Self-assembling asymmetrical tripodal-like peptides as anion receptors

Article abstract of DOI:10.1016/j.tet.2016.04.002

Aspartic and glutamic-acid based molecules T1a-b-T5a-b appended with different moieties were designed and synthesized. These molecules displayed unique self-assembly behavior dependent upon the nature of appended units. Trp favored vesicles, while phenyl unit favored fibrillar assembly. These molecules also showed binding towards anions.

Full text of DOI:10.1016/j.tet.2016.04.002

Tetrahedron 72 (2016) 2900e2905
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Self-assembling asymmetrical tripodal-like peptides as anion
receptors
*
Ishanki Bhardwaj, V. Haridas
Department of Chemistry, Indian Institute of Technology Delhi (IITD), Hauz Khas, New Delhi, 110016, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Aspartic and glutamic-acid based molecules T1aebeT5aeb appended with different moieties were
designed and synthesized. These molecules displayed unique self-assembly behavior dependent upon
the nature of appended units. Trp favored vesicles, while phenyl unit favored fibrillar assembly. These
molecules also showed binding towards anions.
Received 15 February 2016
Received in revised form 31 March 2016
Accepted 1 April 2016
Available online 4 April 2016
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Molecular recognition
Fluorophore
Tryptophan
Self-assembled vesicles
Encapsulation
1. Introduction
tripodal receptors could offer more binding sites for guest and
thereby achieve high binding affinity and selectivity. Appending
Molecular self-assembly is a powerful method for fabricating
supramolecular structures.1 The formation of well-ordered supra-
molecular structures through self-assembly of diverse organic and
inorganic building blocks has drawn much attention owing to their
potential applications in biology and chemistry.2 Peptides possess
hydrogen bonding capability; hence adopt specific structures
through self-assembly. The intermolecular non-covalent in-
teractions are utilized in designing diverse supramolecular struc-
tures such as fibers, vesicles, spheres, rods and helical ribbons. The
potential applications of these assemblies involve their use in drug
delivery, tissue engineering and wound healing.3e10 External
stimuli such as temperature, pH, electric field, chemicals and ions
can bring changes in the structure and solubility characteristics of
assembly.11,12 Such self-assembling molecules have several appli-
cations in the design of stimuli responsive materials.13e15
Designed peptides can act as good receptors for anions as they
have ability to bind guest through hydrogen bonds.16 The binding
efficiency of a host depends upon its geometry and the cavity size.
Designing tripodal receptors containing asymmetrical binding
arms is an interesting area of host-guest chemistry research.17,18
Amino acids with functionalizable side chain seem to be ideal
candidates for the design of tripodal receptors. The arms of the
with fluorophore can help in fluorescence detection of guests.
Amino acids such as threonine, serine, tyrosine, histidine and
tryptophan have OH or NH groups in their side chains, hence are
useful models for designing receptors for anions.19
2. Results and discussion
We report aspartic acid and glutamic acid-cored peptides
T1aebeT5aeb with and without tryptophan residues (Fig. 1).
T1aeb contain benzyl amine appended on Boc-
amino acid. T2aeb and T3aeb are synthesized by coupling p-
nitrobenzoic acid and Boc- -Tryptophan to the N-terminal of T1a
L-Asp and Boc-L-Glu
L
and T1b respectively. T4aeb and T5aeb constitute another class of
scaffolds where Boc-Asp and Boc-Glu amino acid are partially or
fully functionalized with tryptophan residues (Scheme 1, SI,
Schemes S1 and S2).
These diverse classes of molecules with amide functionalities
provide an interesting set of molecules for anion binding and self-
assembling studies. The three arms of aspartic and glutamic acid
serve as branches to functionalize with different moieties to gen-
erate molecules with tripodal-like topology. Tryptophan provides
indole NH as H-bond donor for binding to the guest and also helps
in fluorescent detection of guests.
Keeping in mind this concept, the binding efficiencies of
T1aebeT5aeb towards anions such as Fꢀ, Clꢀ, Brꢀ, Iꢀ, H2PO4ꢀ and
HSOꢀ4 were analyzed by using spectroscopic techniques such as
* Corresponding author. Tel.: þ91 011 26591380; e-mail address: haridasv@iitd.
ac.in (V. Haridas).
http://dx.doi.org/10.1016/j.tet.2016.04.002
0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.

I. Bhardwaj, V. Haridas / Tetrahedron 72 (2016) 2900e2905
2901
and T3aeb (Table 1). T1b showed higher binding compared to T1a
due to same steric reasons. Changing the N-terminal substituent of
T2aeb from p-nitrobenzoyl to Boc- -Tryptophan provided T3aeb.
