1
Bicomponent β-sheet assembly of dipeptide-fluorophores of opposite polarity and sensitive detection of nitro-explosives
Chilakapati Madhu,a# Bappaditya Roy,a# Pandeeswar Makam,a# Thimmaiah Govindarajua*
aBioorganic Chemistry Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O., Bengaluru 560064, Karnataka, India.
*E-mail: [email protected]
Contents
Page No.
Experimental section
1. Materials and methods
2. Synthesis and characterization
3. Experimental results
(i) Concentration dependent absorbance spectra of 1:1 complexes
and Job’s plot.
(ii) pH dependent FL study
(iii) TEM image
(iii) Rheology study
(iv) Absorbance data of dipeptide amphiphiles, and their complexes in
presence and absence of TNB
(v) FTIR study
(vi) Lifetime study
(v) NACs detection
(vii) Limit of detection experiment
(viii) Film-based detection
2-14
2-3
3-9
10-14
10
10
10
11
11
12
12
13
13
14
Reference 14
Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2018
2
Experimental section 1. Materials and methods
Materials. All reagents and solvents were purchased from Sigma Aldrich and used without
any further purification unless mentioned. The solvents were brought as HPLC grade from
Spectrochem, India. Dulbecco’s phosphate-buffered saline (PBS) was purchased from Sigma-
Aldrich. All the experiments were performed in 10 mM PBS buffer of pH 7.4.
Absorbance (UV-vis) spectroscopy. UV-vis spectra were recorded on Perkin Elmer Model
Lambda 900 spectrophotometer. The spectra were recorded in quartz cuvette of 10 mm path
length.
Fluorescence (FL) spectroscopy. Fluorescence spectra were recorded on a PerkinElmer
model LS 55 spectrophotometer. All fluorescence emission spectra were recorded with
excitation wavelength λex = 350 nm, and analysed in quartz cuvette of 10 mm path length.
Circular dichroism (CD) spectroscopy. The CD measurements were carried out on a Jasco J-
815 spectrometer under nitrogen atmosphere. The CD was recorded in quartz cuvette of 1
mm path length with the speed of 100 nm/min and the spectra represent an average of three
scans.
NMR spectroscopy. 1H and 13C NMR spectra were recorded on a Bruker AV-400
spectrometer with chemical shifts reported as ppm (in CDCl3/DMSO-d6 with
tetramethylsilane as internal standard).
Image and microscopy. The digital images of the inverted gel vial were taken in Canon
SX50HX digital camera. Fluorescent images were captured using DMi8 Leica Inverted
fluorescence microscope). The 1:1 complex (of TGM-82 and TGM-83) at 0.25 mM total
concentration was taken in a microscope slide (glass) and covered with a cover slip. FESEM
images were acquired using FEI Nova nanoSEM-600 equipped with a field emission gun
operating at 15 kV. The samples were prepared by drop-casting solutions on to the silicon
(111) surface and dried in air.
Time-correlated single photon counting (TCSPC). Fluorescence decay profiles were
performed using FLSP 920 spectrometer, Edinburgh Instrument.
Fourier-transform infrared spectroscopy (FTIR). The FTIR spectra were recorded on a
Bruker IFS 66/V spectrometer. The measurements were done for the vacuum-dried solid
powders of the derivatives and xerogel at room temperature.
Rheology. The mechanical strength of the hydrogel was measured on MCR 302 rheometer
using P-PTD 200/Air measuring and a plate geometry of 1 mm gap.
3
Hydrogel preparation. The dipeptide derivatives (TGM-82 and TGM-83) were taken in
calculated amounts and prepared clear stock solutions of 4.0 mM concentration. For hydrogel
preparation equivolume solutions of each component were mixed in 10.0 mM PBS buffer
(pH 7.4) and sonicated to get clear solution at 25 ºC. The final total concentration was
maintained at 2.0 mM. After 5 min it formed hydrogel, as confirmed by ‘vial inversion test’
at 25 ºC.
Film-based NACs detection. The peptide-coated thin film was developed on quartz plate
(almost 5 mm diameter and ~1 µm thickness) following drop-casting method. The
fluorescence emission spectra were recorded using front face geometry. For detection, the
analytes were dissolved in acetonitrile solvent and drop-casted 5 µL on the films, and the
emission spectra were recorded.
2. Synthesis and characterisation
All the air and moisture sensitive reactions were carried out under argon or nitrogen
atmosphere. The syntheses of the two dipeptide derivatives (TGM-82 and TGM-83) is
presented in Scheme 1 and 2. All the compounds were thoroughly characterised by NMR and
HRMS studies.
