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Selenocysteine vs. cysteine: Tuning the derivatization on benzene sulfonyl moiety of a triazole linked dansyl connected glycoconjugate for selective recognition of selenocysteine and the applicability of the conjugate in buffer, in serum, on silica gel and in HepG2 cells Sivaiah Areti, Surendra Kumar Verma, Jayesh Bellare, and Chebrolu Pulla Rao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01518 • Publication Date (Web): 16 Jun 2016 Downloaded from http://pubs.acs.org on June 20, 2016

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Analytical Chemistry

Selenocysteine vs. cysteine: Tuning the derivatization on benzene sulfonyl moiety of a triazole linked dansyl connected glycoconjugate for selective recognition of selenocysteine and the applicability of the conjugate in buffer, in serum, on silica gel and in HepG2 cells

Sivaiah Areti,a Surendra Kumar Verma,b Jayesh Bellareb and Chebrolu Pulla Raoa*# a

Bioinorganic Laboratory, Department of Chemistry, bDepartment of Chemical

Engineering, Indian Institute of Technology Bombay, Powai, Mumbai – 400076, India, E-mail: [email protected]

ABSTRACT: A dansyl derivatized triazole linked glucopyranosyl conjugate (NO2L) has been synthesized, characterized and was used in the present study. The conjugate NO2L releases a fluorescent product upon reaction by Cys-SeH in aqueous PBS buffer by exhibiting a ~210 fold fluorescence enhancement even in the presence of twenty other amino acids with a minimum detection limit of (1.5±0.2)×10–7 M. The selectivity of the Cys-SeH to NO2L was further proven by extending the fluorescence study to different other selenium compounds. The role of para nitrobenzenesulfonyl (pNBS) center in NO2 L in the selective recognition of Cys-SeH was confirmed when the fluorescence emission studies were carried out using five different derivatizations possessing two NO2, five fluoro, two fluoro, one fluoro and no fluoro groups. The nucleophilic substitution reaction of Cys-SeH on NO2L has been clearly demonstrated on the basis of 1H NMR, ESI-MS and absorption spectroscopy, and the heat changes were monitored by isothermal titration calorimetry. The application potential of NO2L has been demonstrated by studying its selectivity towards Cys-SeH in aqueous PBS buffer, in bovine serum and on silica gel surface that lead to minimum detection limits of (25±2), (80±5) and (168±16) ppb respectively. The biological applicability of NO2L for Cys-SeH was further demonstrated in HepG2 cells by fluorescence microscopy. Thus, NO2L is aqueous soluble and biologically acceptable probe for Cys-SeH.

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INTRODUCTION Selenocysteine (Cys-SeH) is the 21st amino acid present in proteins and is genetically encoded by UGA in mammalian tissues.1,2 Selenium is a biologically essential element for cancer prevention, immune response and inflammation protection.3-6 Thus, selenium is a constituent of redox active enzymes, such as, glutathione peroxidase (GPx) and thioredoxin reductase (Trx).7-11 Inappropriate levels of Cys-SeH in tissue can lead to a number of diseases, such as, neurodegenerative and cardiovascular type.12-16 Inside the cell, the concentration of cysteine (Cys-SH) is very much greater than that of Cys-SeH17 where the amount of Se content in human body is 13-16 mg.18 This throws a big challenge to the researchers for the development of small molecular probes for the selective detection of Cys-SeH even in the presence of biologically relevant thiols including Cys-SH. It would be doubly better if the same molecular probe can provide even the cell imaging. Owing to a lower pKa of selenol in Cys-SeH (~5.3) as compared to the thiol in Cys-SH (~8.3),19-21 the Cys-SeH is expected to exhibit high reactivity than that of simple Cys-SH.

Indeed, such difference in pKa provides an opportunity to

develop systems for the selective detection of Cys-SeH even in the presence of other biologically relevant thiol molecules (biothiols). However, till date very few fluorescent probes are reported for the detection of Cys-SeH,22-24 instead, some chromatographic methods coupled with fluorometric or mass spectrometric analysis were reported which makes the analysis cumbersome.25-27 Recently, a fluorescent probe BESThio has been reported to discriminate the Cys-SeH from that of Cys-SH at pH 5.8.28 Unfortunately, this was not suitable for detecting or imaging Cys-SeH at physiological pH, which prevents its in vivo application. All this brings in a challenge to develop biologically

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adaptable molecular probe that selectively detects Cys-SeH over the other biothiols under physiological conditions in aqueous medium.

