Synthesis of a novel thiazole based dipeptide chemosensor for Cu(II) in water

Article abstract of DOI:10.1016/S0040-4039(00)01853-0

A thiazole-based novel dipeptide module has been synthesized, which exhibited selective Cu(II) recognition in water in the presence of other competing divalent transition metal ions at micromolar concentrations. This is the first example of an entirely synthetic peptide based chemosensor for Cu(II). (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01853-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 10313–10317
Synthesis of a novel thiazole based dipeptide chemosensor
for Cu(II) in water†
Santanu Bhattacharya* and Mini Thomas
Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India
Received 29 August 2000; revised 10 October 2000; accepted 19 October 2000
Abstract
A thiazole-based novel dipeptide module has been synthesized, which exhibited selective Cu(II)
recognition in water in the presence of other competing divalent transition metal ions at micromolar
concentrations. This is the first example of an entirely synthetic peptide based chemosensor for Cu(II).
© 2000 Elsevier Science Ltd. All rights reserved.
Chemosensors are molecular-sized or larger devices that signal interactions with analytes
reversibly and in real time.1 Fluorescence proffers high sensitivity among the many signal types
available. Therefore this mode of signal transduction has been used widely in the detection of a
number of transition metal ions.2,3 Among these, the sensors targeted toward the detection and
estimation of divalent copper ions are currently receiving a lot of attention.3 Detection of
individual metal ions with high specificity under physiologically relevant conditions is an
important aspect in the design of fluorescent chemosensors for biological and environmental
applications. In this regard peptide based sensors offer several advantages over other systems as
they are biocompatible and also could be conveniently conjugated to appropriate systems of
biological interest such as proteins or nucleic acids.
Herein we report the convenient synthesis of a Cu(II)-binding peptide module 1, which is
based on thiazole derivatives. This represents the first example of a Cu(II)-sensing peptide of
entirely synthetic origin, which is also the smallest peptide motif that acts as a specific
fluorescence chemosensor for a metal ion. Notably, the sensing of Cu(II) using 1 has been
achieved in water under physiologically relevant pH and salt conditions, in the presence of other
competing divalent transition metal ions.
To synthesize the thiazole containing peptide module, we took advantage of the commercially
available 2-amino-4-thiazole acetic acid, 3a as the key starting material. This was first converted
to the corresponding ethyl ester using EtOH and SOCl2 as described in Scheme 1. Removal of
* Corresponding author: Swarnajayanti Fellow. E-mail: sb@orgchem.iisc.ernet.in
†
This paper is dedicated to Professsor Robert A. Moss on the occasion of his 60th birthday.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01853-0

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Scheme 1. Reagents, conditions and yields: (i) SOCl2/EtOH, −5°C, 1 h“rt, 12 h; (ii) 1:1 v/v HOAc/Ac2O, −5°C, 1
h“rt, 6 h, 95%; (iii) 0.5 M NaOH, rt, 30 min“0.5 M HCl, 0°C, 97%; (iv) 3b, DCC, HOBT, DMF, 0°C, 1 h“rt, 6
h (85%); (v) 0.5 M NaOH, rt, 30 min“0.5 N HCl, 0°C, 98%; (vi) DCC, HOSU, DMF, 1 h“rt, 6 h; (vii) Et3N, dry
CHCl3, 0°C, 1 h, 70%; (viii) 3j, dry CHCl3, rt, 2 h (80%); (ix) 3i, dry DMF, rt, 4 h (85%)
excess reagents followed by aqueous work up furnished the ester, 3b in quantitative yield. This
was then reacted with Ac2O/HOAc. Removal of excess reagents and recrystallization from
EtOAc yielded an analytically pure sample of the N-acetylated product, 3c in 95% yield.
Saponification of this key intermediate and subsequent acidification provided the acid, 3d in
97% yield. The coupling of 3d with the amine, 3b was effected by dropwise addition of a solution
of dicyclohexyl carbodiimide (DCC) in DMF to a solution of 3b in DMF in the presence of
1-hydroxybenzotriazole (HOBT). To ensure satisfactory amide coupling slow addition of DCC
was important as bulk introduction of DCC resulted in the formation of the N-acyl isourea
derivative of the corresponding acid. This could be due to low reactivity of the aromatic amine,
3b. DCU was removed by filtration and the product was precipitated by the addition of water.
The amide 3e was obtained in pure form (85%) by successive washing of this solid with EtOAc
and 5% NaHCO3 solution, to remove the unreacted starting materials. Saponification and
subsequent acidification of 3e afforded the corresponding acid, 3f in analytically pure form
(98%) which was subsequently converted to the corresponding N-hydroxysuccinimide ester, 3g
employing DCC and N-hydroxysuccinimide (HOSU). DCU was removed by filtration and the
product was precipitated by the addition of water, and was used as such for further reactions.
In a separate set of reactions, commercially available 5-dimethylaminonaphthalene-1-sulfonyl
chloride (dansyl chloride), 3h and N-methyldiethylenetriamine, 3i (2 equiv.) were reacted in

