1

Supporting information

A novel fluorescence probe for estimation of Cysteine/Histidine in

human blood plasma and recognition of endogenous Cysteine in

live Hct116 cells

Upendar Reddy G.,a Hridesh Agarwalla,

a Nandaraj Taye,

b Suvankar Ghorai,

b

Samit Chattopadhyay,b* Amitava Das

a*

aCSIR-National Chemical Laboratory, Organic Chemistry Division, Pune-

411008, India; E-mail: a.das@ncl.res.in; Tel: +91(0)2025902385; Fax:

+91(0)2025902629; bChromatin and Disease Biology Lab; National Centre for Cell Science; Pune

411007, India, Email: samit@nccs.res.in.

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2014

2

Table of contents: Page

Materials and method S3

Synthetic scheme S4

Synthesis and characterisation of L S4

Synthesis and characterisation of R S5

1H NMR spectra of L S6

13C NMR spectra of L S7

Mass spectra of L S8

Mass spectra of R S9

Mass spectra of R with His S10

Mass spectra of R with Cys S11

Absorbance spectra of L, R and R in presence and absence of Cys and His S12

Benesi-Hildebrand plot for binding studies of Cu2+

towards L S11

Benesi-Hildebrand plot for binding studies of [His] towards R S13

Change of Emission intensity of R at 500 nm as a function of the solution pH: S13

Spectrophotometric interference study of R with Cys in presence of various Amino

Acids

S14

Spectrophotometric interference study of R with Cys in presence of various Amino

Acids

S14

Detection of His in Human blood plasma S14

Pre-treatment of the healthy human blood plasma for estimation of CCys & CHis S15

Evaluation of CCys and CHis, in human blood plasma sample S16

Cell culture and fluorescence imaging S17

MTT assay for evolution of cytotoxicity of the reagent R towards Hct116 cells S18

Lower detection of the probe R for Cys S19

References S20

3

Materials.

Dansyl chloride, Hydrazine Monohydrate (98%), Phthalic anhydride, 2-

Pyridinecarboxaldehyde, Sodium triacetoxyborohydride, Copper (II)perchlorate hexahydrate,

were obtained from Sigma Aldrich and were used as received. Cysteine, Histidine,

Glutathione, Arginine, iso- Leucine, Proline, Methionine, Glycine, Alanine, Serine,

Threonine, Tryptophan, Tyrosine, Valine, Leucine were purchased from SD Fine Chemicals

in India. Solvents used for synthesis of various intermediates and final compounds were of

AR grade (S.D. Fine Chemicals) and were used as received without further purification.

HPLC grade (S.D. Fine Chemicals) solvents were used for various spectroscopic studies.

Analytical Methods:

1H NMR spectra was recorded on a Bruker 500 MHz FT NMR (Model: Avance-DPX 500)

using CDCl3 as the solvent and tetra methyl silane (TMS) as an internal standard. ESI-Ms

measurements were carried out on a Waters QT of-Micro instrument. Uv spectra’s was

recorded with a Shimadzu UV-3101 PC spectrophotometer; while fluorescence spectra were

recorded using an Edinburgh instrument Xe 900 spectrofluorometer.

General experimental procedure for UV-Vis and Fluorescence studies:

1.0 x 10-4

M, solution of the L in aq. HEPES buffer: acetonitrile (96: 4 (v/v)) and R in pure

aq.-HEPES buffer medium was prepared and stored in dark. This solution was used for all

spectroscopic studies after appropriate dilution. 10 mM and pH 7.4 solution of aq. HEPES

buffer was used for all spectroscopic studies unless mentioned otherwise. 1.0 x 10-2

M of

amino acid solutions were prepared in 10 mM HEPES buffer (pH 7.4). Solution of the

compound R was further diluted for spectroscopic titrations, and the effective final

concentration of the solution of compound R used for the fluorescence study was 2.0 x 10-5

4

M, while the final analyte concentration during emission spectral scanning was 4.0 x 10-3

M.

For all luminescence measurements, Ext = 350 nm with an emission slit width of 3 nm. The

relative fluorescence quantum yields (f) were estimated using equation 1 in acetonitrile

medium by using the integrated emission intensity of dansyl amide (f = 0.37) for L and R as

a reference.1

Synthesis:

Scheme 1: Methodologies that were adopted for synthesis of 1, L and R.

