Packing-induced solid-state fluorescence and thermochromic behavior of peptidic luminophores

Article abstract of DOI:10.1039/c6ra24799g

The effect of packing on the solid state fluorescent propensities of peptidic luminophores has been investigated. A series of peptides containing 6-nitro-coumarin-3-carboxylic acid and α-aminoisobutyric acid (Aib) has been synthesized to study the structure-property relationship and stimuli responsive emission properties of the luminophores. In spite of the presence of a coumarin chromophore, peptide 2 exhibits no solid state fluorescence. The alkyl chains of Aib affect the solid state molecular packing modes and optoelectronic properties significantly. However, the fluorescence microscopy confirmed the change of blue fluorescence of peptide 3 in crystal and in amorphous states. The X-ray crystallography sheds some light on the molecular orientation, packing and the diverse degree of π-π stacking orbital overlap of adjacent coumarin chromophores of the peptide 3. Irrespective of coumarin chromophore, the peptides exhibit different packing-induced solid state emission properties varying from one peptide to another, which highlights the effect of Aib side chains.

Full text of DOI:10.1039/c6ra24799g

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PAPER
Packing-induced solid-state fluorescence and
thermochromic behavior of peptidic
luminophores†
Cite this: RSC Adv., 2017, 7, 389
Apurba Pramanik and Debasish Haldar*
The effect of packing on the solid state fluorescent propensities of peptidic luminophores has been
investigated. A series of peptides containing 6-nitro-coumarin-3-carboxylic acid and a-aminoisobutyric
acid (Aib) has been synthesized to study the structure–property relationship and stimuli responsive
emission properties of the luminophores. In spite of the presence of a coumarin chromophore, peptide
2
exhibits no solid state fluorescence. The alkyl chains of Aib affect the solid state molecular packing
modes and optoelectronic properties significantly. However, the fluorescence microscopy confirmed the
change of blue fluorescence of peptide 3 in crystal and in amorphous states. The X-ray crystallography
sheds some light on the molecular orientation, packing and the diverse degree of p–p stacking orbital
overlap of adjacent coumarin chromophores of the peptide 3. Irrespective of coumarin chromophore,
the peptides exhibit different packing-induced solid state emission properties varying from one peptide
to another, which highlights the effect of Aib side chains.
Received 6th October 2016
Accepted 25th October 2016
DOI: 10.1039/c6ra24799g
www.rsc.org/advances
23
pyrene by acetylation. Fraser and co-workers have reported the
polymorphism induced reversible mechanochromic lumines-
Molecular packing directed luminescence is highly important cence for diuoroboronavobenzone. But, controlling molec-
Introduction
24
in crystal engineering, where a solid material exhibits change in ular orientation and diverse degree of orbital overlap of
1
25
photoluminescence colour in response to external stimuli. In adjacent chromophores is still challenging. However, the
a recent review, Yang et al. summarized the effect of alkyl length inuences of packing on the uorescent colors of peptides/
2
6
on the solid-state uorescence and mechanochromic behaviour foldamers are relatively rare.
of organic days. The organic materials with packing directed
2
Coumarin a natural product has been used as gain medium
luminescence are of great interest due to practical applications in blue-green dye lasers and sensitizer for photovoltaic cell.
such as lasers, OLEDs, optical data storage, two-photon pho- Coumarin derivative also have been used to develop array for
toluminescence (PL) microscopy, optical switching and protein sensing. Ting and co-workers have used coumarin
27
3
4
5
28
6
limiting. Various external stimuli and strategies such as uorophore ligation for imaging protein–protein interaction
7
–10
29
chemical modication,
mechanical control of the packing inside living cell. Herein, we have synthesized a series of
have been used to alter the uores- nitropeptides containing 6-nitro-coumarin-3-carboxylic acid
11–16
17,18
modes,
co-crystals,
cence properties. Several reports have shown the inuences of methyl ester, a-aminoisobutyric acid (Aib) and investigated
packing on the uorescent colors of various synthetic organic their structures and optical properties. Aib is conformationally
1
9,20
dyes.