Strategy and design: It is understood from the literature that sensing thiols is achieved through a selective cleavage of fluorescence quencher 2,4-dinitrobenzene via aromatic nucleophilic substitution as shown here and the reaction is pH dependent.29-32 Based on this strategy, we found that the C–S (or C–O) bond involved in the molecular probe can be cleaved by thiolate group with the release of fluorescent moiety (Scheme 1a). Motivated by this, we emerged with a glyco-conjugate based probe in which the moiety possessing this bond can be tuned for its reactivity by appropriate derivatization on benzene sulfonyl unit (Scheme 1b).

Hence the same can be cleaved by Cys-Se-

(selenolate) due to its better nucleophilicity over that of Cys-S- at neutral pH. Such derivatization of the conjugate would result in a probe molecule that is more selective to Cys-SeH over that of Cys-SH. If this probe molecule can pass through the membrane, it will release the fluorescent moiety upon attack by Cys-SeH present in side the cell and hence the cells can be imaged by fluorescence microscopy. All this resulted in the design and development of glyco-based conjugates as given in Scheme 1b.

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Scheme 1: (a) Mechanism of nucleophilic aromatic substitution by thiols. (b) Schematic representation of the generic structure and the labeling of all the derivatives used in the present study.

The syntheses of the derivatives, viz., 2NO2L, 5FL, 2FL 1FL and 0FL, shown in Scheme 1b, has been reported by us recently33,34 and that the synthesis of

NO2

L follows the same

methodology but with the use of appropriately derivatized benzene sulfonyl chloride (pnitrobenzene sulfonyl chloride). The synthesis and characterization of the new conjugate (NO2L) was given in the experimental. In order to understand the sensitivity towards thioand seleno derivatives and to achieve selectivity, appropriate studies were carried out using emission and absorption spectroscopy. The reactivity followed by the product formation was established by 1H NMR and ESI MS. Upon carrying out all the studies extensively, the NO2L was established as a selective probe for Cys-SeH over that of CysSH in aqueous buffer and the same was used for imaging Cys-SeH in biological cells.

EXPERIMENTAL SECTION General information and materials:

1

H and

13

C NMR spectra were measured on a

Bruker NMR spectrometer working at 400 or 500 MHz. The mass spectra were recorded on Q-TOF micromass (YA-105) using electrospray ionization (ESI) method. Steady state fluorescence spectra were measured on Perkin-Elmer LS55. The absorption spectra were measured on Varian Cary 100 Bio. All the amino acids were procured either from Sisco Research Laboratories Pvt Ltd., India or from Sigma Aldrich chem. Co, USA.

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Fluorescence and UV/Vis absorption studies: The fluorescence studies were performed in 20 mM aqueous PBS buffer solution by always using a 25 µl of bulk solution of NO2L of 6 x 10–4 M prepared in PBS buffer. All the fluorescence titrations were carried out in 1 cm quartz cell in buffer solution and the total volume in each measurement was made to 3 mL by adding requisite volume of PBS buffer, to give a 5 µM cuvette concentration of NO2

L. During the titration, the concentration of amino acid were varied accordingly in

order to result in requisite mole ratios of amino acids to NO2L and the total volume of the solution was maintained constant at 3mL in each case by adding appropriate volume of aqueous 20 mM PBS buffer. Fluorescence emission spectra were recorded in 370 – 700 nm range by exciting the solutions at 360 nm. For the absorption studies, the final concentration of NO2L was kept constant at 10 µM.

1

H-NMR titration: The receptor

NO2

L (5 mM) was dissolved in 0.4 mL of D2O and

recorded the 1H NMR spectra in absence of Cys-SeH. The spectra were also measured by adding Cys-SeH to NO2L at different mole ratios up to a highest mole ratio of 10.