Synthesis of L: Synthetic procedure that was adopted for synthesis of 1 from our previous

literature.1

Synthesis of L2: Compound 1 (210 mg, 0.625 mmol) and 2-Pyridinecarboxaldehyde (401

mg, 3.75mmol) were dissolved in 7 ml of 1, 2 dichloromethane and refluxed it for 1 h. To

this reaction mixture, Sodium triacetoxyborohydride (791 mg, 3.75 mmol) in 1,2

dichloromethane (10mL) was added. The reaction mixture was allowed to stirring at room

temperature for 48 h. Progress of the reaction was monitored by checking the TLC and

stopped when no further change was observed. The reaction mixture was treated with

saturated aqueous sodium bi carbonate solution and subsequent extraction using chloroform

was performed. The organic layer was recovered, dried over anhydrous Na2SO4 and

concentrated under reduced pressure. The crude product was subjected to neutral alumina gel

5

chromatography using chloroform: methanol (99.5: 0.5, v/v) as eluent. Major fraction was

collected and dried under vacuum, which afforded a sticky oil solid.

Yield: 270 mg, 61.78 %. ESI- Ms (m/z) calculated for C40H44N8O2S: 700, observed: 701 [M

+ H+].

1H NMR [500 MHz, CDCl3: δ (ppm)]: 8.46 (5H, d, J = 4.0 Hz, ArH); 8.18(1H, d, J =

8.5 Hz, ArH); 8.10 (1H, d, J = 6.5 Hz, ArH); 7.59 (4H, t, J = 8.0 Hz, ArH); 7.47-7.44 (1H, m

, ArH);7.39 (5H, t, J = 12 Hz, ArH); 7.15-7.10 (5H, ArH); 3.67 (8H, s, CH2); 3.39 (4H, t, J =

7.0 Hz, CH2); 2.77 (6H, s, CH3); 2.54 (4H, t, J = 7.0 Hz, CH2). 13

C NMR (500 MHz, CDCl3,

δ (ppm)): 173.60, 164.71, 161.18, 145.80, 138, 137, 135.17, 129, 127.74, 124.24, 119, 50.24,

40.17, 32 and 31.60.

Synthesis of R3: The compound L (120 mg, 0.17 mmol) was dissolved in 7.5 mL methanol,

to this Cu (ClO4)2.6H2O (126 mg, 0.342 mml) was added. Solution colour was changed

immediately. The reaction mixture was stirred for 8 h and then transferred into a beaker and

allowed to evaporation at room temperature to precipitate the desired compound. Light bluish

white solid compound was isolated through filtration and was further carefully washed with

cold Dichloromethane. Yield 120 mg, 42.25%. ESI-Ms (m/z) calculated for C40H44Cu2N8

.2H2O: 862, observed: 863 [M+H+].

6

1H NMR spectra of L

SI Figure 1: 1H NMR spectra of L in CDCl3 medium.

7

13C NMR Spectra of L

SI Figure 2: 13

C NMR spectra of L in CDCl3 medium.

8

Mass spectra of L

SI Figure 3: ESI- Ms spectrum of L in CH3OH.

m/z50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

%

0

100

GUR B311 1 (0.019) 1: TOF MS ES+ 852701.5803

547.4297

502.4030

260.2052

226.2170

212.1951

171.159592.0977

405.3275360.2958

261.2096

348.2660305.2676361.2970

431.2591

443.2944

503.4040

504.4038

548.4365

585.4036

638.5011

587.4025 639.5033

702.5859

703.5826739.5545

740.5488

741.5510 799.5076

742.5302 882.5844

11

L+H+

L + K +

9

m/z50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

%

0

100

GU-B331-2N 5 (0.093) 1: TOF MS ES+ 4.29e3863.2952

382.1575

335.6297

218.0883

212.1358134.1062102.1463

264.6216

316.2366

266.6184

383.1585

384.1579

451.1017

450.1020

449.1024

385.1636

538.1714452.1020

483.0951

484.1063

485.1031

540.1671686.2607

623.2405

562.3132625.2383

659.2242

688.2579690.2594 774.2430 801.3066

865.2966

867.3036

868.3098

869.3044

983.2083

979.2218870.3077

943.2673

R+ H+

Mass spectra of R

SI Figure 4: ESI- Ms spectrum of R in H2O.