mechanochromism triggered uorescent colour switching not only for molecular conformation, but also for controlling
For example, Mei and co-workers have shown the rigid and helicogenic. So, introduction of Aib will be interesting
21
among polymorphs of emodin. Wang et al. explained the molecular orientation (to adjust the steric hindrance) and
polymorphism and pseudo-polymorphism directed uores- crystal packing. Interestingly, the 6-nitro-coumarin-3-carboxylic
cence switching of N,N-di(n-butyl)-quinacridone. Hariharan acid methyl ester 1 exhibits red-green birefringence under
et al. have engineered the solid state packing and colour of cross-polarized light, before and aer heating. However, the
nitropeptide 2 exhibits no birefringence. The nitropeptide 3
22
exhibits green-gold birefringence which disappeared on heat-
Department of Chemical Sciences, Indian Institute of Science Education and Research ing. The uorescence microscopy conrmed the change of blue
Kolkata, Mohanpur, West Bengal 741246, India. E-mail: deba_h76@yahoo.com; uorescence of nitropeptide 3 in crystal and in amorphous
deba_h76@iiserkol.ac.in; Fax: +91 3325873020; Tel: +91 3325873119
state. Finally X-ray crystallography sheds some light on the
†
Electronic supplementary information (ESI) available: Synthesis and
molecular orientation, packing and the diverse degree of p–p
stacking orbital overlap of adjacent coumarin chromophores of
the nitropeptide 3.
1
13
characterization of trisamides, H NMR, C NMR, Fig. ESI S1–S8, Fig. S1–S12.
CCDC 1505407, 1505411 and 1505412. For ESI and crystallographic data in CIF
or other electronic format see DOI: 10.1039/c6ra24799g
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Results and discussion
For nitropeptides 2 and 3 (Fig. 1), the design principle explored
was how to use a chromophore containing aromatic amino acid
with helicogenic Aib to develop rigid foldamers and use as

uorescence probe. The introduction of Aib-moiety will
increase crystallinity. Moreover, to adjust the steric hindrance
Aib is suitable for controlling molecular orientation and crystal
packing. Nitropeptide 3 contains two coumarin chromophores
connected by a peptide bond. The target compound 6-nitro-
coumarin-3-carboxylic acid methyl ester 1 (Fig. 1) was synthe-
sized by following a standard coumarin synthesis protocol
(
8
reux salicyldehyde, dimethyl malonate and piperidine at
0 C for 10 h) followed by nitration using mixed acid. Nitro-
ꢀ
peptides 2 and 3 were synthesized by conventional solution-
phase peptide synthesis methodology using DCC as coupling
reagent. The synthesized compounds were puried and char-
Fig. 2 Concentration dependent UV/Vis spectra of (a) peptide 2 and
(b) peptide 3. (c) The plot showing gradual quenching of emission peak
at 400 nm with increasing nitropeptide 2 concentrations (lex ¼ 330
nm). (d) The plot showing gradual increase of emission peak at 450 nm
with increasing nitropeptide 3 concentrations (lex ¼ 330 nm).
1
13
acterized by H-NMR, C-NMR, FT-IR and mass spectrometry
(MS) analysis.
First we have investigated the assembly of compound 1 and
nitropeptides 2 and 3 in solution phase by different spectroscopic
techniques. The solution state UV/vis spectra shows that there is is assigned for the stretching band of amide I and the bending
30
no change of coumarin spectral positions (275 and 335 nm for p peak of amide II. The peptides 2 and 3 exhibit N–H stretching
ꢁ
1
to p* transition) or intensity of compound 1 in chloroform (ESI vibrations at 3351 and 3338 cm and amide I and amide II
ꢁ
1
30
Fig. 1†). Also the nitropeptide 2 shows two peaks at 275 and peaks at 1617, 1538 and 1614, 1533 cm respectively. Peaks at
3
ꢁ1
35 nm responsible for p to p* transition (Fig. 2a) The nitro- 1720 and 1723 cm are responsible for ester functional group
peptide 3 exhibits three peaks at 260, 300 and 335 nm and (Fig. 3a).