L
The introduction of tryptophan residue was expected to increase
the binding affinity as it can provide an additional NH for binding.
Interestingly, the binding studies showed no considerable increase
in binding affinity (Table 1). This may be due to orientation of
tryptophan group away from the binding cavity, thereby not par-
ticipating in the binding. The stoichiometry of binding for
T1aebeT3aeb and T4beT5aeb with all anions is 1:1, while T4a
binds to sulfate in 1:2 fashion (G:H 1:2) as evident from Job’s plot
(SI, Fig. S2).
In order to enhance the binding, T4aeb and T5aeb with two
and three tryptophan residues were synthesized. UVevis and
fluorescence titration experiments carried out with anions revealed
that T4a efficiently binds to phosphate and sulfate.
Fig. 1. Structural representation of tripodal receptors T1aebeT5aeb.
Fluorescence titration showed increase in fluorescence intensity
at 445 nm upon slot-wise addition of TBAHSO4 in chloroform
(Fig. 2b). 1H NMR titration showed the downfield shift of NHs upon
addition of anion salt (Fig. 3). It is evident from Fig. 3, that amide
NH(b) showed downfield shift from 6.32 ppm to 6.78 ppm while
indole NH’s (d and e) showed downfield shift from 8.11 ppm to
9.12 ppm indicating that these NHs are involved in binding with the
guest. T4b has less binding affinity as compared to T4a (Table 1).
The extra methylene group present in T4b moves the side chains
away from each other, thus decreasing the binding. When the N-
terminal of T4aeb was functionalized by another tryptophan resi-
due to give T5aeb, the observed binding affinity is comparatively
lesser or similar (Table 1). The contribution towards binding is
majorly due to two tryptophan residues. None of these receptors
showed binding towards Brꢀ and Iꢀ ions.
As T1aebeT5aeb contain amide bonds, they can associate by
intermolecular hydrogen bonds. In addition, they contain amino
acid residues with side chains capable of
pep interactions due to
Scheme 1. Synthesis of tripodal peptides T1aebeT5aeb. (i) DCC, HOSu, NEt3, Benzyl
amine, dry CH2Cl2, 0 ꢂC, 24 h (ii) Trifluoroacetic acid, dry CH2Cl2, 0 ꢂC, 4 h (iii) p-ni-
trobenzoyl chloride, NEt3, dry CH2Cl2, 0 ꢂC, 12 h (iv) Trifluoroacetic acid, dry CH2Cl2,
0 ꢂC, 4 h (v) Boc-L-Trp-OH, DCC, HOSu, NEt3, dry CH2Cl2, 0 ꢂC, 24 h (vi) DCC, HOSu, NEt3,
L-Trp-OMe, dry CH2Cl2, 0 ꢂC, 24 h (vii) saturated soln. of HCl in ethyl acetate, 0 ꢂC, 6 h
(viii) Boc-L-Trp-OH, DCC, HOSu, NEt3, dry CH2Cl2, 0 ꢂC, 24 h.
presence of aromatic groups. The presence of these moieties in the
scaffolds can provide distinct self-assembling features. Hence,
these peptides provide interesting candidates for self-assembly
studies. The self-assembling features of T1aebeT5aeb were
studied in 1:1 CH3OH/CHCl3 by several microscopic techniques
such as scanning electron microscopy (SEM), transmission electron
microscopy (TEM) and atomic force microscopy (AFM). The studies
revealed that T1aeb and T2aeb self-assembled into fibers, while
T3aebeT5aeb self-assembled into vesicles (Fig. 4, SI, Figs. S5eS6).
The backbone of T1aebeT5aeb consists of Asp and Glu amino
acids, while the arms are functionalized with different units. Thus,
the self-assembly can be controlled by appended units. A closer
look at the SEM images showed that the average size of vesicles of
UVevis fluorescence and NMR (Figs. 2 and 3, SI, Figs. S1eS4). The
anion binding studies were carried out in chloroform. UVevis and
NMR titrations showed that T2b acts as a receptor for phosphate
and chloride ion (Table 1). The occurrence of one isosbestic point in
UV titration profile of T2b (4.5ꢁ10ꢀ5 M) with phosphate
(4.5ꢁ10ꢀ2 M) is a noteworthy observation (Fig. 2a). Surprisingly,
T2a (with one methylene less than T2b) didn’t show any changes in
the UV spectra when titrated with TBAH2PO4, TBABr, TBAI and
TBAHSO4. This may be due to steric crowding around the two amide
NHs in T2a as compared to T2b. Presence of one additional meth-
ylene in T2b relieves the steric strain and hence increases the
binding. One of the three arms of T1aeb is not functionalized in
comparison to T2aeb and T3aeb. Therefore, T1aeb show differ-
ences in binding selectivity compared to tripodal receptors T2aeb
T3aebeT5aeb is 0.3e0.8
phosphotungstic acid) revealed that T3aebeT5aeb formed vesi-
cles in the range of 0.3e0.9 m.
mm (SI, Fig. S7). TEM (stained with 0.2%
m
After discovering the distinct morphological features of these
peptides, we turned our attention to evaluate the encapsulating
potency of the vesicles formed from self-assembly of
T3aebeT5aeb.