Scheme S1 Synthesis of TGM-82
BocHNHN
OHO
OLiOH
H2O:THF
NH
OHN
ONHBoc
DIPEAHBTUHOBT
Py.Methylaminert, DMF
NH
OHN
ONH2
95% TFA
TGM-79 b
BocHNHN
OMeO
O
BocHNO
OH DIPEA
EDC.HClHOBtACN TGM-80
TGM-82
TGM-79Boc-Ala-OH
H-Ala-OMe
4
Scheme S2 Synthesis of TGM-83
General experimental procedure for dipeptide derivatives TGM-82 and TGM-83
Synthesis of TGM-79
To the solution of Boc-Ala-OH (0.95 g, 5.0 mmol) in DCM, DIPEA (1.92 mL, 11.0 mmol),
EDC. HCl (0.96 g, 5.0 mmol) and HOBt (0.67 g, 5.0 mmol) were added at 0 ºC under
nitrogen atmosphere. The solution was stirred for 10 min. and a mixture of H-Ala-OMe (0.52
g, 5.0 mmol) and DIPEA (870 µL, 5.0 mmol) was added. After completion of the reaction
(TLC analysis), the reaction mixture was washed with 10% Na2CO3 (3´ 50 mL), 5% citric
acid (3´ 50 mL), water (2´ 50 mL) and brine solution (1´ 5 mL). The organic layer was
separated, evaporated and the residue was purified through column chromatography to obtain
the dipeptide (TGM-79) in 85% yield.
Synthesis of TGM-79a
TGM-79 (1.0 g, 3.65 mmol) was subjected to Boc-deprotection in a round bottom flask by
treating with a deprotection-cocktail of TFA, DCM and TIPS (95:3:2), and the reaction
mixture was stirred for 3 h. TFA was evaporated under vacuo and triturated with diethylehter
to obtain TGM-79a in 87% yield.
Synthesis of TGM-79b
TGM-79 (0.27 g, 1.0 mmol) and LiOH (0.05 g, 1.2 mmol) were taken in a round bottom flask
containing THF:H2O (4:6) solvent mixture and stirred at room temperature for 3 h. After
completion, THF was evaporated and 10% Na2CO3 was added. The reaction mixture was
then washed with diethylether (2´ 50 mL) and neutralised with citric acid to obtained pure
5
precipitate of TGM-79b. The precipitate was filtered and dried to get solid product in 90%
yield.
Synthesis of TGM-80
To a solution of TGM-79b (1.30 g, 5.0 mmol) in DMF, DIPEA (1.92 mL, 11.0 mmol),
HBTU (5.0 mmol) and HOBt (0.67 gm, 5.0 mmol) were added under stirring and at nitrogen
atmosphere at 0 ºC. Pyrenemethylamine hydrochloride (1.34 g, 5.0 mmol) was added to the
above reaction mixture and continued for 3 h. The reaction mixture was diluted with water
and the precipitate was collected by washing with 10% Na2CO3 solution, citric acid, and
water. The precipitate was dried and purified in silica gel column chromatography using
CHCl3:CH3OH (97:3) as eluent to obtain TGM-80 in 80% yield.
Synthesis of TGM-81
Pyrene acetic acid (2.0 g, 7.68 mmol) was dissolved in DMF and DIPEA (1.34 mL, 7.68
mmol), HBTU (2.91 g, 7.68 mmol) and HOBt (1.04 g, 7.68 mmol) were added at 0 ºC under
nitrogen atmosphere. A neutralised solution of TGM-79a (1.34 g, 7.68 mmol) with DIPEA
(1.34 mL, 7.68 mmol) was added to the reaction mixture under stirring condition. After
completion (TLC analysis), the reaction mixture was diluted with water to precipitate the
product. The precipitate was washed with 10% Na2CO3 solution, citric acid, and water. The
dried precipice was purified by column chromatography using CHCl3:CH3OH (97:3) as
eluent to obtain TGM-81 in 78% yield.
Synthesis of TGM-82
TGM-80 (0.25 g, 0.53 mmol) subjected to BOC-deprotection in a round bottom flask using 2
mL deprotection-cocktail of TFA, DCM and TIPS (95:3:2). After 3 h of stirring, 50 mL
diethylether was added to the reaction mixture and the precipitated product was collected by
filtration. 1H NMR (400 MHz, DMSO-d6): δ 8.73-8.79 (m, 1H), 8.66 (dd, J = 16.3, 7.5 Hz,
1H), 8.23-8.39 (m, 5H), 8.18 (s, 2H), 8.03-8.12 (m, 4H), 5.05 (q, J = 5.4 Hz, 2H), 4.40-4.47
(m, 1H), 3.85-3.92 (m, 1H), 1.42-1.23 (6H); 13C NMR (100 MHz, DMSO- d6): δ 172.2,
169.8, 158.4, 158.2, 133.1, 131.1, 130.7, 130.6, 128.5, 128.0, 127.8, 127.5, 126.8, 126.7,
125.7, 125.6, 125.2, 124.5, 124.4, 123.6, 48.9, 48.5, 18.8, 17.6; ESI-MS m/z: [M+H]+ calcd.
for C23H23N3O2, 374.1863; found 374.1858.