10

Mass spectra of R with His

.

SI Figure 5: ESI- Ms spectrum of R with His in H2O.

m/z960 980 1000 1020 1040 1060 1080 1100 1120 1140 1160 1180 1200 1220 1240 1260 1280 1300

%

0

100

GUR-B3-33H 12 (0.223) TOF MS ES+ 34.41081.75

1079.75

1078.73

1041.86980.69962.79

962.59 964.79

1017.72983.76 1016.70

984.74997.60

1039.75

1020.57

1066.861042.84

1082.74

1180.84

1180.70

1083.86

1116.62

1114.73

1084.75

1085.78

1086.61

1100.76

1118.751178.68

1119.80

1120.67

1122.67

1157.89

1182.74

1235.88

1233.891183.74

1184.82

1185.85

1228.771186.86

1236.97

1238.05

1238.95

1240.04

1253.92

1280.751289.91

R+2His+ClO4-

11

Mass spectra of R with Cys

SI Figure 6: ESI- Ms spectrum of R with Cys in H2O.

m/z100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400

%

0

100

GUR-B3-33H 12 (0.223) TOF MS ES+ 5.94e3701.68

414.30

110.13

93.11

156.15

317.24

226.24319.23

670.55

414.79

457.48649.59

502.48540.86

702.71

917.81763.66

703.71 798.64

862.65

919.81

937.811081.75 1180.84

1235.88

R-2Cu.H2O+H+

12

0.0 8.0x1010

1.6x1011

2.4x1011

-3.00x10-4

-1.50x10-4

0.00

1/[Cu]2

1/(

F-F

O)

R2= 0.99692

0.00 1.80x10-5

3.60x10-5

5.0x104

1.0x105

1.5x105

I 5

52

[Cu2+

]

a b

240 320 400

0.2

0.4

R

L

Wavelength(nm)

Ab

s(a

.u)

240 320 400

0.0

0.9

1.8

2.7

Wavelength(nm)A

bs(a

.u)

R +Cys

R+His

R

a b

Absorbance spectra of L, R and R in absence and presence of Cys and His

SI Figure 7 (a) Absorbance spectra of L (2.0 x 10-5

M) and R (2 x 10-5

M) (b) Absorbance

spectra of R (2.0 x 10-5

M) in presence of His and Cys were performed in aq HEPES buffer

medium(10 mM, pH 7.4).

Benesi-Hildebrand plot for binding studies of Cu2+

towards L

SI Figure 8 (a) BH plot of L (2 x 10-5

M) for varying [Cu2+

] (0 to 4.6 x 10-5

M) ext = 350 and

Mon = 552 nm. Good linear fit confirms the 1: 2 binding stoichiometry in aq.-HEPES buffer-

CH3CN (96: 4(v/v); 10mM, pH 7.4) medium. (b) Luminescence titration profile.

13

2 4 6 8 10 12 142000

4000

6000

8000

pH

Inte

nsity

0.0 7.0x10-4

1.4x10-3

2.1x10-3

0.0

4.0x107

8.0x107

1/(

F-F

O)

1/[His]2

R2= 0.996

Benesi-Hildebrand plot for binding studies of [His] towards R

SI Figure 9. Benesi-Hildebrand plot of R (2 x 10-5 M) for varying [His] (0 to 3.2x 10

-3 M)

Ext = 350 and Mon = 552 nm. Good linear fit confirms the 1: 2 binding stoichiometry in aq.-

HEPES (10 Mm, pH 7.4) medium.

Change of Emission intensity of R at 500 nm as a function of the solution pH

SI Figure 10. Change in emission intensity at 500 nm with variation in pH of the solution for R

(20 M) by using 10 mM HEPES buffer Ex= 350 nm.

