical shifts of the vari-
ous NHs with temperature in DMSO-d6 in which all the
four NHs exhibited different chemical shifts. Interest-
ingly, the turn like structure is more or less maintained
in the presence of Ca2+ ions.
values (Fig. 2) that are within the Kessler limit14 of
ꢁ3 ppb/K, indicating strong intramolecular H-bonding
and supported the predominant turn like structure of
the peptide. The appearance of a crosspeak for the
hydrogens attached to C-2 and C-11 in NOESY spec-
trum also provided further evidence for the turn like
conformation of the two peptide arms of the enediyne
backbone. The H-bonded conformation was also sup-
ported by the semi-empirical AM1 geometry
optimization.15
2.3. Photophysical behavior
We then turned our attention to the photophysical prop-
erties of 1 and possible perturbation by different metal
ions. The peptide itself in TFE showed emission at kmax
380 nm when excited at 320 nm with a large Stoke shift
(60 nm), a characteristic emission of the enediyne moi-
Of the four amide NHꢀs, one alanine NH and the NH
belonging to the enediynyl amino acid exhibited Dd/DT

4098
A. Basak et al. / Bioorg. Med. Chem. 13 (2005) 4096–4102
8.3
8.2
8.1
8.0
7.9
7.8
7.7
ions are strong quenchers16 of fluorescence and our re-
sults demonstrated, for the first time, the ability of an
enediynyl peptide to show fluorescence enhancement
upon complexation to various transition metal ions sim-
ilar to cryptand-based fluorophores.4c,17 A possible
explanation behind the enhancement of fluorescence
may be that the distance and orientation of the metal
ion entering in relation to the p-system is such that the
spin-orbit coupling which facilitates the S–T intersystem
crossing is minimum. In other words, the metal ion–
fluorophore communication is considerably less than
the metal ion–receptor interaction.18 To quantify the
effect of complexation on fluorescence intensity, fluores-
cence quantum yields (U) were determined. The complex
formation constants (K) were also calculated using
Benesi–Hildebrand19 equation, which revealed a good
correlation between fluorescence quantum yield and
their complex formation constant. The order of K and
U is Co(II) ffi Zn(II) > Cu(II) > Ni(II). Fluorescence
titration experiment (Fig. 4) with Cu2+ ion showed that
the peptide binds more than one Cu2+ ion as is evident
from the nonlinear plot of 1/[I ꢁ I0] versus 1/[C] (inset).
This result was also supported from MALDI TOF mass
spectrometry; the expected molecular ion peak, corre-
sponding to the peptide and two Cu2+ ions, was ob-
served. For the other metal ions, a linear plot revealed
1:1 complex formation (Table 1).
--EndNH
--Ala NH
--Ala NH
--PheNH
300
305
310
315
320
325
330
Temp[K]
Figure 2. Temperature dependence of NH chemical shifts of peptide 1.
ety. The intensity of emission increases with increasing
concentration of the peptide. For better understanding
of the fluorescence phenomenon, we have also observed
the emission at low temperature. No phosphorescence
could be detected in the solvent (TFE) at 77 K, indicat-
ing that the emission arises due to S1 ! S0 fluorescence.
At 77 K a blue shift of about 12 nm is observed in TFE
(Fig. 3). The shape of the band at 77 K in a given solvent
also differs from that of the solution spectrum (298 K).
