Sensing of biomolecules and label-free discrimination of DNA containing a triple T-C/T-G mismatch pair with a fluorescence light-up probe, triazolylpyrene (TNDMBPy)

Article abstract of DOI:10.1016/j.tetlet.2013.03.029

Binding to the minor groove of calf-thymus DNA (ct-DNA) and strong binding in hydrophobic pocket of bovine serum albumin (BSA), switched-on the fluorescence of smart fluorescent probe, triazolylpyrene (^TNDMBPy). Also, a novel label-free strateg

Full text of DOI:10.1016/j.tetlet.2013.03.029

Tetrahedron Letters 54 (2013) 2627–2632
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Sensing of biomolecules and label-free discrimination of DNA containing
a triple T–C/T–G mismatch pair with a fluorescence light-up probe,
triazolylpyrene (TNDMBPy)
⇑
Subhendu Sekhar Bag , Rajen Kundu, Subhashis Jana
Bioorganic Chemistry Laboratory, Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati 781039, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Binding to the minor groove of calf-thymus DNA (ct-DNA) and strong binding in hydrophobic pocket of
bovine serum albumin (BSA), switched-on the fluorescence of smart fluorescent probe, triazolylpyrene
(
Received 24 December 2012
Revised 6 March 2013
Accepted 7 March 2013
Available online 16 March 2013
TNDMBPy). Also, a novel label-free strategy was adopted to detect DNA base mismatch via the generation
of distinct fluorescence signal at visible region utilizing the same probe
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Triazolylpyrene
Fluorescence light-up probe
Sensing biomolecules
ct-DNA
BSA
Label free mismatched DNA detection
Monitoring various structures, functions, and dynamics of bio-
logical molecules such as DNAs and proteins and their surround-
ings with an ideal microenvironment sensitive fluorescence
probe is an emerging research area in biological science and engi-
neering for understanding biological events associated with inter-
biomolecular interactions.1 In such a probing scenario, when the
fluorophore comes into interaction with biomolecules, the fluoro-
phores’ emission property may be so modulated as to enable visual
observation of biomacromolecular species.1e–h Several fluorescent
bioprobes, thus, have been reported based on the fluorophores’
properties like intercalation, stacking, and/or groove binding for
DNA detection with the generation of enhanced fluorescence sig-
nal.2 On the other hand, protein binding abilities of several chro-
mophores also have been studied via enhanced fluorescence
signal generation.3 However, many of the explored fluorophores
were found to suffer from self quenching of emissions that makes
them inefficient biosensors.4 Therefore, there is a need to design
probes which would show low or zero emission in buffer but upon
interaction with biomolecules it could fluoresce drastically.
probe) of various pHs, it showed only a faint ICT emission
(kem = 525 nm). We envisaged that the pseudo aromatic 1,2,3-tria-
zole residue of the probe might significantly enhance the interac-
tion ability via stacking/H-bonding/electrostatic interaction with
the amino acid residues in a protein and/or with the aromatic
bases in DNA. This would allow one to gather information on the
protein’s/DNA’s microenvironments. Our probe consists of an
N,N-dimethyl aminophenyl donor linked triazole functionality
which served as an electron donor to effectively modulate the
emission response of electronically coupled pyrene and also can
provide an interaction site for biomolecules (Fig. 1a). This idea
along with the intense emission of the probe in low polar media
compared to buffer drew our attention to exploit it for the possible
detection of ct-DNA and BSA protein which are easily available bio-
molecules with widespread applications and biological
importance.2,3
Herein, we report our probe triazolylpyrene as an efficient fluo-
rescent light-up biosensor for the detection of DNA and protein.
Before going to study the interactions with DNA/protein, we
wanted to establish that the fluorescence at 525 nm is only due
to ICT emission. Thus, we subsequently studied the spectral
parameters over a broad range of probe concentration. Figure 1
showed the absorption and fluorescence spectra of the probe over
As a part of our ongoing research efforts on the design of solva-
tochromic fluorescent molecules via click reaction, we, recently
have reported triazolylpyrene as a dual emissive highly solvato-
chromic fluorophore (Fig. 1a).5 We have now observed that in buf-
fer (2% DMF was added in each case to completely solubilize the
the concentration range of 10–70 lM in a buffer of pH 7.0. The
absorption, excitation, and fluorescence spectra did not change in
band position. Moreover, the absorbance, and fluorescence intensi-
ties depend linearly on the concentrations of the probe in solution
⇑
Corresponding author. Tel.: +91 361 258 2324; fax: +91 361 258 2349.