intensity of the bands increases with increasing concentration
DSC scans were carried out with a second generation high-
Fig. 2b). The typical emission band of coumarin in chloroform sensitivity METTLER differential scanning calorimeter. For
(
solution at 400 nm also increases (ESI Fig. 2†) with addition of the reproducibility of the results, the experiments were repeated
compound 1. However, the concentration dependence studies thrice with three different samples. Cooling scans yielded
show that the emission band responsible for coumarin unit at curves are different to the heating scans. This is expected due to
4
00 nm has quenched with increasing concentration of nitro- change of packing pattern by thermal stimuli. Therefore, only
peptide 2 (Fig. 2c). But for the nitropeptide 3, the characteristic heating scans are discussed in this work. The rst DSC heating
emission bands at 450 nm increases with increasing concentra- scan of 6-nitro-coumarin-3-carboxylic acid methyl ester 1
ꢀ
tion (Fig. 2d). The results suggest irrespective of coumarin exhibits two peaks at 181.08 and 226.68 C. The rst heating
chromophore, the intermolecular interactions play an important scan DSC experiment (Fig. 3b) clearly shows that the nitro-
ꢀ
part for the luminophore peptides.
peptide 2 has sharp melting temperature at 149.65 C. A closure
Solid state FT-IR spectroscopy is an excellent method to inspection of the rst heating scan reveals a weak endothermic
ꢀ
investigate the structure and assembly pattern of the nitro- transition at 192.58 C (Fig. 3b). There is another weak endo-
ꢁ
1
ꢀ
peptides. The FT-IR region 3500–3200 cm is important for the thermic transition at 218.40 C. This transition is assigned as
ꢁ1
31
N–H stretching vibrations, however the range 1800–1500 cm
melting temperature for nitropeptide 3. However, the rst DSC
cooling scans for nitropeptides 2 and 3 (ESI Fig. 3†) are void of
any crystallization peak which indicates the amorphous nature
31
of melted and cooled samples.
The morphology of the peptides was studied by eld-
emission scanning electron microscopic (FE-SEM) measure-
ments. For FE-SEM experiments, dilute solutions (0.5 mM) of
above reported peptides in chloroform were placed on a micro-
scopic glass slide and then dried under a vacuum for two days.
Fig. 4 depicts the FE-SEM images of the aliphatic–aromatic
backbone hybrid peptides. From Fig. 4a the micrographs show
the ake like morphology for 6-nitro-coumarin-3-carboxylic acid
methyl ester 1 in the self-assembled state. Peptides 2 exhibits
microsphere morphology (Fig. 4b) with a diameter ca. 400 nm.
Peptide 3 exhibits fused microsphere like morphology (Fig. 4,
panel c and d respectively).
Fig. 1 The schematic presentation of 6-nitro-coumarin-3-carboxylic
acid methyl ester 1 and peptides 2 and 3.
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and f). Fig. 5g and j show images of samples of 1 that were cooled
from the melt. The Fig. 5j shows clearly that the 6-nitro-
coumarin-3-carboxylic acid methyl ester from crystal from melt
and the red-green birefringence under polarized light has per-
sisted. The samples of nitropeptide 2 that were cooled from the
melt show amorphous nature and do not show any birefringence
by POM (Fig. 5h and k). The same effect was observed when
nitropeptide 3 was cooled from the melt. This indicates that the
stability of the amorphous state under ambient conditions is
high and the amorphous compound does not exhibit birefrin-
gence under polarized light (Fig. 5i and l).
To explore the relationship between the emission of the
crystals and their amorphous state, uorescence microscopy
32,33
were performed.
methyl ester 1 crystals show blue emission on excitation at
30 nm (Fig. 6a and d). Irrespective of presence of 6-nitro-
The 6-nitro-coumarin-3-carboxylic acid
3
coumarin-3-carboxylic acid chromophore, the crystals of nitro-
peptide 2 do not show any emission on excitation at 330 nm
(Fig. 6b and e). However, the nitropeptide 3 containing two
coumarin chromophores connected by a peptide bond exhibits
blue emission on excitation at 330 nm (Fig. 6c and f). The Fig. 6j
clearly shows that the crystal of 6-nitro-coumarin-3-carboxylic
acid methyl ester 1 obtained from melt shows blue emission.
But the samples of nitropeptide 2 (Fig. 6h and k) and 3 (Fig. 6i
and l) that were cooled from the melt do not show blue emission
on excitation at 330 nm. Sample decomposition is not an issue
and conrmed by NMR, FT-IR and UV/Vis spectroscopy.