Observed changes in the fluorescence spectrum can be
rationalized in terms of the modification of the excited
state (S1) of the solute by interaction with the solvent
molecule. Significant changes in fluorescence intensity
were also observed upon addition of metal ions. Transi-
tion metal ions all caused enhancement of fluorescence
intensity with the extent of enhancement depending
upon the nature of the added metal ion. Although the
precise nature of the interactions between the peptide
and the metal ions is not known, it is possible that the
intrinsic turn like structure allows the accommodation
of the metal ions for binding. Generally, transition metal
The fluorescence behavior in the presence of alkali and
alkaline earth metal ions is even more interesting. When
the ratio of peptide to metal ion was kept at 1:1, fluores-
cence quenching was observed. At peptide–metal ion
ratio of 1:2, enhancement of fluorescence of varying
degree was observed. Interestingly, strongest enhance-
ment was shown for K+ ions while Na+ and Ca2+
showed weak enhancement. This anomalous effect may
be due to the fact that at low metal ion concentration,
coordination occurs through carbonyl oxygen like in
aza-crown ether based cryptand20 due to well-known
hard acid–hard base interaction leaving the lone pair
of electrons on the amide N free to interact with the
8.0
7.5
λmax = 380 nm7.00E-008
2.5
6.00E-008
[Pep 1] : [Cu2+
1 : 2.00
1 : 1.75
1 : 1.50
1 : 1.25
1 : 1.00
1 : 0.50
1 : 0.25
1 : 0.00
]
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
5.00E-008
4.00E-008
3.00E-008
2.00E-008
1.00E-008
B--At 77K
C--At 298K
Inset of
Fig 4
2.0
1.5
1.0
0.5
0.0
B
0
50000 100000
200000 250000
1 /1[5C0]000
C
Enhancement
330 340 350 360 370 380 390 400 410 420 430 440 450
Wave length (nm)
340
360
380
400
420
Figure 4. Fluorescence emission spectra of Cu(II) titration of peptide
1. Excitation wavelength was 320 nm. Initial conc of
1.87 · 10ꢁ5 M in TFE.
Wavelength [nm]
1 was
Figure 3. Fluorescence at 77 and 298 K.

A. Basak et al. / Bioorg. Med. Chem. 13 (2005) 4096–4102
4099
Table 1. Summary of complex formation constants and fluorescence
quantum yields in TFE at 298 K
creased. To the best of our knowledge, uptil now there
has been only two reports22 about the gold-nanoparti-
cle-enhanced emission from the fluorophore.
Host
Guest ions
Formation
constant (K)
Fluorescence
quantum yield (U)
The gold nanoparticles are nonfluorescent and peptide 1
in THF is moderately fluorescent with emission maxima
at 372 nm. The peptide bound to gold nanoparticles
exhibits strong emission bands at 375 nm and the inten-
sity increases as the concentration of nanoparticles in-
creases. The red-shift in the emission peaks parallels
the shift in absorption bands. These new electronic tran-
sitions of the enediyne chromophore become allowed as
the amide nitrogens bind strongly to the gold particle.
No such spectral shift or enhanced emission could be
seen when we added a THF solution of enediynyl pep-
tide containing tetraoctylammonium bromide and trea-
ted with NaBH4 (Fig. 6). The fluorescence quantum
yield of surface-bound peptide is as high as 0.32 at a
gold concentration of 30 · 10ꢁ5 mM. On the basis of
the absorption as well as steady-state emission, it is con-
cluded that surface binding has a significant effect on the
fluorescence yield, but it has no observable effect on the
intersystem crossing efficiency and thus the enhance-
ment of fluorescence was observed. The increase in the
fluorescence yield reflects the suppression of the nonra-
diative decay processes upon adsorption of peptide on
to the surface of gold nanoparticles. The photoinduced
electron transfer between the lone pair of N of amide
functional groups and the enediyne moiety competes
with the radiative and nonradiative decay of the singlet
excited state. Upon binding of the N-lone pairs to the
gold surface, the electron-donating ability of the N is de-
creased and this in turn suppresses the electron transfer
from its lone pair to the enediyne moiety. A similar che-
lation-enhanced fluorescence has been reported earlier
by Czarnick3 and de Silva et al.23 They were able to
demonstrate the suppression of intramolecular quench-
ing by binding metal cations to the amine functional
groups of probes (e.g., anthracene).
None
Co(II)
Zn(II)
Cu(II)
Ni(II)
0.0087
0.49
0.40
1.01 · 105
9.57 · 104
4.52 · 104
2.51 · 104
Peptide 1
0.38
0.25
fluorophore via the PET mechanism thus causing
quenching. With increase in concentration of the metal
ion, binding also took place with the amide N; the
unavailability of the lone of N for PET caused fluores-
cence enhancement.