14

0.0

5.0x104

1.0x105

1.5x105

Cys+

Ala

Cys+

Pro

Cys+

Val

Cys+

Trp

Cys+

Arg

Cys+

Ser

Cys+

Tpy

Cys+

Ile

Cys+

Met

Cys+

GS

H

Cys+

Hcy

Cys+

Gly

Cys

Inte

nsity

0.0

5.0x104

1.0x105

1.5x105

His

+G

ly

His

+T

rp

His

+V

al

His

His

+M

et

His

+T

yr

His

+A

la

His

+S

er

His

+A

rg

His

+P

ro

His

+G

SH

His

+H

cy

His

+Ile

Inte

nsity

Interference study for interaction of Cys with R in presence of various Amino Acids (AAs)

SI Figure 11. Spectrophotometric interference study of R(2 x 10-5

M) with Cys (2.8 x 10-3

M)

in presence of various metal ions (2.8 x 10-3

M) in HEPES buffer by using lExt = 350 and lMon

= 552 nm.

Interference study for interaction of His with R in presence of various AAs

SI Figure 12. Spectrophotometric interference study of R(2 x 10-5

M) with His (3.2 x 10-3

M)

in presence of various metal ions (3.2 x 10-3

M) in HEPES buffer by using lExt = 350 and lMon

= 552 nm.

15

2.0x10-4

4.0x10-4

6.0x10-4

0

4

8

I-I0

O BP+NEM+200M His

O BP+NEM+300M His

[His] (M)

Detection of His in human blood plasma

SI Figure 13. Plot of DI = (I0- I) vs. [His], where I0 and I are emission intensities of receptor

R at 552 nm (Ext = 350 nm) in presence of known [His] and blood plasma samples spiked

with a known [His] + 10 mM NEM.

Pretreatment of the healthy human blood plasma for estimation of CCys & CHis

4

Fresh and human blood samples (5 mL) with added Lithium anticoagulant were centrifuged

in a vacutainer tube at 3000 rpm for 15 min. The supernatant solution (plasma), which

contains proteins and amino acids, was collected. 2 ml of collected plasma was vigorously

mixed with appropriate amount of NaBH4 and incubated for 5 minutes at room temperature in

order to hydrolyse the disulphide bond. Proteins present in the sample after reduction were

precipitated by the addition of methanol, followed by centrifugation (18 500g) of the sample

for 15 minutes. The supernatant liquid, which contained Cys and His in blood plasma, was

used for the spectroscopic studies.

16

Evaluation of CCys and CHis, in human blood plasma sample

Methodology 1:

Calculation of the CHis in Human Blood Plasma (HBP) sample that contains Cys and His. To

the 20M HBP sample 10 mM NEM was added. NEM is known to react irreversibly with

Cys and yield a non fluorescent compound (Scheme 2 shown below). Then this resultant

solution was used for quantitative analysis of His.

Scheme 2

Concentration of His in HBP is low and thus to avoid any error in its detection, HBP samples

(pre-treated with excess NEM) were spiked with two known concentration of His (100M

and 200M, respectively). Thus fluorescence intensity of such HBP samples spiked with 200

and 300 M of His were IHBP+200 and IHBP+300, respectively. Fluorescence intensities for pure

aqueous HEPES buffer solution having pH of 7.4 was evaluated for [His] of 200 and 300 M

and these values were I200 and I300, respectively.

Thus, the difference between IHBP+200 and I200 should lead to the actual concentration of His

([His]1) in HBP sample.

Similarly, the difference between IHBP+300 and I300 should lead to the actual concentration of

His ([His]2) in HBP sample.

Arithmetic mean of [His]1 and [His]2 led us to the value ((23 2.1) M) for CHis in HBP

sample. Please note that the data reported for CHis is an average of three independent

evaluations for [His]1 and [His]2.

17

Cell

i ivii iii

1 mM NEM + 10M R

DAPI MergeCell

viivi viiiv10M R DAPI MergeCell

Emission intensity measurements for HBP samples treated with the reagent R directly gave

the summation of concentration of CCys and CHis (CT = CCys + CHis).

Thus, CT – CHis = CCys.

Methodology 2:

CCys was also evaluated from the experiments with NEM, that reacted specifically with Cys.

Emission intensity for HPB samples treated with R was evaluated (IT), which reflects the

total concentration of Cys and His present in HBP sample. Then this solution was treated

with NEM. NEM reacted with Cys present in HBP sample and the product was non emissive.

The emission intensity (INEM) for the resulting solution was evaluated and the difference in

intensities (DI = IT – INEM) was used for evaluation of [Cys] in HBP sample. Value evaluated

for [Cys] in HBP sample, following this methodology, agreed well with the value that was

evaluated by adopting the methodology 1.