Fluorescence microscopic analysis of T3aebeT5aeb revealed
that the vesicles can be seen in blue color (lex¼380e450 nm), while
the
rhodamine
B
entrapped
vesicles
appeared
red
(
lex¼510e560 nm) (Fig. 5a,b) clearly indicating the encapsulation
potency of rhodamine B by the vesicles. Addition of 0.2 equiv of
sulfate salt to the dye entrapped vesicles resulted in the disruption
of vesicles (Fig. 5c), since we could observe the reappearance of
blue fluorescence of T3b (Fig. 5d). The vesicular morphology is lost
due to anion binding, thus disrupting the self-assembly.
The influence of anion binding on self-assembly of
T1aebeT5aeb was studied by microscopic imaging. Experiments
were performed by adding the anion to the self-assembled system
Fig. 2. (a) UV titration profile for T2b (4.5ꢁ10ꢀ5 M) with (0e40 equiv) TBAH2PO4
(4.5ꢁ10ꢀ2 M) in CHCl3 (b) Fluorescence titration profile for T4a (1.2ꢁ10ꢀ4 M) with
TBAHSOꢀ4 (1.08ꢁ10ꢀ1 M) in CHCl3.

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I. Bhardwaj, V. Haridas / Tetrahedron 72 (2016) 2900e2905
Fig. 3. Partial 1H NMR (300 MHz, CDCl3) of T4a (3.9ꢁ10ꢀ3 M) with and without addition of various amounts of TBAHSO4 (1.5ꢁ10ꢀ1 M) (0e7.2 equiv) in CDCl3.
Table 1
morphology is due to the anion binding on the self-assembled T3b,
Binding affinities of T1aebeT5aeb with TBA anions in chloroform (NB¼not
resulting in the removal of some T3b molecules from vesicles
resulting in voids, which is observed in SEM as wiffle-ball. Addition
of 2.0 equiv of anion to the self-assembled system completely de-
stroys the vesicular assembly and formation of particles of in-
definite shape is observed (Fig. 6c).
binding)
Receptor
Anion T1a T1b T2a T2b T3a T3b T4a
T4b
T5a
T5b
Fꢀ
2300 NB
NB NB
NB NB 640
61,900 15,478 450
NB
630
CIꢀ
NB 4800 NB 1000 360 NB NB
290
NB
NB
NB
1300
NB
H2POꢀ4 NB
HSOꢀ4 NB
NB
NB
NB 1000 NB 22 8400
NB NB
NB NB 2.6ꢁ105 NB
3. Conclusions
In summary, we have designed and synthesized aspartic and
glutamic acid based branched peptides. They displayed varied self-
assembling architectures, such as fibers and vesicles. The anion
binding property of these molecules is used to fabricate wiffle-ball
like assembly. The vesicles as well as wiffle-ball like supramolecular
self-assemblies offer opportunities to utilize these systems as
of T1aebeT5aeb in 1:1 CH3OH/CHCl3. Addition of 0.5 equiv of
anion salt resulted in the formation of holes on the surface of
vesicles. Addition of 1.0 equiv resulted in the formation of wiffle-
ball like structure (Fig. 6aec). The formation of wiffle-ball like
Fig. 4. SEM images of (a) T1a (3.1 mM) (b) T1b (3.4 mM) (c) T2a (2.8 mM) (d) T2b (2.9 mM) (e) T3a (3.1 mM) (f) T3b (3.4 mM) (g) T4a (2.8 mM) (h) T4b (2.9 mM) (i) T5a (2.8 mM) (j)
T5b (2.9 mM) in 1:1 CH3OH/CHCl3 respectively.

I. Bhardwaj, V. Haridas / Tetrahedron 72 (2016) 2900e2905
2903
(multiplet). High resolution mass spectra (HRMS) were recorded in
Bruker MicrO-TOF-QII model using ESI technique. UV-visible
spectra were recorded in Shimadzu double beam spectrophotom-
eter, UV-2450. The emission spectra were recorded in HORIBA
JOBIN YVON Scientific, fluoromax-4 spectrofluorometer with a slit-
width of 5 nm. The experimental procedure and spectral data of
T4b and T5b are reported in ref. 2(b).