2.4. Fluorescence perturbation by gold nanoparticle
Observing the perturbation of fluorescence behavior of 1
by mono- and divalent metal cations, we became inter-
ested to know whether zero valent metal is also able to
have similar effect. For this purpose, we studied the
changes in fluorescence intensity upon addition of
Au(0) nanoparticle21 in dry THF solvent. Interestingly,
we observed enhancement of the fluorescence intensity
(Fig. 5) as the concentration of nanoparticle was in-
20
[Pep 1] : [Au(0)]
λmax = 375 nm
1 : 10.00
1 : 8.00
1 : 4.00
1 : 2.00
1 : 1.50
1 : 1.00
1 : 0.75
1 : 0.50
1 : 0.00
18
16
14
12
10
8
Enhancement
6
4
2
3. Conclusion
340
360
380
400
420
440
Wave Length (nm)
We have for the first time developed a small peptide
fluorescence chemosensor capable of binding strongly
the transition metal ions selectively with enhancement
of fluorescence intensity, which can be used as small
Figure 5. Fluorescence emission spectra of Au(0) titration of peptide 1.
Excitation wavelength was 320 nm. Initial concd of Au(0) was 10ꢁ3 M
in THF.
O
O
R2
N
Br
N
Br
N
Br
N
R2
R1
N
Br
N
Br
Au
Au
X
+
Br
Br
..
..
N
N
N
R1
Br
Br
N
N
H
H
1
Gold Nanoparticle
Non fluorescent
Bound Enediynyl Pentapeptide
Enhanced Fluorescence
Free Enediynyl Pentapeptide
Moderately Fluorescent
Figure 6. Probable mode of fluorescence enhancement by gold nanoparticle.

4100
A. Basak et al. / Bioorg. Med. Chem. 13 (2005) 4096–4102
molecule receptors for transition metal cations, for
example, Zn2+ in zinc finger protein, and for molecular
recognition study especially for DNA/ATP recognition
or continuous DNA-cleavage assay.24 The design of b-turn
receptors for large bio-molecules with bigger residues
between the two arms of the enediynyl amino acid is
currently under investigation. Efforts are also on toward
making water-soluble systems to study possible fluores-
cence enhancement with transition metal ions.
(50 mL), and dried over Na2SO4. Evaporation of the
solvent in vacuum gave an oil from which the title com-
pound (5) was isolated by column chromatography (Si
gel, PE–EA = 10:1) as a brown oil (1232 mg, 60%). mmax
(KBr): 3454, 2931, 2405, 1731, 1443, 1162, 698; dH: 2.69
(2H, t, J = 9.0 Hz, CH2CH2OH), 2.82 (4H, q, J = 4.3,
11 Hz, CH2CH2COOCHPh2), 3.77 (2H, t, J = 8.9 Hz,
CH2OH), 6.93 (1H, s, CHPh2), 7.20–7.39 (10H, m,
Ar–H); dC: 10.39, 18.86, 28.74, 55.70, 71.21, 85.26,
86.71, 121.90, 122.37, 122.71, 123.29, 126.34, 126.83,
134.80, 165.78; dC (DEPT 135): 10.39, 18.85, 28.73,
55.70, 71.12, 121.89, 122.39, 122.73, 123.30, 126.34,
126.84; Mass (EI) m/z 408 (M+).
4. Experimental
4.1. General remarks
4.2.3.
5-[2-(4-Methanesulfonyloxy-but-1ynyl)-phenyl]-
Melting points (mp) were recorded on a Toshniwal hot-
coil stage melting point apparatus and are uncorrected.
Among the spectra, NMR spectra were recorded on a
200 MHz spectrometer (Bruker) unless mentioned
otherwise. IR spectra were recorded on Perkin–Elmer
883 using KBr pellet for solids and neat for liquids.
Mass spectra were obtained from CRIM (Clinical Re-
search Institute of Montreal), Canada. All solvents for
chromatography were distilled prior to use. In most of
the column chromatographic purifications, ethyl acetate
(EA) and petroleum ether (PE) of boiling range 60–
80 ꢁC were used as eluents. Columns were prepared with
silica gel (60–120 mesh, S.D. Fine chemicals).
pent-4-ynoic acid benzhydryl ester (6). To a solution of
5 (1200 mg, 2.94 mmol) in CH2Cl2 (30 mL), metha-
nesulfonyl chloride (273 lL, 3.53 mmol), and triethyl-
amine (491 lL, 3.53 mmol) were added at 0 ꢁC. The
reaction mixture was stirred for 15 min after which it
was poured into water and CH2Cl2 (50 mL each). The
organic layer was washed with water (2 · 50 mL), dried,
and then evaporated. The title compound 6 was then
isolated by column chromatography (Si gel, PE–
EA = 10:1) as a brown oil (1214 mg, 85%); and subse-
quently used for the azide formation.
