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: tgraju@jncasr.ac.in

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)