Confocal microscopy studies using HepG2 cells: After trypsinization, the HepG2 cells were seeded on to a 12 well plate at a density of 12000 cells per well and incubated overnight. The medium was removed and the cells were washed twice with pre-warmed PBS buffer at 37°C. The samples for the confocal laser scanning microscopy (CLSM) were prepared on the cover slip and were scanned under the laser source at 365 nm excitation using Olympus IX. The images were analyzed by Fluoview Viewer.

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Preparation of samples on silica gel strips: TLC plate coated with silica gel was cut into small strips of 3 × 1 cm2. Thin layer of the compound of

NO2

L was prepared by drop

casting 10 µL of 10 µM solution of the probe molecule using silica gel strips and the solvent was dried by leaving these at room temperature. The dried strips were used as such for fluorescence measurements. Each of these experiments was repeated for three times in order to get error bars.

Isothermal Titration Calorimetry (ITC): The calorimetric titrations were performed at 25 °C with a microcal ITC 200 isothermal titration calorimeter procured from MicroCal (Northampton, MA, USA). All the solutions were degassed for half an hour before performing the experiment. Titration was carried out by adding 0.5 mM solution of CysSeH or Cys-SH to 200 µL of a 0.05 mM solution of the ligand (2NO2L,

NO2

L and

0F

L) in

the ITC cell by twenty successive injections of 2 µL of Cys-SeH/Cys-SH each, while maintaining 200 seconds between each addition for a total addition of 40 µL of CysSeH/Cys-SH. The ITC data were fitted with the origin software package provided by MicroCal by using curve fitting model for one set of sites. experiment is being carried out without taking the molecule (viz.,

Each time, a control 2NO2

L,

NO2

L and

0F

L)

and the corresponding data are subtracted from the main titration data and the resultant data was subjected to the curve fitting.

Synthesis and characterization of

NO2

L: To a solution of

NO2

P (860 mg, 1 mmol) in 3

mL of MeOH was added an in situ prepared methyl acetate containing the liberated HCl. This is being prepared by adding 0.2 ml of acetyl chloride into 1 ml of methanol. The

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reaction mixture was stirred at room temperature until the starting material was consumed as evidenced by TLC analysis. The solution was neutralized with sodium bicarbonate and was filtered. The solvent was then removed in vacuum and obtained a solid product (0.48g) in 68% yield as yellow solid.

1

H NMR (400 MHz, CD3OD, δ ppm): 2.76 (s,

6H), 3.49-3.6 (m, 2H), 3.76 (dd, J= 3.1 and 6.3 Hz, 1H), 3.84 (t, J= 13.8 Hz, 1H), 3.93 (d, J= 6.4 Hz, 1H), 4.15-4.21(m, 2H), 5.37 (s, 2H), 5.4 (d, J= 8.4 Hz, 1H), 7.66-7.71 (m, 4H), 7.86 (d, J= 8.6 Hz, 1H), 8.02-8.05 (m, 2H), 8.19 (s, 1H), 8.42 (dd, J= 7.5, 3.6 Hz, 1H), 8.5 (d, J= 8.3 Hz, 1H).

13

C NMR (100 MHz, CD3OD, δ ppm): 30.81, 44.96,

45.82, 61.70, 62.42, 70.91, 73.99, 78.62, 81.26, 89.60, 116.69, 119.06, 124.52, 124.90, 125.90, 130.30, 130.38, 130.91, 130.93, 133.83, 133.97, 134.59, 144.11, 144.91, 151.74, 153.51. HRMS: Chemical Formula: C27H30N6O11S2 [M+Na+], calculated: 701.1306, found 701.1309.

RESULS AND DISCUSSION Synthesis and characterization: The conjugate,

NO2

L has been synthesized by using

precursor P5 which has been developed in three steps as reported by us recently.33,34 In order to demonstrate the role of benzene sulfonyl unit of NO2L in the recognition of CysSeH, the

NO2

L has been synthesized. The precursors and

NO2

L, as given in Scheme 1,

were well characterized by different spectral and analytical techniques, such as, 1H and 13

C-NMR, ESI-MS and FTIR (S01-S03 in ESI).