4.2.4. 5-[2-(4-Azido-but-1ynyl)-phenyl]-pent-4-ynoic acid
benzhydryl ester (7). To a solution of the mesylate 6
(1200 mg, 2.47 mmol) in dry DMF (20 mL), NaN3
(241 mg, 3.71 mmol) was added and stirred for 18 h at
room temperature. The mixture was then partitioned be-
tween EtOAc and water (50 mL each). The organic layer
was thoroughly washed with water (3 · 50 mL), dried,
filtered, and then evaporated. The titled compound 7
was isolated by column chromatography (Si gel, PE–
EA = 25:1) as a pale brown oil (855 mg, 80%). mmax
(neat): 3446, 3063, 2927, 2410, 2102, 1738, 1635, 1158,
754; dH: 2.70 (2H, t, J = 10.3 Hz, CH2CH2N3), 2.81
(4H, q, J = 4.15, 10.9 Hz, CH2CH2COOCHPh2), 3.44
(2H, t, J = 10.2 Hz, CH2N3), 6.92 (1H, s, CHPh2),
7.18–7.37 (10H, m, Ar–H); Mass (EI) m/z 433 (M+).
4.2. Synthesis of pentapeptide 1
4.2.1. 5-(2-Bromophenyl)-pent-4-ynoic acid benzhydryl
ester (4). To a solution of o-dibromobenzene (3)
(1980 mg, 8.39 mmol) and Pd(PPh3)4 (291 mg,
0.252 mmol) in dry, degassed triethyl amine (30 mL),
4-pentynoic acid benzhydryl ester
9
(2215 mg,
8.39 mmol) was added followed by Cu2Br2 (73 mg,
0.252 mmol) and the solution was refluxed for 4 h under
argon. The reaction mixture was concentrated in vac-
uum, taken in diethyl ether (50 mL) and the organic
layer was washed with saturated NH4Cl (50 mL), then
with water (50 mL), and dried over Na2SO4. Evapora-
tion of the solvent in vacuum gave oil from which the
title compound (4) was isolated by column chromato-
graphy (Si gel, PE–EA = 30:1) as greenish oil
(2109 mg, 60%). mmax (neat): 3452, 2936, 2864, 1726,
1533, 1475, 1443, 1359, 1269, 1122, 1028, 765; dH: 2.82
(4H, m, 2 · CH2), 6.93 (1H, s, CHPh2), 7.08–7.26 (2H,
m, Ar–H), 7.43 (1H, dd, J = 1.8, 7.5 Hz, Ar–H), 7.54
(1H, dd, J = 1.4, 7.8 Hz, Ar–H); dC: 12.35, 30.65,
79.35, 80.14, 91.75, 125.34, 126.33, 127.15, 128.40,
129.82, 130.72, 131.03, 134.35, 143.79, 172.52; Mass
(EI) m/z 419 (M+).
4.2.5. 5-[2-(4-Amino-but-1ynyl)-phenyl]-pent-4-ynoic acid
benzhydryl ester (2). To a solution of the azide 7
(850 mg, 1.96 mmol) in THF (25 mL), PPh3 (771 mg,
2.94 mmol), and water (250 lL) were added and stirred
at room temperature for 24 h. Evaporation of the sol-
vent in vacuum gave oil from which the title compound
2 was isolated by column chromatography (Si gel, 10%
CH3OH in CH2Cl2) as a brown oil (678 mg, 85%). mmax
(neat): 3025, 2928, 2229, 1735, 1642, 1251, 754; dH: 2.76
(6H, m, 3 · CH2), 3.13 (2H, t, J = 9.8 Hz, CH2COO-
CHPh), 4.01 (1H, br s, NH2), 6.85 (1H, s, CHPh2),
7.12–7.38 (15H, m, Ar–H).