This indicates that irrespective of the presence of coumarin
Fig. 3 (a) The solid state FT-IR spectra of nitropeptides 2 (red) and 3
(
blue). (b) The DSC stack plot of (black) 6-nitro-coumarin-3-carboxylic
acid methyl ester 1, (red) nitropeptide 2 and (blue) nitropeptide 3.
Fig. 4 The FE-SEM images (a) flake like morphology of compound 1,
b) microsphere morphology of peptide 2 and (c) and (d) fused
(
microsphere like morphology of peptide 3.
The thermal effect and change of optical properties of the
nitropeptides were studied by polarized optical microscopy Fig. 5 The optical microscopic images of (a) compound 1, (b) nitro-
peptide 2 and (c) nitropeptide 3. (d) Compound 1, (e) nitropeptide 2
(
POM). The 6-nitro-coumarin-3-carboxylic acid methyl ester 1
and (f) nitropeptide 3 under polarized light. The optical microscopic
images of solid obtained by cooling of melt of (g) compound 1, (h)
nitropeptide 2 and (i) nitropeptide 3. The solid obtained by cooling of
melt of (j) compound 1, (k) nitropeptide 2 and (l) nitropeptide 3
crystals exhibit red-green birefringence under polarized light
Fig. 5a and d). The crystals of nitropeptide 2 do not show any
birefringence by POM (Fig. 5b and e). However, the nitropeptide 3
(
exhibits green-gold birefringence under polarized light (Fig. 5c respectively under polarized light.
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Fig. 6 The fluorescence microscopic images of (a) compound 1, (b)
nitropeptide 2 and (c) nitropeptide 3. (d) Compound 1, (e) nitropeptide
Fig. 7 (a) Solid state packing structure of compound 1 showing p–p
stacking interactions between the coumarin units. (b) Packing diagram
of nitropeptide 2 showing parallel arrangement of the molecules. (c)
Crystal packing of nitropeptide 3 showing face to face p–p stacking
interaction.
2
and (f) nitropeptide 3 on excitation at 330 nm. The fluorescence
microscopic images of solid obtained by cooling of melt of (g)
compound 1, (h) nitropeptide 2 and (i) nitropeptide 3. The solid ob-
tained by cooling of melt of (j) compound 1, (k) nitropeptide 2 and (l)
nitropeptide 3 on excitation at 330 nm.
chromophore, the uorescence properties of the nitro peptides
depend on the packing of the molecules in crystal and amor-
phous state.
Fig. 4†). There are two molecules in the asymmetric unit (ESI
34
Fig. 6†). There is an intra molecular hydrogen bond between
coumarin ring C]O and Aib NH that impose an overall planarity
and stability of the molecule. In higher order packing the mole-
cules are in parallel arrangements (Fig. 7b). There is no inter
molecular hydrogen bond or p–p stacking interaction. The
To explore the relationship between the emission of the solid
systems and crystal structure, single crystal X-ray diffraction
analysis were performed. The monoclinic light yellow crystals of 6-
nitro-coumarin-3-carboxylic acid methyl ester 1 were obtained
from methanol solution by slow evaporation (ESI Fig. 4†). There is
˚
centroid to centroid distance is 7.16 A. Though the nitropeptide 2
contains coumarin chromophore, due to non communication
between the coumarin units, the compound does not show uo-
rescence in solid state. The structure of the crystals used for
measuring uorescence is identical with that used for X-ray
crystallographic analysis. The triclinic light yellow crystals of
nitropeptide 3 were obtained from methanol solution by slow
evaporation (ESI Fig. 4†). There is only one molecule in the
34
only one molecule in the asymmetric unit (ESI Fig. 5†). In higher
order packing the molecules are in anti parallel arrangements and