Optimizing the parameters for fluorescence study: In order to determine the optimum reaction time and pH on the reaction of

NO2

7

L by Cys-SeH, fluorescence study was



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performed in aqueous PBS buffer (Figure 1a and Figure S04 in ESI). The fluorescence intensity at 550 nm sharply increases with increasing incubation time from 0 to 5 min and remains unaltered thereafter. It is observed that the intensity is high and unaltered during pH 7 to 10. Therefore, all the studies reported in this paper corresponds to pH = 7.4 in PBS buffer after an incubation period of 5 min.

Differentiating Cys-SeH from Cys-SH and also from naturally occurring amino acids: To show its biological relevance, the

NO2

L (5 µM) was reacted with amino acids, low

molecular weight thiols and Cys-SeH. Reaction of

NO2

L with Cys-SeH, results in the

enhancement of fluorescence intensity as a function of added Cys-SeH concentration (Figure 1b). A plot of fluorescence intensity as a function of added [Cys-SeH]/[NO2L] mole ratio (Figure 1c, middle inset) shows ~210 fold enhancement at ≥10 equivalents, while Cys-SH showed no change even at higher concentration. The data given in Figure 1c clearly suggests that the Cys-SeH is more reactive than that of Cys-SH due to its better nucleophilic character and hence Cys-SeH can be easily distinguished from Cys-SH and/or biologically relevant thiols. Similar studies were carried out with other amino acids, but found no significant change in the fluorescence intensity of

NO2

L (Figure 1c).

From the concentration dependence studies, the detection limit was derived to be (1.5±0.2)×10–7 M (25±2 ppb) for Cys-SeH (Figure S05 in ESI).35 The color changes from non-fluorescent to greenish yellow only in the presence of Cys-SeH. This suggests that an effective cleavage takes place at the electron-withdrawing pNBS moiety of the weakly fluorescent probe

NO2

L and releases L4 and L5. The release of L4 and L5 was

proven based on 1H NMR and ESI MS as reported in this paper. Thus,

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NO2

L clearly

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distinguishes the biologically important Cys-SeH from those of other naturally occurring amino acids (Figure 1c top inset). This suggests that the

NO2

L is a reactive probe that is

selective to Cys-SeH as compared to the naturally occurring amino acids including Cys-

I/IO at 550 nm

Val

Tyr

Pro

L

Ser Thr Trp

Ile Lys Lue Met Phe

L

I/IO at 550 4

5

6

7

1

2

3

4

5

L

3

6

0F

2

+Cys-SeH

L

+BzSeH

1

NO2

F

0

0

L

50 2F

50 +DBDS

Se-Cys

100

+(SeCys)2

Cys

20

150

+SeO2

DTT

Cys-SeH

DDT

GSH

Cys-SH

HCys

GSH

MPA

Hcys

TAA

L

AMP

L

NO2

TTA

0

16

200

100

50

12

20

250

150

100

5 10 15 Mole ratio

Gly His

150

0

(f)

(e)

NO2

I/IO at 550 nm

200

8

L

(d)

200

4

5F

4

+Na2SeO3

250

2

L

0

L

2

50

NO2

2

Cys-SeH Cys-SH Asp Arg Asn Ala Gln

100

250 200 150 100 50 0

(c)

2NO2

150

250 200 150 100 50 0

I/IO at 550 nm

100 µM 250 (b) Cys-SeH 200 150 NO 0 { L+Cys-SeH} 100 NO L 50 0 480 540 600 660 6 8 10 12 pH Wavelength (nm)

200 (a)

Intensity (a.u)

Intensity at 550 nm

SH and biothiols owing to its unique interaction.

I/IO at 550 nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. (a) Plot showing the effect of pH on the fluorescence emission intensity upon reaction of

NO2

L (5 µM) by Cys-SeH (100 µM) when excited at 360 nm in PBS buffer.

(b) Fluorescence spectra obtained during the titration of [NO2L] (5 µM) with Cys-SeH (up to 100 µM) in PBS buffer (pH = 7.4) at λex = 360 nm. (c) Histogram showing the relative fluorescence intensity (I/I0) at 550 nm for the reaction of [NO2L] with amino acids including Cys-SeH. The upper inset: Vials exhibiting colour for the reaction mixtures of NO2

L (10 µM) with different amino acids (200 µM) under 365 nm UV lamp. The lower

inset: Relative fluorescence intensity plot (I/I0) as a function of [Cys-SeH]/[NO2L] mole ratio. (d) Bar diagrams showing the relative fluorescence intensity (I/I0) at 550 nm for the reaction of –SH molecules and Cys-SeH by NO2L (5 µM, at λex = 360 nm, pH = 7.4).