4.2.2. 5-[2-(4-Hydroxy-but-1-ynyl)-phenyl]-pent-4-ynoic
acid benzhydryl ester (5). To a solution of 4 (2109 mg,
5.033 mmol) in dry, degassed Et3N (30 mL), Pd(PPh3)4
(174 mg, 0.151 mmol), homopropargyl alcohol 10
(419 lL, 5.54 mmol) and Cu2Br2 (44 mg, 0.151 mmol)
were added in succession and refluxed for 12 h. The
reaction mixture was concentrated in vacuum, taken in
diethyl ether (50 mL) and the organic layer was washed
with saturated NH4Cl (50 mL), then with water
4.3. General procedure for peptide coupling
To a solution of N-protected amino acid or peptide in
dry CH2Cl2, 1-[3-dimethyl aminopropyl]-3-ethylcarbo-
diimide hydrochloride (EDCÆHCl) (1 equiv) and HOBT
(1 equiv) were added and the reaction mixture was stir-
red for 1 h at 0 ꢁC. The free amine or the tosylate salt of

A. Basak et al. / Bioorg. Med. Chem. 13 (2005) 4096–4102
4101
the C-protected amino acid or peptide (1 equiv), dis-
solved in CH2Cl2 or DMF, was added dropwise fol-
lowed by DIPEA (2 equiv). The reaction mixture was
stirred for another 6 h at 0 ꢁC to room temperature.
After partitioning between CH2Cl2 and water (50 mL
each), the organic layer was washed with NaHCO3, then
with dil HCl, dried over Na2SO4, and evaporated. From
the oily residue, the coupled peptides were isolated pure
by column chromatography.
1.89 (2H, m, Pro–CH2), 2.65 (2H, m, CH2CH2NH),
2.78 (4H, m, CH2CH2COOCH2Ph), 3.46 (4H, m, Pro–
NCH2 and NCH2CO), 4.25 (1H, m, Pro–Ha and Ala–
Ha), 4.40 (2H, m, Fmoc–CH, Fmoc–CH2), 6.78 (1H,
d, J = Hz, NHCO), 6.89 (1H, s, CHPh2), 7.14–7.38
(18H, m, Ar–H), 7.56 (2H, d, J = 7.2 Hz, Fmoc–Ar–
H), 7.78 (2H, d, J = 7.4 Hz, Fmoc–Ar–H); Mass (EI)
m/z 797 (M+); HRMS calcd for C51H47N3O6 + H+
798.3555. Found 798.3516.
4.4. 2-(1-Benzhydryloxycarbonyl-ethylcarbamoyl)-pyrol-
idine-1-carboxylic acid 1-(9H-fluoren-9-ylmethyl)ester
(13)
4.8. 2-{1-[4-(2-4-[1-(1-Benzhydryloxycarbonyl-2-phenyl-
ethylcarbamoyl)-ethylcarbamoyl]-but-1-ynyl-phenyl)-but-
3-ynylcarbamoyl]-ethylcarbamoyl}-pyrolidine-1-carbox-
ylic acid-9H-fluoren-9-yl methyl ester (1)
mmax (KBr): 3654, 3034, 2889, 1744, 1681, 1528, 1189,
750; dH: 1.40 (3H, d, J = 6.0 Hz, CH3), 1.93 (2H, m,
Pro–CH2), 2.19 (2H, m, Pro–CH2), 3.52 (2H, m, Pro–
NCH2), 4.25 (1H, m, Pro–Ha), 4.31(1H, t, J = 11.1 Hz,
Fmoc–CH), 4.38 (2H, d, J = 7.5 Hz, Fmoc–CH2), 4.67
(1H, t, J = 10.77, Ala–Ha), 6.77 (1H, d, J = Hz, NHCO),
6.86 (1H, s, CHPh2), 7.28 (4H, m, Fmoc–Ar–H), 7.57
(2H, d, J = 7.2 Hz, Fmoc–Ar–H), 7.74 (2H, d,
J = 7.4 Hz, Fmoc–Ar–H); Mass (EI) m/z 574 (M+);
HRMS calcd for C36H34N2O5 574.2469. Found
574.2467.