4.2. Microscopy methods
4.2.1. Optical microscopy. A drop of solution of the compound was
put on a glass slide, the solvent was allowed to evaporate in air. It
was then viewed using Nikon Eclipse TS100 optical microscope
system.
4.2.2. Transmission electron microscopy. Around 3.0 mM solution of
the sample in HPLC grade methanol/chloroform (1:1) was used for
TEM. All the sample solutions were filtered through a nylon syringe
filter (0.2 mm). About 2 ml aliquot of the sample solution was placed
Fig. 5. Fluorescence microscopic image (in 1:1 CH3OH/CHCl3) of (a) T3b (2.54 mM)
alone (lex¼380e450 nm) (b) T3bþ0.02 equiv rhodamine B (lex¼510e560 nm) (c) (b)
on a 200 mesh copper grid and stained with 2% wt. phospho-
tungstic acid in water for 2 min and the grid was allowed to dry in
atmosphere. Samples were viewed using a Philips CM 12 trans-
mission electron microscope.
þ2.0 equiv TBAHSO4
lex¼380e450 nm).
(
lex¼510e560 nm) (d) (b) þ2.0 equiv TBAHSO4
(
4.2.3. Scanning electron microscopy. Around 3.0e4.0 mM solution
of compound in HPLC grade methanol/chloroform (1:1) was pre-
pared and one drop of the solution was put on the glass cover slip
pasted on a carbon tape mounted on a stub, dried under sodium
lamp and coated with w10 nm of gold. Samples were analyzed
using scanning electron microscope ZEISS EVO 50 SEM.
4.2.4. Atomic force microscopy. Around 3.0e4.0 mM solution of
compound in HPLC grade methanol/chloroform (1:1) was prepared
and one drop of the solution was put on silicon wafer and dried in
atmosphere. Samples were analyzed using Dimension Icon AFM
operating at tapping mode in air. Images were recorded in air at
room temperature and data analysis was performed using nano-
scope 5.31r software.
4.3. Anion binding studies
4.3.1. UVevis titration experiments. Stock solutions of compounds
T1aebeT5aeb were prepared in UV spectroscopy grade chloro-
form with concentration (10ꢀ4e10ꢀ5 M). Solutions of anion salts
were prepared in UV spectroscopy grade chloroform with con-
centration (10ꢀ1e10ꢀ2 M). The absorbance of blank compound
solution and upon gradual addition of anion salt was recorded
using Shimadzu double beam UVevis spectrophotometer model
UV-2450.
Fig. 6. SEM images of T3b (a) 0.5 equiv (b) 1.0 equiv (c) 2.0 equiv of TBAHPO4. All
experiments were done in 1:1 CH3OH/CHCl3.
containers for guest encapsulation and release. The present study
also demonstrates the successful execution of the principle of
molecular recognition to fabricate wiffle-ball like structure from
vesicular assembly.
4. Experimental section
4.3.2. Fluorescence titration experiments. Stock solutions of com-
pounds T1aebeT5aeb were prepared in UV spectroscopy grade
chloroform with concentration (10ꢀ4e10ꢀ5 M). Solutions of anion
salts were prepared in UV spectroscopy grade chloroform with
concentration (10ꢀ1e10ꢀ2 M). The fluorescence emission spectra of
blank compound solution and upon gradual addition of anion salt
were recorded using Horiba Jobin Yvon Scientific, fluoromax-4
spectrofluoremeter with slit width of 5 nm.
4.1. Materials and methods
All organic solvents employed in the synthesis were distilled
and dried from appropriate drying agents. Reactions were moni-
tored by silica gel-based thin layer chromatography (TLC). Silica gel
(100e200 mesh) was used for purification by column chromatog-
raphy. Fisher-Scientific melting point apparatus was used for re-
ꢀ ꢀ
cording melting points. Nicolet, Protege 460 spectrometer was used
4.3.3. NMR titration experiments. Stock solutions of compounds
T1aebeT5aeb were prepared in CDCl3 with concentration 10ꢀ3 M.