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(e) The reaction of different selenium compounds with NO2L. (f) The reaction of Cys-SeH with different conjugates (100 µM, at λex = 360 nm, pH = 7.4).

Reaction of thio- and seleno- compounds towards

NO2

L

&

2NO2

L by fluorescence

spectroscopy: In order to demonstrate the reactivity of different biologically relevant -SH containing molecules, mercaptopropionic acid (MPA), thioacetic acid (TAA), homocysteine (Hcy), dithiothreitol (DTTred) and GSHred were studied for their reaction on NO2

L possessing one p-NO2 group and found no fluorescence enhancement (Figure 1d

and Figure S06 in ESI), however, Cys-SeH exhibits large fluorescence enhancement as reported in the previous section.

To study the reactivity of the di-nitro derivative, the

2NO2

L was treated with various

biologically relevant molecules, such as, amino acids and low molecular weight thiols and all these resulted in the enhancement of fluorescence intensity as a function of added concentration of these (Figure S07a in ESI). Similar derivative with penta-fluorobenzene group present in

5F

L reacted with various anions and molecules and found fluorescence

enhancement in presence of CN– ion. This result suggests that

2NO2

L and

5F

L cannot

differentiate thiols, anions and selenol, and hence these turn out to be non-selective molecules. Thus

2NO2

L and

L were more selective towards Cys-SH and CN– ion

5F

respectively, as reported by us recently,33,34 though both go through similar nucleophilic attack at benzene sulfonyl center in the respective probe molecule.

In order to compare the reactivity of different selenium compounds, viz., Cys-SeH, CysSe-Se-Cys, SeO2, Na2SeO3, PhCH2Se-SeCH2Ph (DBDS) and BzSeH were studied for

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their reaction on

NO2

L.

Reaction of

NO2

L by Cys-SeH and BzSeH results in the

enhancement of fluorescence intensity by 210 and 195 fold respectively and all others showed no significant change in the fluorescence intensity (Figure 1e and Figure S07 in ESI). These results clearly support the fact that the Cys-SeH is more reactive towards NO2

L than all the other selenium compounds (Figure 1e). The involvement of –SeH has

been further confirmed when the titration of NO2L were carried out with the oxidized (-SeSe-) form and found no change in the fluorescence intensity of

NO2

L, since the oxidized

form does not possess any free –SeH (Figure S07c in ESI).

This was further

demonstrated by checking the visual fluorescent color (under UV light) in the presence of the native (Cys-Se-Se-Cys) and the reduced (Cys-SeH), where a strong fluorescent colour is noticed in case of the latter due to the presence of free –SeH group.

Reactivity of Cys-SeH towards all the conjugates: A number of differently derivatized conjugates given in Scheme 1b were synthesized owing to the variation in their electronic and steric factors and these were studied for their reactivity by Cys-SeH in aqueous PBS buffer at pH = 7.4. In case of

2F

L, only 20 ± 2 fold of enhancement was observed with

Cys-SeH because of the presence of partially fluorinated moiety suggesting that the necessity of electron withdrawing group. Thus the fold of enhancement with NO2L is 210, while it is only 20 ± 1 in case of 0F

2F

L (Figure 1f). The fluorescence intensity of

1F

L and

L remains unaltered during the reaction with amino acids, thiols and Cys-SeH owing to

the absence of any electron with drawing moiety (Figure 1f and Figure S08 in ESI). All these results suggests that

NO2

L discriminates selenol from that of thiol, because of the

higher nucleophilicity of selenol group (-SeH). Thus, these results suggests that among

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all the conjugates,

NO2

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L showed remarkable fluorescence enhancement with Cys-SeH.

Hence further studies pertinent to 1H NMR, ESI MS, absorption and cell imaging were carried out for the reaction of NO2L by Cys-SeH.