Synthesis of TGM-83
TGM-81 (0.39 g, 1.0 mmol) was taken in a round bottom flask, LiOH (50 mg, 1.2 mmol) was
added in THF:H2O (4:6) solvent mixture and stirred at room temperature for 3 h. After
6
completion of the reaction, THF was evaporated and 10% Na2CO3 was added. The reaction
mixture was washed with diethylether (2´ 50 mL) and neutralised with 5% HCl solution to
precipitate the product. The precipitate was filtered and dried to obtain TGM-83 in 75%
yield. 1H NMR (400 MHz, DMSO-d6): δ 12.56 (b) 8.39-8.46 (m, 2H), 8.16-8.30 (m, 7H),
8.02-8.09 (m, 2H), 4.34-4.41 (q, J = 7.1 Hz, 1H), 4.16-4.30 (m, 3H), 1.16-1.27 (m, 6H); 13C
NMR (100 MHz, DMSO-d6): δ 174.6, 172.4, 170.2, 131.5, 131.3, 130.8, 130.1, 129.5, 129.1,
127.8, 127.6, 127.3, 126.6, 125.5, 125.3, 125.2, 124.6, 124.4, 48.4, 47.9, 18.9, 17.6; ESI-MS
m/z: [M] calcd. for C24H22N2O4, 402.1580; found 403.1651.
7
1H and 13C NMR spectra of TGM-82
(Mill
ions
)0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
X : parts per Million : 1H
10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0
8.77
78.
754
8.74
08.
726
8.69
18.
673
8.65
18.
632
8.35
18.
323
8.30
78.
284
8.26
58.
258
8.18
08.
098
8.04
58.
026
5.06
95.
055
5.04
25.
029
4.45
74.
440
4.42
24.
404
3.92
13.
904
3.88
73.
869
3.85
1
1.37
01.
355
1.33
81.
314
1.29
61.
238
6.02
4.96
4.34
1.98
1.95
1.01
1.00
0.98
0.97
(Tho
usan
ds)
01.
02.
03.
04.
0
X : parts per Million : 13C
210.0 200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0
172.
186
169.
773
158.
465
158.
159
133.
066
130.
755
130.
602
128.
524
127.
999
127.
824
127.
532
126.
884
126.
745
125.
746
125.
651
125.
156
124.
368
123.
574
48.9
7248
.542
18.8
2617
.682
NH
O HN
ONH2
8
1H and 13C NMR spectra of TGM-83
(Tho
usan
ds)
010
0.0
200.
030
0.0
400.
050
0.0
600.
070
0.0
X : parts per Million : 1H
13.0 12.0 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0
12.5
60
8.41
78.
395
8.30
38.
298
8.28
48.
254
8.23
58.
225
8.20
18.
172
8.15
68.
092
8.07
38.
041
8.02
1
4.40
74.
390
4.37
14.
353
4.33
64.
302
4.26
54.
255
4.23
24.
214
4.19
54.
177
4.15
9
1.27
11.
250
1.23
11.
199
1.18
11.
164
7.19
6.26
2.82
2.13
1.99
1.00
0.67
(Tho
usan
ds)
01.
02.
03.
04.
0
X : parts per Million : 13C
200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0
174.
606
172.
375
170.
195
131.
476
131.
280
130.
828
130.
150
129.
457
129.
107
127.
846
127.
620
127.
263
126.
614
125.
506
125.
345
125.
178
124.
587
124.
383
48.3
5947
.864
18.9
1417
.645
HN
NHO
O
O
OH
9
HR-MS (ESI) spectra of (a) TGM-82 and (b) TGM-83
(a)
(b)
10
3. Experimental results
Fig. S1 (a) Concentration dependent absorbance spectra of 1:1 complex (of TGM-82 and TGM-83) at pH 7.4 and and at 25 ºC; (b) Job’s plot was derived from the intensity of excimer formation at 0.25 mM total concentration at pH 7.4 and at 25 ºC.
Fig. S2 The pH dependent FL study was carried out with the 1:1 complex (of TGM-82 and TGM-83) at 0.25 mM total concentration and at 25 ºC.