E-mail address: ssbag75@iitg.ernet.in (S.S. Bag).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.03.029

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S. S. Bag et al. / Tetrahedron Letters 54 (2013) 2627–2632
4x106
0.75
10 µM
10 µM
20 µM
30 µM
40 µM
50 µM
60 µM
70 µM
(b)
(c)
20 µM
30 µM
40 µM
50 µM
60 µM
70 µM
3x106
2x106
0.50
(a)
N
0.25
0.00
1x106
0
370
470
570
670
260
360
460
560
N
N
Wavelength (nm)
Wavelength (nm)
N
5x106
4x106
3x106
2x106
0.9
0.6
(d)
(e)
1(TNDMBPy)
Triazolylpyrene
0.3
0.0
1x106
0
0
15 30 45 60 75 90
0
15 30 45 60 75 90
Concentration (µM)
Concentration (µM)
Figure 1. (a) Chemical structure of the probe 1 (TNDMBPy). UV–visible (b) and emission spectra (c) of various concentrations of 1 in phosphate buffer of pH 7.0. Plots of
absorbance (d) and emission intensity (e) versus concentration of the probe (kex = 345 nm).
(Fig. 1d and e) suggesting that the triazolylpyrene existed in a
monomeric state and no aggregation formed. We have also studied
the absorption of the probe of various concentrations in the pres-
ence of a fixed amount of biomolecule, BSA and plotted the absor-
bance versus concentration of the probe. The plot showed a good
linear relationship (see Supplementary data) that would allow us
to fit fluorescence titration curve data quantitatively to derive
the association constants of probe-protein/DNA complex with no
harm.
The strong emission at a long wavelength region (520 nm) in
the presence of biomolecule in buffer again might be confused
with the possibility of aggregation induced emission.6 However,
our observation in emission behavior of a dioxane solution of the
probe when titrated with water did not reflect any aggregation in-
duced emission (see Supplementary data). Thus, increasing % of
water led to a decrease in emission along with a red shift of the
band at 520 nm of the probe. The band totally died down at 20%
water and the LE band at 380 nm started appearing. The LE band
intensity reached maximum at 70% water content. When a large
amount (80% and 100%) of water, a poor solvent of the probe,
was added to the dioxane solution (with the final concentration
we checked this event and we could say that the long wavelength
emission at around 520 nm was due to ICT emission and not due to
the characteristic excimer emission of pyrene. The proof of our
assignment came from a study of concentration dependent emis-
sion of both the bands in 10% water in dioxane solvent and evalu-
ation of the changes in intensity of both the LE (ꢀ380 nm) and ICT
(ꢀ520 nm) bands. This solvent system was so chosen as to get both
the emissions prominently. However, in this solvent system, the
ICT band was appearing at 570 nm and the LE band centered at
393 nm. If the emission at 570 nm was excimer emission, it should
increase compared with the emission at 393 nm at higher concen-
trations. However, we observed that the intensity of both the emis-
sion bands increased linearly as the concentration of the probe
increased supporting our assignment of the band at 570 as ICT
emission (see Supplementary data).
After establishing the long wavelength emission band as ICT
emission and linear dependency of absorption and emission to
the probe concentration either in the absence or in the presence
of biomolecule, we next turned our attention to exploit this fluoro-
phore’s emission property to the study of possible interaction with
protein/DNA biomolecules.
of the mixture being adjusted to 10
l
M) the LE band intensity
To examine the biomolecule sensing ability of our probe, first,
we investigated the interaction behavior with ct-DNA by UV–visi-
ble and fluorescence spectroscopy in a buffer (pH 7.0) at 25 °C.
Thus, the absorption maxima of triazolyl pyrene located at
355 nm showed a clear hyperchromicity with a minimal shift in
absorption wavelength as ct-DNA was added gradually. This indi-
cated a probable external groove binding characteristics of our
probe (see Supplementary data).8 From the fluorescence titration
spectra it was evident that upon addition of increasing concentra-
tions of ct-DNA, the fluorescence intensity (kem = 525 nm) of triaz-
olyl pyrene significantly increased and reached to a maximum at
again started to decrease with a negligible appearance of only LE
band in 100% water under identical measurement conditions.