Reactivity of Cys-SeH on

NO2

L by 1H NMR and ESI MS: The 1H NMR spectra were

measured for the addition of 0 to 10 equiv of Cys-SeH and these are given in Figure 2a. As the added Cys-SeH increases, the 1H NMR signals corresponding to

NO2

L starts to

diminish, while those corresponding to L4 and L5 starts to appear (Figure 2a). Thus, the spectrum (iii) and (iv) in Figure 2a corresponds to a mixture of all the three. However, in case of spectrum (v), only small quantity of NO2L is present along with L4 and L5, while it is completely L4 and L5 in case of spectrum (vi) supporting the complete conversion of NO2

L. The ESI MS of the corresponding reaction mixtures were recorded and found m/z

peaks corresponding to L4 and L5 at 516.15 [L4+Na]+ and 312.14 [L5+H]+ respectively in the spectrum shown in Figure 2b and c measured upon addition of 5 and 7.5 equiv of Cys-SeH respectively. The results obtained from ESI MS study tally with that observed in case of 1H NMR study. Thus, both the 1H NMR and mass spectra supported the nucleophilic attack of Cys-SeH on

NO2

L releasing L4 and L5. However, when similar

studies were carried out with Cys-SH, no significant changes in the chemical shifts were observed in 1H-NMR spectra of NO2L (Figure S09 in ESI)).

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(a)

(b)

(c)

(d)

Figure 2: (a) 1H NMR spectra measured during the titration of

NO2

L [5 mM] with Cys-

SeH [up to 50 mM] in D2O to result in, (i) 0, (ii) 1 (iii) 2.5, (iv) 5, (v) 7.5, and (vi) 10 equiv. The peaks with the symbol (▲) correspond to NO2L, (●) correspond to L4 and (■) correspond to L5. ESI MS spectra obtained for the reaction mixture when NO2L is treated with, (b) 5 and (c) 7.5 equiv of Cys-SeH. (d) The products formed in the reaction of CysSeH on NO2L.

13




NO2

L by absorption spectroscopy: In order to further support

NO2

the reaction of Cys-SeH on

NO2

L, the absorption studies were carried out. The

L

shows absorption bands at 360, 220 along with a shoulder at ~263 nm. Upon the reaction of

NO2

L by Cys-SeH, increase in the absorbance is observed at 360 and 220 nm, and a

decrease is observed at ~295 and ~550 nm (Figure 3a). All this resulted in isosbestic points at 325 and 450 nm. However, when

NO2

spectra remain similar to that of the untreated

L is treated with other amino acids, the

NO2

L (Figure S10 in ESI). Therefore, the

spectral changes observed in case of Cys-SeH are attributable to its unique reactivity towards NO2L. The involvement of selenol function (-SeH) has been ascertained from the negative result obtained when treated with the oxidized Cys-SeH, viz., Cys−Se−Se−Cys having no free −SeH group (Figure 3b).

0.30

(a)

0.6 0.5 0.4 0.3 0.2 0.1

(b)

0.4 0.3 0 2 4 6 8 10

0.2

0.15

0.02

300 400 500 Wavelength (nm) 0

(e)

-100

0.00

-30

-400 -500

-0.06 0

1000 2000 Time (Sec)

3000

1000 2000 Time (Sec)

2NO2

L

1

NO2

L

2

3000

0F

L 3

(f)

-300

-0.04

-40

0

-200

-0.02

(d)

-30

kcal/mol

-20

Kcal/sec

-10

-20 (c)

-40

0.0

330 440 550 Wavelength (nm) 0

-10

0 2 4 6 8 10 Mole ratio

0.1

0.00

0

0.25 0.20 0.15 0.10 0.05

Kcal/sec

Absorbance

0.45

Kcal/sec

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0

1000 2000 Time (Sec)

14

3000


Cys-SeH Cys-SH

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Figure 3: (a) Absorption spectra obtained during the titration of NO2L (10 µM), with Cys-

SeH (100 µM) in PBS buffer at pH = 7.4. Inset: shows plots of absorbance vs. {[CysSeH]/[NO2L]} for three different bands. titration of

NO2

(b) Absorption spectra obtained during the

L (10 µM) with (Cys-Se-Se-Cys) (200 µM).