Solutions of anion salts were prepared in CDCl3 with concentration
10ꢀ1 M. The spectra were collected on a Bruker DRX 300 (300 MHz)
NMR spectrometer. Anion binding titrations were carried out by
monitoring changes in the aromatic proton (ArH) and amide NH
for recording FT-IR. Compounds were used as KBr pellets. 1H NMR
spectra were recorded on Brucker-DPX-300 spectrometer. Tetra-
methylsilane (1H) was used as an internal standard. Coupling
constants are reported in Hz and data are reported as s (singlet),
d (doublet), br (broad), t (triplet), dd (double doublet) and m

2904
I. Bhardwaj, V. Haridas / Tetrahedron 72 (2016) 2900e2905
signal of receptors as a function of added tetrabutylammonium
halide concentration.
followed by addition of p-nitrobenzoyl chloride (0.083 g,
0.44 mmol) in dry CH2Cl2 (20 mL) and stirred for 12 h. The reaction
mixture was diluted with 100 mL of CH2Cl2, washed sequentially
with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution.
The organic layer was dried over Na2SO4, and concentrated under
reduced pressure. The crude product was chromatographed on
a column of silica gel using ethyl acetate/hexane as eluents to ob-
tain the pure product T2a as off-white solid. Mp: 223e225 ꢂC.
Yield: 37%. Rf (4% methanol/chloroform): 0.6. 1H NMR (300 MHz,
4.3.4. Calculation of binding constants and stoichiometry. The
binding constants were calculated by UVevis titration experiments
using Benesiehildbrand plot as standardized for 1:1 and 1:2 stoi-
chiometry. Stoichiometry of binding was calculated by Job’s plot
from UVevis absorbance data.
4.4. Synthesis of T1a
DMSO-d6)
1H,
eCH), 7.20e7.24 (m,10H, ArH), 8.08 (d, 2H, J¼7.5 Hz, ArH), 8.31
(d, 2H, J¼8.0 Hz, NH), 8.52 (br s, 1H, NH), 8.97 (br s, 1H, NH).
13C NMR (75 MHz, DMSO-d6)
22.14, 31.34, 42.91, 53.17, 121.05,
123.45, 126.60, 126.77, 126.98, 127.21, 128.29, 136.15, 139.32, 139.51,
140.89, 158.12, 170.96, 172.50. IR (KBr) 1000, 1025, 1641, 2258, 2360,
2851, 2921, 3433 cmꢀ1. HRMS calcd for C25H24N4O5Na m/z 483.1639
found 483.1630.
d 2.73 (m, 2H, Asp (CH2)), 4.27 (br s, 4H, ArCH2), 4.87 (m,
a
To an ice-cooled and stirred solution of 1a (0.500 g, 2.13 mmol)
in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added N-hydrox-
ysuccinimide (HOSu) (0.538 g, 4.68 mmol), DCC (0.965 g,
4.68 mmol) and stirred for 10 min. To the reaction mixture was
added mixture of triethylamine (0.65 mL, 4.68 mmol), benzyl
amine (0.51 mL, 4.68 mmol), dry CH2Cl2 (20 mL) and stirred for 24 h
at room temperature. The reaction mixture was filtered and the
filtrate was diluted with 100 mL of CH2Cl2, washed sequentially
with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution.
The organic layer was dried over Na2SO4, and concentrated under
reduced pressure. The crude product was chromatographed on
a column of silica gel using ethyl acetate/hexane as eluents to ob-
tain the pure product T1a as white solid. Mp: 160e162 ꢂC. Yield:
91%. Rf (60% ethyl acetate/hexane): 0.6. 1H NMR (300 MHz, DMSO-
d
4.7. Synthesis of T2b
To T1b (0.229 g, 0.53 mmol) in dry CH2Cl2 (4.0 mL) at 0 ꢂC, added
trifluoroacetic acid (1.2 mL, 9.0 mmol) drop-wise and stirred for 4 h.
Solvent was evaporated under high vacuum to give N-deprotected
T1b (0.175 g). Yield: quantitative. To an ice-cooled and stirred so-
lution of free amine (0.175 g, 0.53 mmol) in 100 mL of dry CH2Cl2
and 0.3 mL DMF, was added triethylamine (0.07 mL, 0.53 mmol)
followed by addition of p-nitrobenzoyl chloride (0.083 g,
0.44 mmol) in dry CH2Cl2 (20 mL) and stirred for 12 h. The reaction
mixture was diluted with 100 mL of CH2Cl2, washed sequentially
with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution.