These results suggested that the emission was not from an aggre-
gate state but from the monomeric probe (see Supplementary
data).
We attributed further that the emission at 520 nm was due to
the ICT emission. However, the structure-less emission at longer
wavelength could also be a characteristic to the excimer emission
of pyrene.7 Therefore, to establish the ICT emission we measured
emission spectra of the probe of varying concentrations and plot-
ted emission intensities versus concentrations at two different
wavelengths of emission. If the emission at 520 nm was due to
excimer emission, only this emission should increase compared
with the emission at 380 nm (monomer emission) at the higher
concentration; otherwise both the emissions would increase. Thus,
300 lM concentration of ct-DNA compared to 50 lM probe con-
centration. This result indicated a well-defined binding of the
probe with ct-DNA (Fig. 2a). The association constant of triazolyl
pyrene with ct-DNA was determined by a Benesi–Hildebrand plot
(Fig. 2b) which was found to be 7.2 Â 103 MÀ1 with free energy of

S. S. Bag et al. / Tetrahedron Letters 54 (2013) 2627–2632
2629
Figure 2. (a) Fluorescence titration of probe with various concentrations of ct-DNA. (b) Benesi–Hildebrand plot. (c) Fluorescence image under UV-transilluminator (254 nm)
of only probe, in the presence of ct-DNA. All the experiments were carried out in 5 mM phosphate buffer, 5 mM NaCl, pH 7.0, rt, kex = 345 nm.
binding,
D
G = À5.24 kcal/mol. The enhancement of fluorescence
bound to ct-DNA. Therefore, both resonance energy transfer and re-
lease of Hoechst from ct-DNA probably were the main cause of de-
crease in emission intensity of Hoechst. Finally, the competition
between the probe and hoechst for association with ct-DNA indi-
cated that the probe triazolyl pyrene bound to the DNA minor
groove.12 The groove binding event of the probe was further
supported by an insignificant change of fluorescence anisotropy/
polarization (Fig. 3b).13 Amber⁄ optimized geometry of the model
DNA-probe complex also supported the minor groove binding
event of the probe (see Supplementary data).14
emission was also clear from a change of color of the probe solu-
tion from that of ct-DNA-probe solution when irradiated at
254 nm under a transilluminator (Fig. 2c).
Thermal denaturation experiment indicated a little destabiliza-
tion of ct-DNA upon binding with the probe possibly because of ste-
ric clash near the groove. However, CD spectral measurement in the
presence of the probe revealed an unperturbed B-form DNA confor-
mation of ct-DNA. These results indicated that the probe possibly
bind to a groove of ct-DNA (see Supplementary data).8 To investi-
gate the binding mode of the probe with ct-DNA, whether it is an
intercalator or a groove binder, both the ethidium bromide (EB)
and Hoechst 33258 displacement experiments were carried out.
Ethidium bromide is a well-known example of an intercalating
agent.9 Fluorescence intensity of EB increases when it binds with
DNA because of intercalation. In a competing scenario, if our probe,
TNDMBPy, intercalates with ct-DNA, then it would be a competitive
binding agent with EB and the fluorescence intensity of EB bound
with ct-DNA would decrease with an increase in probe concentra-
tion. However, we did not observe any significant fluorescence
change of EB-ct-DNA complex upon increase in probe concentra-
tion. Thus, this observation implied that the probe may be a groove
binder (see Supplementary data).10 To establish the groove binding
property of the probe, fluorescence titration experiment with Hoe-
chst 33258 was carried out. Hoechst 33258 is a minor groove bind-
ing agent to double-helical DNA.11 Like EB the fluorescence
intensity of Hoechst 33258 increased upon binding with DNA. The
crystal structure of Hoechst 33258 bound with DNA complex
showed that Hoechst 33258 bound to a minor groove of DNA. If
the probe binds in minor groove, then again it would be a compet-
itive binding agent of Hoechst 33258 and it would replace Hoechst
33258. Thus, the fluorescence intensity of Hoechst 33258 bound
with ct-DNA would decrease with an increase in probe concentra-
tion. Unlike with EB, we observed a significant decrease in fluores-
cence intensity (kem = 468 nm) of hoechst 33258 of hoechst 33258-
ct-DNA complex with an increase in probe concentration (Fig. 3a).