stabilized by multiple face to face, face to edge and edge to edge
p–p stacking interactions (Fig. 7a). We observed a p–p stacking
˚
distance of 3.25 A. The closest C–C distance between two
˚
coumarin units is 3.78 A. But the centroid to centroid distance is
˚
34
5
.08 A. Hence, the week p–p interactions between the coumarin
asymmetric unit (ESI Fig. 7†). There are two intra molecular
units are responsible for the blue emission in solid state. Wang
et al. have reported that the crystal phases with stronger p–p
interactions will exhibit maximum emission at a longer wave-
length region, however the crystal phases with relatively weaker
p–p interactions will show emission at a shorter wavelength
hydrogen bonds between coumarin(2) ring C]O and Aib NH and
coumarin(1) ring C]O and coumarin(1) NH. Moreover the 179
ꢀ
torsion angle between two coumarin units and placement of two
lactone units in opposite direction make the molecule almost
planer. In higher order packing the nitropeptide 3 molecules are
in anti parallel arrangements (Fig. 7c) and stabilized by face to
face p–p stacking interactions. The centroid to centroid distance
22
region. The triclinic yellowish green crystals of nitropeptide 2
were obtained by slow evaporation of methanol solution (ESI
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Table 1 Backbone torsion angles (deg) of Aib residue for peptide 2 and benzotriazole (DCC/HOBt). The products were puried by
peptide 3
column chromatography using silica (100–200 mesh size) gel as
a stationary phase and an n-hexane–ethyl acetate mixture as an
u
1
f
1
j
1
eluent. The intermediates and nal compounds were fully
1
Peptide 2
Molecule A
Molecule B
Peptide 3
characterized by 500 MHz and 400 MHz H NMR spectroscopy,
ꢁ156.83 125 MHz
1
3
ꢁ167.56
172.38
ꢁ179.78
ꢁ178.50
ꢁ51.77
55.02
C NMR spectroscopy, FTIR spectroscopy and
mass spectrometry. The peptides were characterized by X-ray
crystallography.
ꢁ44.39
43.83
(a) Synthesis of coumarin-3-carboxylic acid methyl ester (4).
2
mL (18.6 mmol) of salicyldehyde, 2.18 mL (19 mmol) of
dimethyl malanoate and 200 mL of piperidine were taken in a 50
mL round bottom ux and reuxed at 80 C for 10 h with
continuous stirring. The reaction mixture was cooled to room
temperature. Aer that ethyl acetate and water was added and
shaken vigorously. The ethyl acetate layer was collected and
˚
˚
is 3.68 A and centroid to plane distance is 3.49 A. There is no inter
molecular hydrogen bond. Hence, the p–p interactions between
the coumarin units are responsible for the blue emission of
nitropeptide 3 in solid state. The crystals used for X-ray analysis is
identical with that used for measuring uorescence. However, on
melting and cooling the packing pattern changed, the nitro-
peptide 3 became amorphous in nature and lost its blue uo-
rescence. The backbone torsion angles around Aib residue are
listed in Table 1. The lauric acid appended 6-amino-coumarin-3-
carboxylic acid methyl ester shows no crystallinity and change
of uorescence on heating (ESI Fig. 8†). Hence, Aib is suitable for
making uorescent crystals.
ꢀ
2 4
dried over anhydrous Na SO . The products were puried by
column chromatography using silica (100–200 mesh size) gel as
a stationary phase and an ethyl acetate : n-hexane (1 : 3) as an
eluent. Yield: 3.12 gm (15.20 mmol, 88.25%).
1
H NMR (CDCl 400 MHz, d in ppm): 8.47–8.44 (s, 1H, Ar-H),
3
,
7
.53–7.50 (m, 2H, Ar-H), 7.20–7.24 (m, 2H, Ar-H), 3.81 (s, 3H,
1
3
–
OCH
3
). C NMR (100 MHz, DMSO-d
6
) 162.66, 158.13, 155.08,
1
47.99, 143.59, 128.67, 126.13, 119.23, 118.12, 117.78, 52.73.
+
HR-ESI-MS (m/z): [M + H] calculated for C11
found 205.19.