Cell culture and fluorescence imaging

SI Figure 14.Confocal laser fluorescence microscopic images of Hct116 cells treated with 10

M of R in HEPES buffer and various reagents mentioned in the Figs. iv & viii are overlay

of the merged images and confirmed the intracellular fluorescence.

18

0

25

50

75

100

20

0M

50

0M

10

0M

80M

60M

40M

20M

10M

5M

1M

0.1M

Ce

ll V

iab

ility

Ctr

l

Concentration of R in M

Hct116 cells (3 x 105) were seeded on coverslips placed in 6 well plates. After 24 hours cells

were treated with R (10μM) for 30 minutes or pre-treated with N-Ethyl Maleimide (NEM, a

thiol specific blocking reagent (1mM) for 30 minutes before adding R (10μM) for 30

minutes. Cells were then washed thrice with Phosphate Buffer Saline (1X PBS) and fixed

with 4% PFA for 20 minutes and washed again with 1X PBS. Permeabilization of the cells

was done using 0.2% Triton X 100 for 5 minutes. Again three washes were given and then

coverslips mounted using Fluor shield with DAPI (Sigma) mounting medium. Nail paints

was used to seal the coverslips mounted on the glass slides. Images were acquired in

Olympus Fluoview Microscope.

MTT assay for evolution of cytotoxicity of the reagent R towards Hct116 cells

SI Figure 15. MTT assay to determine the cell viability percentage in Hct116 colon cancer

cells. The concentration of the R ranges from 0.1- 500μM and treated for 12 hours. IC50 has

been calculated to be 200 μM.

The in vitro cytotoxicity of R on Hct116 cells (Colon cancer cell) were determined by

conventional MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, a yellow

19

tetrazole) assay. Hct116 colon cancer cells (7 x 103) were seeded in each well of a 96 well

plate and cultured in a 37°C incubator supplied with 5% CO2. Cells were maintained in

DMEM medium, supplemented with 10% Fetal Bovine Serum and 100 Units of Penicillin

Streptomycin antibiotics. After 24 hours the cells were treated with different concentrations

of the R in triplicates for 12 hours. After treatment cells were added with 0.5μg/ml of MTT

reagent. The plate was then incubated for 4 hours at 37°C and then later added to each well

with 100 μl of Isopropyl Alcohol. The optical density was measured at 570nm using

Multiskan Go (Thermo Scientific) to find the concentration of the cell inhibition. IC50 value

has been calculated to be 200μM.

The formula used for the calculation of the MTT assay for evaluation of the cell viability is as

follows:

Cell viability (%) = (means of Absorbance value of treated group/ means of Absorbance

value of untreated control) X 100.

Determination of detection limit:5

The detection limit was calculated based on the fluorescence titration. To determine the

S/N ratio, the emission intensity of R without Cys was measured by 10 times and the

standard deviation of blank measurements was determined. The detection limit (DL) of R for

Cys was determined from the following equation:

DL = K * Sb1/S

Where K = 2 or 3 (we take 2 in this case);

Sb1 is the standard deviation of the blank solution;

S is the slope of the calibration curve.

From the graph we get slope = 4.85x 104, and Sb1 value is 0.0004

Thus using the formula we get the Detection Limit = 1.64x 10-8

M.

20

i.e. R can detect Cys in this minimum concentration by fluorescence techniques.

References

(1) U. Reddy G, R. Lo, S. Roy, T. Banerjee, B. Ganguly and A. Das, Chem. Commun., 2013,

49, 9818. (2) U. Reddy G, P. Das, S. Saha, M. Baidya, S. K. Ghosh and A. Das, Chem.

Commun., 2013, 49, 255-257. (3) M. Ikeda, A. Matsu-ura, S. Kuwahara, S.S. Lee and Y.

Habata, Org. Lett., 2012, 14, 1564-1567. (4) P. Das, A. K. Mandal, U. Reddy G., M. Baidya,

S. K. Ghosh, A. Das, Org. Biomol. Chem., 2013, 11, 6604-6614. (5) S.Goswami, S.Das, K.

Aich, B. Pakhira, S. Panja, S. Kanti Mukherjee and S. Sarkar, Org. Lett., 2013, 15, 5412.