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CHCl3 in presence of Et3N as described in Scheme 1. The monodansylated amine, 3j was
isolated in 70% yield after column chromatography on neutral alumina. Aminolysis of the
activated ester, 3g with 3j in dry CHCl3 furnished the dipeptide chemosensor, 1 in 80% yield
after column chromatography on neutral alumina. Similarly aminolysis of 3g with 3i (0.5 equiv.)
furnished the tetrapeptide, 2 in 85% isolated yield after column chromatography on neutral
alumina. All numbered intermediates and final compounds were characterized by their IR,
NMR, mass spectra and elemental analysis.4
Fluorescence emission spectra of 1 were recorded upon excitation at 330 nm. A linear decrease
in the fluorescence intensity was observed until one equiv. of Cu(II) was added (Fig. 1A).
Further addition of Cu(II) produced only a nominal decrease in fluorescence intensity. The
fluorescence quenching of 1 by Cu(II) was tested over a range of concentrations (2–20 mM of 1)
and was found to be linear with the added metal ion concentration. When 1 equiv. of each of
Mn(II), Fe(II), Co(II), Ni(II) and Zn(II) were first added to a solution of the peptide, 1 in tris
buffer, only a marginal quenching of the sensor fluorescence was observed (Fig. 1B, curve ‘b’).
Addition of 1 equiv. of Cu(II) to the above solution however, produced a drastic decrease in the
Figure 1. (A) Effect of addition of Cu(II) on the emission spectra (uex=330nm) of 1 in 20 mM tris buffer containing
150 mM NaCl (pH 7.4). Curve ‘a’, free probe (4.4 mM). 4.4 mM of Cu(II) was added in ten equal aliquots. Curve ‘k’
corresponds to 4.4 mM of Cu(II). (B) Effect of addition of Cu(II) in presence of other divalent transition metal ions.
Curve ‘a’ is due to the free probe (5.4 mM), curve ‘b’ shows the effect of addition of 5.4 mM each of Mn(II), Fe(II),
Co(II), Ni(II), Zn(II); curve ‘c’ shows the effect of addition of 5.4 mM of Cu(II). (C) ESI-MS of 1/Cu(II) complex.
(D) Possible coordination scheme for the 1/Cu(II) complex

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fluorescence intensity (Fig. 1B, curve ‘c’). This experiment confirms that the peptide, 1 can act
as a specific sensor for Cu(II) even in the presence of elevated levels of other competing divalent
transition metal ions. The Cu(II) induced quenching was found to be reversible with the
addition of excess EDTA.
To discern the nature of the complex formed between 1 and Cu(II) in solution, we examined
the system by electrospray ionization mass spectrometry (ESI-MS) (Fig. 1C). At 1:1 metal/lig-
and ratio, the signal obtained at a mass/charge (m/z) unit of 734.2 could be assigned to a species
corresponding to [M−2H+Cu]·H+. The presence of the peak at m/z of 673.2 is likely to be due
to the free peptide formed by decomplexation under ESI-MS conditions as further addition of
Cu(II) did not increase the intensity of the bound species. The corresponding tetrapeptide, 2
exhibited a signal corresponding to [M−4H+2Cu]H+ in ESI-MS, suggesting that two indepen-
dent co-ordination centers of similar nature as in 1 are present. Direct involvement of the central
tertiary amino group and the dansyl group in metal-complexation is less likely. We propose a
co-ordination scheme (Fig. 1D) that explains the ESI-MS results. Work is underway to obtain
crystals of the Cu(II) complexes of 1 and 2 to establish firmly the structure of the metal ligation
site.
The present system 1, which could be easily synthesized in solution phase, represents a simple,
yet unexplored class of peptide sensors for Cu(II) with high selectivity. As this motif selectively
targets Cu(II) ions, intramolecular quenching of the covalently tethered fluorophore represents
a convenient mechanism for signaling of Cu(II) binding. Functional devices based on 1 could be
designed for environmental monitoring of Cu(II) ions. We are now exploring the incorporation
of this peptide module into other peptides/proteins5a,b and minor groove binders6a,b for genera-
tion of affinity cleavage agents and chemical nucleases.6c–e
Acknowledgements
The work was supported by the Department of Science and Technology, Govt. of India. We
thank Professor P. Balaram and C. Das of the Molecular Biophysics Unit of this institute for
ESI-MS spectra.
References
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1
4. Selected spectroscopic data 1: H NMR (300 MHz, CDCI3): l (ppm) 2.25 (s, 3H); 2.30 (br s, 2H); 2.37 (br s, 2H);
2.89 (s, 6H); 3.01 (br s, 2H); 3.13 (br s, 2H); 3.59 (s, 2H); 3.87 (s, 2H); 6.59 (s, 1H); 6.77 (s, 2H); 6.80 (s, 2H); 7.16
(d, 1H, J=7.5 Hz); 7.41 (t, 1H, J=8.1 Hz); 7.53 (t, 1H, J=8.1 Hz); 8.26 (d, 1H, J=7.2 Hz); 8.34 (d, 1H, J=8.7
Hz); 8.56 (d, 1H, J=8.4 Hz). ESI-MS: calcd for C29H37N8O5S3 (MH+) 673.3, found: 673.3. 2: 1H NMR (500 MHz,
CDCI3+DMSO-d6): l (ppm) 2.09 (s, 6H); 2.16 (s, 3H); 2.41 (t, 4H, J=7.6 Hz); 3.15–3.23 (m, 4H); 3.49 (s, 4H);
3.74 (s, 4H); 6.69 (s, 2H); 6.76 (s, 2H); 7.72 (br s, 2H); 11.96 (br s, 2H). ESI-MS: calcd for C29H36N11O6S4 (MH+)
762.3, found: 762.2.
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