H
9
O
4
¼ 204.17,
Conclusions
(b) Synthesis of 6-nitro coumarin-3-carboxylic acid methyl
In conclusion, the packing-induced solid-state emission
propensities and thermoresponsive behaviour of peptides con-
taining 6-nitro-coumarin-3-carboxylic acid and a-amino-
isobutyric acid has been reported. The alkyl chains of Aib affect
signicantly the solid state molecular packing modes of peptide
ester (1). 3.00 g (15.78 mmol) of compound 4 was dissolved in
ꢀ
7
.90 mL of conc. H SO and stirred at 0 C for 15 min. Then
2
4
mixture of 3.00 mL (55.46 mmol) nitric acid and 3.65 mL (58.65
mmol) H
2
SO
4
was added dropwise and stirred for 1 h at the
ꢀ
temperature range 0–5 C. Then the reaction mixture was
poured into ice-water and ltered. The residue was washed with
fresh water repetitively and dried. The product was puried by
column chromatography using silica (100–200 mesh) gel and
ethyl acetate : hexane (1 : 2) as an eluent. Yield: 3.65 gm
2
and modulated the extent of p–p interactions between the
coumarin units. Fluorescence spectroscopy and microscopy
conrmed the uorescence switch of peptide 3 in crystal and in
amorphous state. The X-ray crystallography reveals the molec-
ular orientation, packing and the diverse degree of p–p stacking
orbital overlap of adjacent coumarin chromophores of the
peptide 3. The results indicate that apart from the conjugated
backbone of the coumarin chromophore, the adjacent alkyl side
chain of Aib and linking position have major impact on the
solid-state molecular packing as well as the solid-state optical
properties. The information obtained here may be helpful to
tune the uorescence probe for biological and diagnostic
imaging.
(
14.58 mmol, 92.82%).
1
H NMR (CDCl3, 400 MHz, d in ppm): 8.94–8.91 (s, 2H, Ar-H),
8
3
1
.54–8.53 (m, 1H, Ar-H), 7.66–7.64 (m, 1H, Ar-H), 3.86–3.83 (s,
1
3
H, –OCH
3 3
). C NMR (100 MHz, CDCl ) 162.83, 158.10, 148.95,
45.94, 128.62, 126.09, 121.65, 118.19, 117.64, 111.03, 52.31.
+
HR-ESI-MS (m/z): [M + H] calculated for C11
found 250.18.
H
8
NO
6
¼ 249.17,
(c) Synthesis of 6-nitro coumarin-3-carboxylic acid (5). To
5.00 g (20.06 mmol) of compound 1, 80 mL MeOH and 2(M) 32
mL NaOH were added and the progress of saponication was
monitored by thin layer chromatography (TLC). The reaction
mixture was stirred. Aer 10 h, methanol was removed under
vacuum; the residue was dissolved in 150 mL of water and
washed with diethyl ether (2 ꢂ 70 mL). Then the pH of the
aqueous layer was adjusted to 2 using 1 M HCl and it was
extracted with ethyl acetate (3 ꢂ 80 mL). The extracts were
pooled, dried over anhydrous sodium sulfate, and evaporated
under vacuum to obtain compound as a yellowish powder.
Experimental
General
The Aib was purchased from Sigma chemicals. HOBt (1-
hydroxybenzotriazole) and DCC (dicyclohexylcarbodiimide)
were purchased from SRL.
Peptide synthesis
The peptides were synthesized by conventional solution-phase Yield: 4.38 gm (18.63 mmol, 92.87%).
1
methods using racemisation free fragment condensation
6
H NMR (DMSO-d , 400 MHz, d in ppm): 13.90–12.54 (br, 1H,
strategy. The C-terminus was protected as a methyl ester. –COOH), 8.93–8.78 (m, 2H, Ar-H), 8.50–8.37 (m, 1H, Ar-H), 7.66–
1
3
6
Coupling was mediated by dicyclohexylcarbodiimide/1-hydroxyl 7.51 (m, 1H, Ar-H). C NMR (DMSO-d , 125 MHz, d in ppm):
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1
1
1
64.71, 158.20, 147.60, 143.82, 128.94, 126.53, 124.96, 120.68,
18.35, 117.56.
H NMR (CDCl
3
, 400 MHz, d in ppm): 9.38–9.34 (s, 1H, –NH),
9.07–9.01 (s, 1H, Ar-H), 8.89–8.86 (s, 1H, Ar-H), 8.73–8.70 (m,
(
d) Synthesis of 6-nitro coumarin-Aib-OMe (2). 2.38 g (10.12 1H, Ar-H), 8.68–8.59 (m, 1H, Ar-H), 8.27–8.23 (s, 1H, –NH), 8.19–
mmol) of O N-Cum-OH was dissolved in 20 mL DCM in an ice- 8.10 (m, 1H, Ar-H), 7.95–7.84 (m, 1H, Ar-H), 7.64–7.62 (m, 1H,
2
water bath. H-Aib-OMe was isolated from 3.06 g (20 mmol) of Ar-H), 7.48–7.43 (m, 1H, Ar-H), 3.81–3.72 (s, 3H, –OCH
3
), 3.69–
1
3
the corresponding methyl ester hydrochloride by neutralization 3.58 (s, 6H, Aib-H). C NMR (DMSO-d
6
, 125 MHz, d in ppm):
and subsequent extraction with ethyl acetate and the ethyl 170.12, 165.51, 161.10, 161.00, 155.79, 147.01, 145.26, 141.25,
acetate extract was concentrated to 10–12 mL. It was then added 137.24, 136.21, 132.64, 131.49, 131.00, 126.27, 121.01, 120.00,
to the reaction mixture, followed immediately by 2.09 g (10.15 118.13, 116.16, 56.41, 53.52, 26.14.