The organic layer was dried over Na2SO4, and concentrated under
reduced pressure. The crude product was chromatographed on
a column of silica gel using ethyl acetate/hexane as eluents to ob-
tain the pure product T2b as white solid. Mp: 216e218 ꢂC. Yield:
30%. Rf (4% methanol/chloroform): 0.4. 1H NMR (300 MHz, DMSO-
d6)
4.28e4.36 (sþm, 5H, 4 ArCH2þ1
7.27e7.33 (m, 10H, ArH), 8.31 (br s, 2H, NH). 13C NMR (75 MHz,
DMSO-d6) 27.60, 37.03, 41.55, 51.14, 77.67, 126.01, 126.11, 126.33,
d
1.40 (s, 9H, eCH (CH3)3), 2.50e2.68 (m, 2H, Asp (CH2)),
a
eCH), 6.95 (d, 1H, J¼7.8 Hz, NH),
d
126.57,127.53,127.60,138.75,138.87,154.53,168.86,170.79. IR (KBr)
1049, 1086, 1166, 1243, 1309, 1444, 1532, 1573, 1628, 1693, 2850,
2926, 3032, 3320 cmꢀ1. HRMS calcd for C23H29N3O4Na m/z
434.2050 found 434.2049.
4.5. Synthesis of T1b
d6)
d
1.95e2.21 (m, 2H, Glu (CH2)), 2.28 (m, 2H, Glu (CH2)), 4.27 (dd,
eCH),
To an ice-cooled and stirred solution of 1b (0.250 g, 1.01 mmol)
in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added HOSu (0.253 g,
2.2 mmol), DCC (0.453 g, 2.2 mmol) and stirred for 10 min. To the
reaction mixture was added triethylamine (0.31 mL, 2.2 mmol) and
benzyl amine (0.24 mL, 2.2 mmol) and stirred for 24 h. The reaction
mixture was filtered and the filtrate was diluted with 100 mL of
CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated NaHCO3
solution and brine solution. The organic layer was dried over
Na2SO4, and concentrated under reduced pressure. The crude
product was chromatographed on a column of silica gel using ethyl
acetate/hexane as eluents to obtain the pure product T1b as white
solid. Mp: 123e125 ꢂC. Yield: 93%. Rf (100% ethyl acetate): 0.7. 1H
4H, J1¼5.4 Hz, J2¼15 Hz, PhCH2), 4.43e4.46 (m, 1H,
a
7.202e7.32 (m, 10H, ArH), 8.14 (d, 2H, J¼8.4 Hz, ArH), 8.31 (d, 2H,
J¼8.7 Hz, ArH), 8.37 (t,1H, J¼6 Hz, NH), 8.55 (t,1H, J¼6 Hz, NH), 8.96
(d, 1H, J¼7.5 Hz, NH). 13C NMR (75 MHz, DMSO-d6)
d 26.86, 31.45,
41.50, 53.21, 122.76, 126.10, 126.44, 126.59, 127.64, 128.50, 138.83,
138.92, 139.91, 148.50, 164.28, 170.60, 171.03. IR (KBr) 1027, 1076,
1234, 1641, 2258, 2360, 2851, 2921, 3433 cmꢀ1. HRMS calcd for
C
26H26N4O5Na m/z 497.1795 found 497.1794.
4.8. Synthesis of T3a
To an ice-cooled and stirred solution of Boc-L-Tryptophan
NMR (300 MHz, CDCl3)
Glu (CH2)), 2.10e2.36 (m, 2H, Glu (CH2)), 4.13e4.30 (m, 1H,
d
1.41 (s, 9H, eCH (CH3)3), 1.68e2.10 (m, 2H,
eCH),
(0.122 g, 0.40 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was
added HOSu (0.55 g, 0.48 mmol), DCC (0.100 g, 0.48 mmol) and
stirred for 10 min. To the reaction mixture was added triethylamine
(0.07 mL, 0.48 mmol) and N-deprotected T1a (0.151 g, 0.48 mmol)
in dry CH2Cl2 (20 mL) and stirred for 24 h. The reaction mixture was
diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N
H2SO4, saturated NaHCO3 solution and brine solution. The organic
layer was dried over Na2SO4, and concentrated under reduced
pressure. The crude product was chromatographed on a column of
silica gel using ethyl acetate/hexane as eluents to obtain the pure
product T3a as off-white solid. Mp: 193e195 ꢂC. Yield: 34%. Rf
a
4.41 (d, 4H, J ¼ 5.4 Hz, 4 ArCH2), 5.71 (d, 1H, NH), 6.32 (br s, 1H, NH),
6.89 (t, 1H, J¼6 Hz, NH), 7.26e7.31 (m, 10H, ArH). 13C NMR (75 MHz,
DMSO-d6)
d 27.54, 31.40, 32.81, 41.51, 53.78, 77.59, 126.15, 126.47,
126.62, 127.65, 138.89, 139.04, 154.82, 171.03, 171.40. IR (KBr) 1051,
1087, 1166, 1243, 1275, 1312, 1445, 1530, 1574, 1629, 1688, 2851,
2926, 3032, 3323 cmꢀ1
. HRMS calcd for C24H31N3O4Na m/z
448.2260 found 448.2197.