Fig. S3 TEM images of the 1D nanofibers of 1:1 complex (of TGM-82 and TGM-83) prepared at 0.25 mM total concentration, at 25 ºC.
300 400 5000
1
2
3
e, 10
4 M
-1cm
-1
Wavelength (nm)
100 µM 250 µM 500 µM 750 µM 1000 µM
0.0 0.2 0.4 0.6 0.8 1.00.2
0.4
0.6
0.8
1.0
I@48
5 nm
Mole fraction of 82
0 2 4 6 8 10 120
1
2
3
I ex
/ IM
pH
(a) (b)
11
Fig. S4 Rheology experiment for oscillation stress and %strain determination of the bicomponent hydrogel at 2.0 mM total concentration at 25 ºC.
Fig. S5 The absorbance study was carried out with the 1:1 complex (of TGM-82 and TGM-83) at 0.25 mM total concentration and in the presence of 100 µM TNT at 25 ºC.
0.1 1 1010-1
100
101
102
Mod
ulus
(Pa)
Oscillator stress (Pa)
G' G''
0.1 1 10 10010-1
100
101
102
Mod
ulus
(Pa)
% Strain
G' G''
300 400 5000.0
0.6
1.2
Abs
. (a.
u.)
Wavelength (nm)
TGM-82
TGM-83
1:1 complex
300 400 5000.0
0.6
1.2
Wavelength (nm)
Abs
. (a.
u.)
1:1 complex 1:1 complex + TNT
12
Fig. S6 FTIR spectra of the individual components TGM-82 and TGM-83, and their hydrogel in the dried state (xerogel).
Fig. S7 Time-correlated Single Photon Counting (TCSPC) study of the hydrogel in presence and absence of TNT.
3500 3000 1800 1750 1700 1650 1600 1550 1500 1450 1400
1675
% T
Wavenumber (cm-1)
TGM-82 TGM-83 1:1 complex 1634
3278
0 40 80 1200.01
0.1
1
Coun
ts
Time (ns)
Only hydrogel, t = 6.82 with TNT, t = 5.94
13
Fig. S8 The change in fluorescence intensity of 1:1 complex (250 µM) in the presence of different NACs (100 µM).
Fig. S9 Detections of TNB (13.4 µM) and TNT (17.8 µM) in the solution of the 1:1 complex (of TGM-82 and TGM-83) at 0.25 mM and 25 ºC (see the cali. Determination of the detection limit1 The calibration curve was obtained from the plot of fluorescence intensity ratio of complex (H) in complex-quencher (HQ) system in absence and presence of TNB/TNT. The regression curve equation was then obtained as- The limit of detection (LOD) = 3 × S.D. / k Where S.D. represents the standard deviation of H system in the absence of Q and ‘k’ is the slope of the curve.
450 500 550 6000
250
500
750
1000
Inte
nsity
(a.u
.)
Wavelength (nm)
Control NM Benzene Toluene Aniline DCB Phenol BA NP NB DNT Cl-DNB TNT TNB
0 20 40 60 80 1002
4
6
8
10
12
14
Conc. of TNB (µM)
I o /
I
0 20 40 60 80 1002
4
6
8
10
I o /
I
Conc. of TNT (µM)
14
Fig. S10 Film-based detections of TNT and TNB at 25 ºC: (a-b) FL intensity change with the addition of NACs (TNT and TNB); (c-d) the relative intensity changes with the NACs (TNT and TNB) concentrations (the marked points signify >5 fold relative intensity change). Reference
1. B. Ma, F. Zeng, F. Zheng, and S. Wu, Chem. Eur. J., 2011, 17, 14844.
450 500 550 600 6500
In
tens
ity
Wavelength (nm)
Controlled 500 aM 1 fM 100 fM 500 fM 1 pM 10 pM 100 pM 500 pM 10 nm 100 nm 500 nm 1 µM 10 µM 100 µM 500 µM
1.0TNT
400 450 500 550 6000
Inte
nsity
Wavelength (nm)
Controlled 250 pM 500 pM 1 nM 2 nM 3 nM 4 nM 5 nM 10 nM 25 nM 50 nM 75 nM 100 nM 150 nM 250 nM
1.0TNB
0 100 200 300 400 500
0.0
0.2
0.4
0.6
0.8
(I 0-I)
/ I 0
TNT concentration
100 nM
TNT
0 10 20 30 40 50
0.0
0.2
0.4
0.6
0.8
(I 0-I)
/ I 0
TNB conc. (nM)
5 nM
TNB
(a) (b)
(c) (d)