mmol) dicyclohexylcarbodiimide (DCC) and 1.53 g (10.00 mmol)
of HOBt. The reaction mixture was allowed to come to room
temperature and stirred for 48 h. DCM was evaporated and the
residue was dissolved in ethyl acetate (60 mL) and dicyclohex-
ylurea (DCU) was ltered off. The organic layer was washed with
NMR experiments
All NMR studies were carried out on a Br u¨ ker AVANCE 500 MHz
and Jeol 400 MHz spectrometer at 278 K. Compound concen-
trations were in the range 1–10 mM in CDCl and (CD ) SO.
3
3 2
2
(M) HCl (3 ꢂ 50 mL), brine (2 ꢂ 50 mL), 1 M sodium carbonate
3 ꢂ 50 mL) and brine (2 ꢂ 50 mL) and dried over anhydrous
sodium sulfate. It was evaporated in a vacuum to yield O N-Cum-
Aib-OMe as a white solid. Yield 2.86 g (8.56 mmol, 85.62%).
(
FTIR spectroscopy
All reported solid-state FTIR spectra were obtained with a Per-
2
1
H NMR (CDCl , 400 MHz, d in ppm): 9.08–9.04 (s, 1H, Ar-H), kin Elmer Spectrum RX1 spectrophotometer with the KBr disk
3
8
1
1
.98–8.9 (s, 1H, Ar-H), 8.57–8.46 (m, 1H, Ar-H), 7.62–7.53 (m, technique.
H, Ar-H), 7.49–7.46 (s, 1H, –NH), 3.79–3.71 (s, 3H, –OMe), 1.69–
.60 (s, 6H, Aib-H).
Mass spectrometry
(
e) Synthesis of 6-amino coumarin-Aib-OMe (6). 2.86 gm
Mass spectra were recorded on a Q-Tof Micro YA263 high-
resolution (Waters Corporation) mass spectrometer by positive-
mode electrospray ionization.
(
8.56 mmol) of compound 2 was taken in a 250 mL round
bottomed ask. The compounds were poured into 150 mL
distilled water. Then 1.43 gm (25.68 mmol) of iron powder and
1
.83 gm of NH Cl (34.24 mmol) were added into the solution.
4
Field emission scanning electron microscopy
Then the mixture was manually stirred for 1.5 h at heating
condition. Aer the color change of the solution from yellowish
to red, the heating and stirring were stopped. Then the reaction
mixer was allowed to come down to room temperature and the
amine was precipitated out. Aer that the solutions were
Morphologies of the reported peptides were investigated using

eld emission-scanning electron microscopy (FE-SEM). A small
amount of solution of the peptide was placed on a clean silicon
wafer and then dried by slow evaporation. The material was

ltered by gauche funnel using cilite and the residue was dis-
ꢀ
then allowed to dry under vacuum at 30 C for two days. The
solved in mild hot acetone. The solution was dried by rotary
evaporator to obtain reddish yellow solid as a pure product.
materials were gold-coated, and the micrographs were taken in
an FE-SEM apparatus (Jeol Scanning Microscope-JSM-6700F).
Yield: 2.41 gm (7.92 mmol, 92.52%).
1
3
H NMR (CDCl , 400 MHz, d in ppm): 9.32–9.28 (s, 1H, Ar-H),
Single crystal X-ray diffraction study
8
1
3
.76–8.72 (s, 1H, Ar-H), 7.24–7.20 (m, 1H, Ar-H), 7.05–6.97 (m,
Intensity data of peptides were collected with MoKa radiation
using Bruker APEX-2 CCD diffractometer. Data were processed
using the Bruker SAINT package and the structure solution and
renement procedures were performed using SHELX97. CCDC
H, Ar-H), 6.89–6.82 (s, 1H, –NH), 5.33–5.28 (s, 2H, –NH
.71 (s, 3H, –OMe), 1.69–1.51 (s, 6H, Aib-H).