Mp 195 ꢁC; mmax (KBr): 3295, 3068, 2235, 1747, 1639,
1537, 1352, 1120, 753; dH (500 MHz): 1.25 (3H, d,
J = 12.2 Hz, Ala–CH3), 1.35 (3H, d, J = 11.8 Hz, Ala–
CH3), 1.8–2.1 (4H, m, Pro–CH2), 2.45 (2H, m, 2 · H-
3), 2.60 (2H, m, H-10), 2.70 (2H, m, 2 · H-2), 3.10
and 3.20 (2H, m, Phe–CH2), 3.48 (4H, m, 2 · H-11,
Pro–NCH2), 4.24 (2H, m, Fmoc–CH, Pro–Ha), 4.47
(2H, m, Fmoc–CH2), 4.55 (1H, m, Ala–Ha), 4.90 (1H,
m, Phe–Ha), 6.90 (2H, m, b and c-NH), 7.03 (1H, d,
J = 14.5 Hz, d-NH), 7.10 (1H, t, J = 16.5 Hz, a-NH),
7.18–7.35 (23H, m, Ar–H), 7.6 (2H, d, J = 14.4 Hz,
Fmoc–Ar–H), 7.8 (2H, d, J = 14.8 Hz, Fmoc–Ar–H);
dC (DMSO-d6, 50 MHz): 8.36, 9.25, 15.14, 18.30,
19.70, 34.07, 36.29, 37.94, 41.14, 45.35, 46.64, 47.72,
48.14, 50.50, 52.50, 62.62, 76.94, 79.24, 79.96, 92.57,
93.58, 120.61, 125.21, 126.42, 126.50, 127.06, 127.65,
128.11, 128.34, 129.01, 131.59, 135.46, 136.27, 136.85,
137.83, 140.12, 140.65, 143.79, 148.24, 172.27, 172.35,
177.35; Mass MALDI TOF m/z 1039.74 (M+Na+);
HRMS calcd for C63H61N5O8 + H+ 1016.4601. Found
1016.4581.
4.5. Synthesis of 2-(1-carboxy-ethylcarbamoyl)-pyrol-
idine-1-carboxylic acid 9H-fluoren-9-ylmethyl ester (14)
To a solution of 13 (1000 mg, 2.46 mmol) in dry MeOH,
30% Pd–C (30 mol %) was added and stirred for 12 h
under H2 atmosphere. The reaction mixture was filtered
and the residue obtained upon evaporation of the filtrate
was recrystallized from CHCl3/petrol to yield the desired
acid in pure form. mmax (KBr): 3435, 2932, 2240, 1658,
1537, 1122, 752; dH: 1.39 (3H, d, J = 6.9 Hz, CH3),
1.91 (2H, m, Pro–CH2), 2.19 (2H, m, Pro–CH2), 3.52
(2H, m, Pro–NCH2), 4.25 (1H, m, Pro–Ha), 4.31(1H,
t, J = 11.1 Hz, Fmoc–CH), 4.39 (2H, d, J = 7.5 Hz,
Fmoc–CH2), 4.52 (1H, t, J = 10.5, Ala–Ha), 6.79 (1H,
d, J = Hz, NHCO), 7.27–7.55 (4H, m, Fmoc–Ar–H),
7.73 (2H, d, J = 7.2 Hz, Fmoc–Ar–H), 7.91 (2H, d,
4.9. Fluorescence measurement
The fluorescence titration was carried out in 1:1 TFE–
water at pH 6.8. A stock solution (1.87 · 10ꢁ5 M) of
peptide 1 in TFE and various metal salt solutions in
water each of 1 mM concentration were prepared. All
solutions were centrifuged for 15 mins at 10,000 rpm in
order to precipitate all suspended particles. The clear
supernatant liquid at the top was used for each fluores-
cence measurements. The fluorescence measurements
were recorded on a scan mode using a FLUOROLOG
spectrofluorometer. All studies were conducted in a cuv-
ette of 3 mL capacity. The total volume of each sample
was restricted to 200 mL. For metal binding studies the
sample solutions containing different concentrations of
metal salts were incubated at 37 ꢁC for 12 h. The emis-
sion fluorescence was scanned for every 2 nm from 330
to 500 nm with a 1 min shaking before each measure-
ment. To quantify the effect of complexation on fluores-
cence intensity, fluorescence quantum yield (U) were
determined by the integration of corrected fluorescence
spectra; quinine sulfate in 1 N H2SO4 was used for cor-
rection of fluorescence spectra as a fluorescence stan-
dard (U = 0.55). The complex formation constants
were calculated using Benesi–Hildebrand equation.
