supporting information a novel fluorescence probe for
TRANSCRIPT
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: [email protected]; Tel: +91(0)2025902385; Fax:
+91(0)2025902629; bChromatin and Disease Biology Lab; National Centre for Cell Science; Pune
411007, India, Email: [email protected].
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.