The Multi Gaussian fitting spectra clearly shows the decrease in
Hoechst emission with an increase in probe concentration
(Fig. 3b). This result indicated that the probe was likely to be a min-
or groove binder of ct-DNA. As the excitation wavelengths (kex
ꢁ340 nm) of both hoechst 33258 and the probe were quite same,
we observed another emission band (kem ꢁ 520 nm) dominated
over the parent hoechst’s band (kem ꢁ 468 nm) in high concentra-
Motivated by the detection of ct-DNA with an enhanced fluores-
cence signal and to explore the versatility of our probe we next
investigated the detection of protein, BSA spectroscopically.1,3
Thus, upon addition of increasing amounts of BSA to the probe’s
solution, we observed a hyperchromicity with very little (7 nm)
blue shift of the absorbance band as was clear from the UV–vis.
spectra (see Supplementary data). To get further insight into the
binding properties and to investigate the protein sensing ability
of the probe, we then investigated the fluorescence changes upon
addition of various concentrations of BSA to the solution of the
probe upon excitation at 344 nm. Thus, with increasing concentra-
tions of BSA, both the fluorescence intensity and quantum yield of
the fluorophore increased with a blue shift of 41 nm of emission
maximum (Fig. 4a). The fluorescence enhancement was maximized
upon addition of 2.5 equiv of BSA. This result indicated a clear
binding of the probe in more hydrophobic pocket of BSA which
was also evident from the intrinsic emission behavior of the probe
in various solvents of varying dielectric constants (see Supplemen-
tary data).3,5,15 The binding constant of triazolyl pyrene with BSA
was also determined fluorimetrically by a Benesi–Hildebrand plot
which was found to be 5.1 Â 104 MÀ1 with an experimental free
energy of binding,
D
G = À6.41 kcal/mol (Fig. 4b). The blue shift of
emission wavelength was also clear from a change of color of probe
solution from yellowish green to bright blue color of probe-BSA
solution when irradiated at 254 nm under a transilluminator
(Fig. 4c). An increase in the %
a-helicity, as was indicated by CD
spectra of BSA in the presence of our probe, could be attributed
to conformational adjustments on complex formation (see Supple-
mentary data).
The lipophilic nature of the probe and the above results indi-
cated that it would preferentially bind to the hydrophobic sub-
domain of BSA, thus might involve in energy transfer with Trp
unit.15 The possibility of energy transfer process from Trp residue
of BSA to the probe molecule was satisfied primarily by the spec-
tral overlap between the fluorescence emission spectrum of BSA
and the UV–visible absorption spectra of the probe (see Supple-
mentary data). Thus, upon excitation at donor wavelength
(280 nm of Trp of BSA), we observed quenching of fluorescence
tions of the probe (40–50 lM). The appearance of this band was
due to the binding of the probe with ct-DNA and possibly because
of energy transfer between Hoechst and the probe triazolyl pyrene
(see Supplementary data).11b Hoechst had a significantly lower
quantum yield when free in solution, in comparison to that when

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S. S. Bag et al. / Tetrahedron Letters 54 (2013) 2627–2632
40
30
0.3
0.2
40
Hoechst
Hoechst+
ct-DNA
10 µM
Hoechst
(c)
(a)
(b)
Hoechst+
Anisotropy
Polarization
ct-DNA
BSA
30
20 µM
10 µM
30 µM
10 µM
20 µM
40 µM
50 µM
20
10
0
20
20 µM
30 µM
30 µM
40 µM
40 µM
50 µM
50 µM
0.1
0.0
10
0
Anisotropy
Polarization
Hoechst
Hoechst
ct-DNA
400
500
600
700
0
50
100
150
360
460
560
660
Concentration (µM)
Wavelength (nm)
Wavelength (nm)
Figure 3. (a) Emission spectra of hoechst, hoechst-ct-DNA complex, and hoechst-ct-DNA complex titrated with various concentrations of TNDMBPy (kex = 340 nm). (b) Multi
Gaussian fitting of spectra ‘a’ to show clear decreases in Hoechst emission with an increase in [TNDMBPy]. Solid lines and dotted lines are the contribution of Hoechst’s and
TNDMBPy’s emission respectively in each concentration; [Hoechst] = 10 M and [TNDMBPy] = 10, 20, 30, 40, and 50
lM, [ct-DNA] = 50 l lM. (c) Fluorescence anisotropy change of
TNDMBPy in the presence of various concentrations of ct-DNA and BSA. All the experiments were carried out in 5 mM phosphate buffer, 5 mM NaCl, pH 7.0, rt, kex = 345 nm.