J = 7.4 Hz,
C23H24N2O5 408.1686. Found 408.1687.
Fmoc–Ar–H);
HRMS
calcd
for
4.6. 2-(2-tert-Butoxycarbonylamino-propionylamino)-3-
phenyl-propionic acid benzhydryl ester (11)
mmax (KBr): 3869, 3263, 2973, 1895, 1720, 1662, 1252,
1024, 854; dH: 1.24 (3H, d, J = 9.16 Hz, CH3), 1.41
(9H, s, t-butyl–H), 3.13 (2H, t, J = 7.0, CH2Ph), 4.12
(1H, m, Ala–Ha), 4.93 (1H, m, Phe–Ha), 4.99 (1H, d,
J = 7.67 Hz, NHBoc), 6.54 (1H, d, J = 7.8 Hz, NHCO),
6.99 (1H, s, CHPh2), 7.12–7.37 (15H, m, Ar–H); Mass
(EI) m/z 502 (M+).
4.7. 2-(1-{4-[2-(4-Benzhydryloxycarbonyl-butynyl)-phen-
yl]-but-3-ynylcarbamoyl}-ethylcarbamoyl)-pyrolidine-1-
carboxylic acid-9H-fluoren-9-ylmethyl ester (8)
mmax (KBr): 2928, 2400, 2351, 1655, 1539, 1117, 753; dH:
1.38 (3H, d, J = 6.2 Hz, CH3), 1.58 (2H, m, Pro–CH2),

4102
A. Basak et al. / Bioorg. Med. Chem. 13 (2005) 4096–4102
8. (a) Ribrioux, S.; Kleymann, G.; Haase, W.; Heitmann, K.;
Acknowledgements
Ostermeier, C.; Michel, H. J. Histochem. Cytochem. 1996,
44, 207; (b) Hainfeld, J. F.; Furuya, F. R. J. Histochem.
Cytochem. 1992, 40, 177.
The financial support from DST, New Delhi, India
(Amit Basak) and Canadian Foundation of Innovation
and Ontario Innovation Trust, Canada (Ajoy Basak)
is gratefully acknowledged. S.S.B. is thankful to CSIR,
Government of India, New Delhi, for research fellow-
ship. We also thank Prof. C. Palomo, University of
the Basque Country at San Sebastian, Spain for helping
to record 2D NMR.
9. (a) Zheng, Y.; Cao, X.; Orbulescu, J.; Konka, V.;
Andreopoulos, F. M.; Pham, S. M.; Leblanc, R. M. Anal.
Chem. 2003, 75, 1706; (b) Zheng, Y.; Huo, Q.; Kele;
Andreopoulos, P. F.; Pham, M. S. M.; Leblanc, R. M.
Org. Lett. 2001, 3, 3277.
10. (a) Berg, J. M.; Shi, Y. Science 1996, 271, 1081; (b) Berg, J.
M. J. Biol. Chem. 1990, 265, 6513; (c) Torrado, A.;
Walkup, G. K.; Imperiali, B. J. Am. Chem. Soc. 1998, 120,
609; (d) Walkup, G. K.; Imperiali, B. J. Am. Chem. Soc.
1996, 118, 3053.
References and notes
11. Basak, A.; Rudra, K. R.; Bag, S. S.; Basak, A. J. Chem.
Soc., Perkin Trans. 1 2002, 15, 1805.
12. Basak, A.; Bag, S. S.; Bdour, H. M. M. J. Chem. Soc.,
Chem. Commun. 2003, 20, 2614.
13. Sneider, J. P.; Kelly, J. W. Chem. Rev. 1995, 95, 2169.
14. Kessler, H. Angew. Chem., Int. Ed. Engl. 1982, 21,
512.
15. The AM1 calculation was carried out using MOPAC 2002
Version 2.20, CAChe 5.04 (a Fujitsu group limited).
16. (a) Lackowicz, J. R. Principle of Fluorescence Spectro-
scopy; Plenum: New York, 1983; (b) Varnes, R. B.;
Dodson, A. W.; Wehry, E. L. J. Am. Chem. Soc. 1972, 94,
946, and references cited therein; (c) Kemlo, J. A.;
Shepherd, T. M. Chem. Phys. Lett. 1977, 47, 158.