4.6. Synthesis of T2a
(100% ethyl acetate): 0.7. 1H NMR (300 MHz, DMSO-d6)
eCH (CH3)3), 2.58 (br s, 2H, Asp (CH2)), 2.87e3.20 (m, 2H, Trp
eCH), 4.62 (m, 1H, eCH),
d 1.26 (s, 9H,
To T1a (0.231 g, 0.53 mmol) in dry CH2Cl2 (4.0 mL) at 0 ꢂC, added
trifluoroacetic acid (1.2 mL, 9.0 mmol) drop-wise and stirred for 4 h.
Solvent was evaporated under high vacuum to give N-deprotected
T1a (0.175 g). Yield: quantitative. To an ice-cooled and stirred so-
lution of free amine (0.175 g, 0.53 mmol) in 100 mL of dry CH2Cl2
and 0.3 mL DMF, was added triethylamine (0.07 mL, 0.53 mmol)
(CH2)), 4.02e4.26 (m, 5H, 4Ph (CH2)þ1
a
a
6.90e7.32 (m, 15H, ArH), 7.55 (d, 1H, J¼7.5 Hz, NH), 8.12 (t, 1H,
J¼3.9 Hz, NH), 8.31 (d, 1H, J¼7.9 Hz, NH), 8.42 (t, 1H, J¼3.9 Hz, NH),
10.80 (br s, 1H, indole NH). 13C NMR (75 MHz, DMSO-d6)
d 27.23,
28.01, 36.89, 42.04, 50.00, 55.50, 78.26, 110.03,111.19, 118.07, 118.28,

I. Bhardwaj, V. Haridas / Tetrahedron 72 (2016) 2900e2905
2905
120.72, 123.72, 126.50, 126.60, 126.79, 127.11, 127.60, 127.28, 128.04,
128.12, 136.01, 139.09, 139.20, 155.45, 169.40, 170.54, 171.74. IR (KBr)
1172, 1248, 1533, 1650, 1688, 2930, 2974, 3318, 3406 cmꢀ1. HRMS
calcd for C34H39N5O5Na m/z 620.2843 found 620.2849.
triethylamine (0.04 mL, 0.30 mmol) and N-deprotected T4a
(0.160 g, 0.30 mmol) in dry CH2Cl2 (20 mL) and stirred for 24 h. The
reaction mixture was filtered and the filtrate was diluted with
100 mL of CH2Cl2, washed sequentially with 0.2 N H2SO4, saturated
NaHCO3 solution and brine solution. The organic layer was dried
over Na2SO4, and concentrated under reduced pressure. The crude
product was chromatographed on a column of silica gel using ethyl
acetate/hexane as eluents to obtain the pure product T5a as off-
white solid. Mp: 151e153 ꢂC. Yield: 50%. Rf (100% ethyl acetate):
4.9. Synthesis of T3b
To an ice-cooled and stirred solution of Boc-L-Tryptophan
(0.170 g, 0.56 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was
added HOSu (0.69 g, 0.56 mmol), DCC (0.115 g, 0.56 mmol) and
stirred for 10 min. To the reaction mixture, was added triethyl-
amine (0.07 mL, 0.56 mmol) and N-deprotected T1b (0.152 g,
0.47 mmol) in dry CH2Cl2 (20 mL) and stirred for 12 h. The reaction
mixture was diluted with 100 mL of CH2Cl2, washed sequentially
with 0.2 N H2SO4, saturated NaHCO3 solution and brine solution.
The organic layer was dried over Na2SO4, and concentrated under
reduced pressure. The crude product was chromatographed on
a column of silica gel using ethyl acetate/hexane as eluents to ob-
tain the pure product T3b as light yellow solid. Mp: 155e157 ꢂC.
Yield: 73%. Rf (100% ethyl acetate): 0.5. 1H NMR (300 MHz, DMSO-
0.6. 1H NMR (300 MHz, CDCl3)
(m, 2H, Asp (CH2)), 3.11e3.49 (m, 6H, Trp (CH2)), 3.54e3.80 (sþs,
6H, eOCH3), 4.64e4.85 (m, 4H, eCH), 5.30 (br s, 1H, NH),
6.80e7.65 (m, 18H, 15ArHþ3 NH), 8.14 (br s, 1H, NH), 8.46 (br s, 1H,
NH), 8.57 (br s, 1H, NH). 13C NMR (75 MHz, DMSO-d6)
22.51, 25.01,
d 1.41 (s, 9H, eCH (CH3)3), 2.18e2.60
a
d
26.99, 28.23, 29.63, 49.10, 52.27, 52.33, 52.89, 79.00, 109.34, 111.42,
118.31, 119.37, 121.89, 123.77, 127.10, 127.31, 136.08, 155.22, 170.30,
171.50, 172.29. 173.90, 174.090. IR (KBr) 1169, 1225, 1368, 1438, 1517,
1663, 1736, 2360, 2927, 3411 cmꢀ1. HRMS calcd for C44H49N7O9Na
m/z 842.3484 found 842.3472.