Figure 4. (a) Fluorescence titration of probe with various concentrations of BSA. (b) Benesi–Hildebrand plot. (c) Fluorescence image under UV-transilluminator (254 nm) of
only probe, in the presence of BSA. All the experiments were carried out in 5 mM phosphate buffer, pH 7.0, rt, kex = 345 nm.
of BSA and enhancement of fluorescence of the acceptor probe at
long wavelength (490–514 nm). This result indicated the occur-
rence of a FRET process (see Supplementary data). We calculated
the Förster distance of energy transfer which was found to be
R0 = 24.4 Å with an energy transfer efficiency of 62.3%.15
and quantum yield. The same explanation possibly holds true in
case of ct-DNA induced fluorescence enhancement of the probe
where the probe experienced restricted radiationless channel in-
side the groove of ct-DNA.
To explore the versatility of our probe, we also studied the
detection ability of short DNA duplex containing triple mis-
matched base pairs. DNA base mismatch is frequent among the
most common forms of DNA lesions which when left unrepaired
by the base mismatch repair machinery can lead to deleterious
mutations that are dangerous to cellular survival and replication.18
Therefore, the detection and targeting of such specific small defor-
mation in DNA are of paramount importance for the design of new
diagnostics and chemotherapeutics. Several methods have been
developed to detect mismatch DNA with fluorescent probe.19 How-
ever, conceptual design of label-free detection which we present
here is a novel, simple, cost effective, and rapid strategy (Fig. 5).
Spectroscopic evidences of minor groove binding of the probe
with ct-DNA and Amber⁄ optimized geometry of the probe with
a model DNA sequence (see Supplementary data) using Macro-
Model led us to think of the detection of short oligonucleotide du-
plex with our probe via just ‘Mix & Read’ strategy. Our concept
relies on the groove binding of the probe assisted by strong inter-
action possibly via triazolyl nitrogens in the vicinity of a deformed/
mismatched position of the DNA duplex. We envisaged that the
triazolyl pyrene might involve in stacking interaction along the
groove close to the deformed (mismatched) site (Fig. 6a). Such kind
of groove binding and recognition of DNA base mismatch were also
shown by imidazole–imidazole pair.19a Because of such groove
binding we expected that the probe would experience restricted
radiationless channel inside the groove that would lead to the
The occurrence of FRET process was also supported by a molec-
ular docking calculation which clearly showed that the probe was
located in the vicinity of tryptophan (Trp-237) and remained sur-
rounded by other hydrophobic amino acids of the hydrophobic
pocket of sub-domain IIA in site-I of BSA (see Supplementary data).
The free energy of binding (D
G) of TNDMBPy–BSA complex obtained
from the docking calculation was found to be in (À7.72 kcal/mol)
good agreement with our experimental value. That the probe in-
volved in tight binding inside the hydrophobic pocket of BSA and
experienced a highly restricted rotational motion, was further evi-
dent from an enhancement of fluorescence anisotropy of the probe
from 0.018 in free aqueous buffer to 0.201 in the presence of
2.5 equiv of BSA (Fig. 3b).16,17 This large fluorescence enhancement
in case of BSA is quite good for practical applications.
Therefore, it is clear from the above findings that the probe tria-
zolylpyrene was efficient in sensing biomolecules via the genera-
tion of enhanced fluorescence intensity. The low fluorescence
intensity of the probe in phosphate buffer in the absence of bio-
molecules was not because of poor solubility of the probe in buffer
but might be attributed to radiationless channel assisted by inter-
molecular hydrogen bonding present in aqueous solution. How-
ever, in the presence of biomolecules the non-radiative channels
were possibly blocked to a great extent and less effective as the
probe bound more and more with protein’s hydrophobic pocket
leading to a fluorescence switch-on signal of enhanced intensity

S. S. Bag et al. / Tetrahedron Letters 54 (2013) 2627–2632
2631
Figure 5. Schematic presentation of our concept of mismatch DNA detection.