2
), 3.79–
(
f) Synthesis of 6-nitro coumarin-coumarin-Aib-OMe (3).
1
.00 g (4.25 mmol) of O N-Cum-OH was dissolved in 20 mL
2
1505407, 1505411 and 1505412 contains the crystallographic
DCM and 1 mL DMF in an ice-water bath. H-Cum-Aib-OMe was
isolated from 1.82 g (6.00 mmol) of the corresponding methyl
ester was added to the reaction mixture, immediately followed
by 877 mg (4.25 mmol) dicyclohexylcarbodiimide (DCC) and
data for the compound 1, peptide 2 and peptide 3 respectively.
Acknowledgements
612 mg (4.00 mmol) of HOBt. The reaction mixture was allowed
We thank the IISER-Kolkata, India, for nancial assistance. A.
Pramanik acknowledges the C.S.I.R, India for research
fellowship.
to come to room temperature and stirred for 48 h. DCM was
evaporated and the residue was dissolved in ethyl acetate (60
mL) and dicyclohexylurea (DCU) was ltered off. The organic
layer was washed with 2(M) HCl (3 ꢂ 50 mL), brine (2 ꢂ 50 mL),
1
M sodium carbonate (3 ꢂ 50 mL) and brine (2 ꢂ 50 mL) and
Notes and references
dried over anhydrous sodium sulfate. It was evaporated in
a vacuum to yield O N-Cum-Aib-OMe as a white solid. Puri-
2
1 (a) X. Wang, O. S. Woleis and R. J. Meier, Chem. Soc. Rev.,
2013, 42, 7834–7869; (b) M. D. Allendorf, C. A. Bauer,
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cation was done by silica gel column (100–200 mesh size) with
an ethyl acetate and hexane mixture 1 : 4 as the eluent. Yield:
868 mg (1.66 mmol, 39.05%).
394 | RSC Adv., 2017, 7, 389–395
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instrument. The structures were solved by a direct method
2
and rened by least-squares calculations on F for all
35
independent reections (SHELXL-2013). Crystal data of
compound 1: C H N O , M ¼ 249.18, I2/a, a ¼ 20.178(2)
1
1
7
1
6
w
˚
˚
˚
ꢀ
ꢀ
A, b ¼ 4.0966(4) A, c ¼ 25.538(2) A, a ¼ 90 , b ¼ 99.66(9) ,
ꢀ
˚
3
ꢁ3
g ¼ 90 , V ¼ 2081.1(3) A , Z ¼ 8, dm ¼ 1.591 Mg m , K ¼
293, R
2s(I). Crystal data of nitropeptide 2: C15
1
¼ 0.0641 and wR ¼ 0.1721 for 2014 data with I >
2
H
14
N
2
O
7
, M
˚
w
¼
ꢀ
˚
334.28, P1, a ¼ 7.8470(3) A, b ¼ 14.2490(8) A, c ¼
3
˚
˚
1
1
2
2
2
15.0925(8) A, V ¼ 1488.03(15) A , Z ¼ 4, dm ¼ 1.492 Mg
ꢁ3
m
, K ¼ 153, R
1
¼ 0.0373 and wR ¼ 0.0967 for 5228 data
2
with I > 2s(I). Crystal data of nitropeptide 3: C H N O ,
2
5
19 3 10
ꢀ
˚
˚
Mw ¼ 521.43, P1, a ¼ 7.8580(8) A, b ¼ 11.5608(12) A, c ¼
3
˚
˚
13.8689(11) A, V ¼ 1188.1(2) A , Z ¼ 2, dm ¼ 1.458 Mg
ꢁ3
m
, K ¼ 293, R
1
¼ 0.0726 and wR ¼ 0.1887 for 4050 data
2
1 M. Li, Q. Zhang, J.-R. Wanga and X. Mei, Chem. Commun.,
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contains the crystallographic data for the crystals
respectively.
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This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 389–395 | 395