17. Ghosh, P.; Bharadwaj, P. K.; Roy, J.; Ghosh, S. J. Am.
Chem. Soc. 1997, 119, 11903.
18. Ramachandram, B.; Samanta, A. J. Chem. Soc., Chem.
Commun. 1997, 1037.
19. Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949,
71, 2703.
1. (a) Jones, R. G.; Bergman, R. G. J. Am. Chem. Soc. 1972,
94, 660; (b) Bergman, R. G. Acc. Chem. Res. 1973, 6, 25;
(c) Lockhart, T. P.; Bergman, R. G. J. Am. Chem. Soc.
1981, 103, 4091; (d) Basak, A.; Mandal, S.; Bag, S. S.
Chem. Rev. 2003, 103, 4077.
2. (a) Evenzahav, A.; Turro, N. J. J. Am. Chem. Soc. 1998,
120, 1835; (b) Kraft, B. J.; Coalter, N. L.; Nath, M.;
Clark, A. E.; Siedle, A. R.; Huffman, J. C.; Zaleski, J. M.
Inorg. Chem. 2003, 43, 1463.
3. (a) Czarnik, A. W. Acc. Chem. Res. 1994, 27, 302; (b)
Fluoroscent Chemosensors for Ion and Molecule Recogni-
tion; Czarnik, A. W., Ed.; American Chemical Society:
Washington, DC, 1993; (c) Czarnik, A. W. Chem. Biol.
1995, 2, 423.
4. (a) de Silva, A. P.; Gunaratne, H. Q. N.; McCoy, C. P.
Nature 1993, 364, 42; Chem. Commun. 1996, 2399; (b)
Iwata, S.; Tanaka, K. J. Chem. Soc., Chem. Commun.
1995, 1491; (c) Ghosh, P.; Bharadwaj, P. K.; Mandal, S.;
Ghosh, S. J. Am. Chem. Soc. 1996, 118, 1553.
5. (a) Hickman, J. J.; Ofer, D.; Laibinis, P. E.; Whitesides, G.
M.; Wrighton, M. S. Science 1991, 252, 688; (b) Chen, S.;
Ingram, R. S.; Hostetler, M. J.; Pietron, J. J.; Murray, R.
W.; Schaaff, T. G.; Khoury, J. T.; Alvarez, M. M.;
Whetten, R. L. Science 1998, 280, 2098; (c) Elghanian, R.;
Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C.
A. Science 1997, 277, 1078.
20. de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.;
Nieuwenhuizen, M. J. Chem. Soc., Chem. Commun. 1996,
1967.
21. (a) Brust, M.; Walker, M.; Bethell, D.; Schffrin, D. J.;
Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801;
(b) Fink, J.; Kiely, C.; Bethell, D.; Schiffrin, D. J. Chem.
Mater. 1998, 10, 922.
6. (a) Templeton, A. C.; Cliffel, D. E.; Murray, R. W. J. Am.
Chem. Soc. 1999, 121, 7081; (b) Fitzmaurice, D.; Rao, S.
N.; Preece, J. A.; Stoddart, J. F.; Wenger, S.; Zaccheroni,
N. Angew. Chem., Int. Ed. 1999, 38, 1147; (c) Han, W.; Li,
S.; Lindsay, S. M.; Gust, D.; Moore, T. A.; Moore, A. L.
Langmuir 1996, 12, 5742; (d) Makarova, O. V.; Ostafin, A.
E.; Miyoshi, H.; Norris, J. R.; Meisel, D. J. Phys. Chem. B
1999, 103, 9080.
22. (a) Thomas, K. G.; Kamat, P. V. J. Am. Chem. Soc. 2000,
122, 2655; (b) Wang, T.; Zhang, D.; Xu, W.; Yang, J.;
Han, R.; Zhu, D. Langmuir 2002, 18, 1840.
23. de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.;
Huxley, A. J. M.; McRoy, C. P.; Rademacher, J. T.; Rice,
T. E. Chem. Rev. 1997, 97, 515.
24. Biggins, J. B.; Prudent, J. R.; Marshall, D. J.; Ruppen, M.;
Thorson, J. S. Proc. Natl. Acad. Sci. U.S.A. 2000, 97,
13537.
7. Peng, X.; Hisao, Y. Chem. Mater. 1999, 11, 2626.