d6)
s, 2H, Glu (CH2)), 2.58e2.94 (m, 2H, Trp (CH2)), 4.18e4.45 (m, 6H,
4Phe (CH2)þ2 eCH), 6.83e7.32 (m, 15H, ArH), 7.59 (d, 1H, J¼7.8 Hz,
NH), 8.05 (d, 1H, J¼7.5 Hz, NH), 8.34 (m, 2H, NH), 10.89 (br s, 1H,
indole NH). 13C NMR (75 MHz, DMSO-d6)
25.14, 28.02, 31.64,
d 1.28 (s, 9H, eCH (CH3)3), 1.85e2.20 (m, 2H, Glu (CH2)), 2.21 (br
Acknowledgments
a
We thank DST (Grant No. SB/S1/OC-23/2014), New Delhi for fi-
nancial assistance. We acknowledge Department of Textile engi-
neering, IITD for SEM images. We thank Dr. A. K. Panda, NII, New
Delhi for providing fluorescence microscopic facility. IB thanks
CSIR, New Delhi for the fellowship.
d
32.51, 41.99, 52.33, 55.31, 79.32, 110.13,111.16,118.05,118.38,120.70,
123.59, 126.58, 126.98, 127.07, 128.14, 135.99, 139.11, 139.52, 155.18,
171.43, 171.85, 172.60. IR (KBr) 1168, 1247, 1536, 1644, 1692, 2929,
2976, 3303, 3401 cmꢀ1
. HRMS calcd for C35H41N5O5Na m/z
Supplementary data
634.3000 found 634.3007.
Supplementary data (1H NMR, 13C NMR and HRMS spectral data,
microscopic images and binding studies data is available in the
supplementary file provided with this manuscript.) associated with
this article can be found in the online version, at http://dx.doi.org/
10.1016/j.tet.2016.04.002.
4.10. Synthesis of T4a
To an ice-cooled and stirred solution of 1a (0.200 g, 0.86 mmol)
in 100 mL of dry CH2Cl2 and 0.3 mL DMF, was added HOSu (0.216 g,
1.88 mmol), DCC (0.388 g, 1.88 mmol) and stirred for 10 min. To the
reaction mixture was added triethylamine (0.26 mL, 1.88 mmol)
and Trp-OMe.HCl (0.477 g, 1.88 mmol) in dry CH2Cl2 (20 mL) and
stirred for 24 h. The reaction mixture was filtered and the filtrate
was diluted with 100 mL of CH2Cl2, washed sequentially with 0.2 N
H2SO4, saturated NaHCO3 solution and brine solution. The organic
layer was dried over Na2SO4, and concentrated under reduced
pressure. The crude product was chromatographed on a column of
silica gel using ethyl acetate/hexane as eluents to obtain the pure
product T4a as white solid. Mp: 108e110 ꢂC. Yield: 55%. Rf (100%
References and notes
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(CH3)3), 2.30e2.78 (m, 2H, Asp (CH2)), 3.22 (m, 4H, Trp (CH2)),
3.44e3.60 (sþs, 6H, eOCH3), 4.54 (m, 1H, eCH), 4.80 (m, 2H,
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24.84, 27.43, 28.14,
d 1.37 (s, 9H, eCH
a
a
d
33.80, 49.10, 52.27, 52.89, 80.29, 109.20, 111.36, 118.31, 119.35,
121.88, 123.54, 127.30, 136.11, 155.61, 170.73, 171.16, 171.97. IR (KBr)
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2977, 3004, 3057, 3386 cmꢀ1. HRMS calcd for C33H39N5O8Na m/z
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4.11. Synthesis of T5a
To T4a (0.909 g, 0.25 mmol), added saturated solution of HCl in
ethyl acetate (10.0 mL) at 0 ꢂC and stirred for 4 h. Solvent was
evaporated under high vacuum to give N-deprotected T4a (0.160 g).
Yield: quantitative. To an ice-cooled and stirred solution of Boc-L-
Trp-OH (0.76 g, 0.25 mmol) in 100 mL of dry CH2Cl2 and 0.3 mL
DMF, was added HOSu (0.35 g, 0.30 mmol), DCC (0.62 g, 0.30 mmol)
and stirred for 10 min. To the reaction mixture, was added
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