(a)
50000
(c)
Probe
Probe + ODN 1/2
Probe + ODN 1/3
Probe + ODN 1/4
(b) Sequence of ODNs used
ODN 1:5'-CGCAATTTAACGC-3'
25000
ODN 2:5'-GCGTTCCCTTGCG-3'
ODN 3:5'-GCGTTGGGTTGCG-3'
ODN 4:5'-GCGTTAAATTGCG-3'
0
440
540
640
Wavelength (nm)
Figure 6. (a) Amber⁄ optimized geometry of probe-mismatched DNA showing groove binding. (b) Sequences of ODNs used. (c) Fluorescence response of TNDMBPy in the
presence of various mispaired ODNs at pH 7.0, 50 mM sodium phosphate, 0.1 M sodium chloride, room temperature. Excitation wavelength was, kex = 345 nm. Concentration
of each single strand and the probe, TNDMBPy was 2.5
lM.
generation of enhanced fluorescence signal. Thus, we could detect
deformed DNA duplex with a light-up fluorescence response from
bare fluorescent probe, TNDMBPy.
in aqueous media. The probe bound efficiently to the minor groove
of ct-DNA and the hydrophobic pocket of BSA protein. These modes
of binding were supported by fluorescence anisotropy experiment.
Also, the probe was able to detect DNA base mismatch with a gen-
eration of distinct fluorescence signal. All the spectral evidences
open up a multitude of possibilities for using our probe, tria-
zolylpyrene as a fluorescent light-up bioprobe. Furthermore, be-
cause the groove binding event in DNA and strong interaction
with BSA are important in consideration of drug-DNA/drug-protein
interaction, our probe might find application in studying such
events. Our preliminary study on the label-free fluorescent light-
up sensing and discrimination of DNA base mismatches revealed
that the probe would be very useful and might find future applica-
tions for the detection of and targeting DNA lesions with a less
laborious and cost-effective label-free way. We are currently
exploring a detailed study in the context of sensing G-quadruplex
DNA with the same probe.
We also surmised that depending on the sequence at the de-
formed (mismatched) site the probe might be able to generate
a distinct fluorescence signal allowing one to possible discrimina-
tion of different mismatched duplex DNAs. Because of quenching
nature of G-bases the fluorescence of probe in a triple T/G con-
taining short DNA duplex might not show any enhancement.
However, the fluorescence enhancement could be the result when
the probe would bind to a triple T/C mismatch containing DNA.
Therefore, we could discriminate T/C mismatch DNA from T/G
mismatch DNA via the generation of a distinct and enhanced fluo-
rescence signal (Fig. 5).
From the fluorescence spectra it was evident that an enhance-
ment of fluorescence compared to probe’s emission was observed
upon addition of 2.5 lM of probe in duplex ODN 1/2 containing tri-
ple T/C mispair (Fig. 6b). On the other hand, duplex ODN 1/3 con-
taining triple T/G mispair or a fully matched duplex ODN 1/4 led to
no change in emission of bare fluorescent probe, TNDMBPy. These re-
sults indicated that our probe was able to discriminate triple T/C
mispair from a triple T/G mispair duplex via the generation of an
enhanced and distinct fluorescence signal (Fig. 6c). Though the dis-
crimination was not high, our preliminary result might be an inspi-
ration to the design of such fluorescent small molecule to bring
efficient discrimination. Currently we are focusing in this aspect.
In conclusion, we have successfully shown, for the first time,
that the click chemistry derived triazolyl fluorophore serves as a
versatile fluorescent light-up probe for protein and DNA detection
Acknowledgments
Authors thank the Department of Science and Technology [DST:
SR/SI/OC-69/2008], Govt. of India, for a financial support.
Supplementary data
Supplementary data (photophysical spectra, docking/macro-
model structures) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.03.
029.

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Biol. Macromol. 2007, 41, 173; (d) Nafisi, S.; Bonsaii, M.; Maali, P.; Khalilzadeh,
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