Inset: shows plots of

absorbance vs. {(Cys-Se-Se-Cys)/[NO2L]} for three different bands. In both the cases, the colour of the inset plots correspond to, blue for 220, red for 295 and black for 360 nm bands respectively. ITC thermograms of baseline-corrected raw data for heats of reaction vs injection time for the addition of Cys-SeH (red line spectra) and Cys-SH (black line spectra) in case of (c)

2NO2

L, (d)

NO2

L and (e)

0F

L respectively in aqueous PBS buffer at

pH 7.4. (f) Histogram shows heats of reaction of Cys-SeH and Cys-SH with 2NO2L, NO2L and 0FL derivatives.


NO2

L by Isothermal titration calorimetry: The fluorescence

emission studies given in this paper clearly differentiates the higher nucleophilicity of Cys-SeH over that of simple Cys-SH or even other S- and Se- containing compounds. Thus the –SeH are more reactive as compared to –SH. This would also mean that in case of stronger nucleophile, its reaction with the probe molecule is expected to bring greater heat changes. In order to understand the heat changes in the reactions of Cys-SeH and Cys-SH by

2NO2

L,

NO2

L and

0F

L, ITC studies were performed and the corresponding

thermograms were given in Figure 3c-f (Figure S11, in ESI). The interaction of Cys-SeH and Cys-SH with

2NO2

L is exothermic by -506 and -425 kcal/mol respectively and hence

shows the feasibility of the reaction in these cases (Figure 3c). On the other hand, the ∆H values observed in the reactions of Cys-SeH (-382 kcal/mol) and Cys-SH (-45 kcal/mol) with of

0F

NO2

L clearly suggests only a least reactivity in case of Cys-SH (Figure 3d). In case

L no significant heat changes were observed with either of these supporting that

these will not react with this derivative (Figure 3e). The corresponding features can be

15



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Page 16 of 28

comparatively understood from Figure 3f. The feasibility of the reaction of all the conjugates towards Cys-SeH and Cys-SH follows a trend, viz.,

2NO2

L >

NO2

L >>

0F

L,

however, in case of NO2L, the reactivity of Cys-SeH is far greater than Cys-SH. Thus, the NO2

present study showed that

L probe is more reactive as compared to all other

derivatives towards selenols over thiols, and the reaction is also more thermodynamically favorable with the former.

Application potential of the present study: Having clearly showed the selectivity of NO2L towards Cys-SeH over Cys-SH and other thiols relevant to biological systems, its practical applications including the detection of Cys-SeH in serum and on silica gel strips, and the fluorescence imaging of biological cells were demonstrated.

(a) Detection of Cys-SeH by applicability of

NO2

NO2

L in serum by fluorescence measurements: Biological

L has been further addressed by carrying out the fluorescence

titrations in presence of fetal bovine serum (1 mL of serum + 2 mL of PBS buffer). The NO2

L shows 175 ± 15 fold enhancement of fluorescence intensity as a function of the

concentration of added Cys-SeH (Figure 4a and Figure S12, in ESI) and the minimum detection limit of Cys-SeH is (5 ± 0.4)×10–7 M, which is (80 ± 5) ppb in presence of serum. Thus NO2L is sensitive enough to detect Cys-SeH even in the biological fluids.

(b) Detection of Cys-SeH by practical utility of

NO2

NO2

L coated silica gel strips: In order to demonstrate the

L, the detection of Cys-SeH using silica gel coated with

NO2

L was

carried out. Increasing concentrations of Cys-SeH were added to the silica gel strips

16


Page 17 of 28

coated with NO2L resulting in mole ratios [Cys-SeH]/[NO2L] of 0 to 10 and the strips were allowed to dry. The corresponding fluorescence spectra and the color exhibited by these samples under UV light are shown in Figure 4b. The conjugate

NO2

L exhibits weak

fluorescence emission centered at 540 nm which increases upon addition of increasing concentration of Cys-SeH and shows up to 75±5 fold of fluorescence enhancement when the mole ratio of [Cys-SeH]/[NO2L] is 10 (Figure 4c Figure S13 in ESI). Similar studies were carried out with Cys-SH showed no significant change either in the color (under UV light) or in the fluorescence intensity of

NO2

L (Figure S13c in ESI). The fluorescence

intensity ratio plot is fairly linear in the [Cys-SeH] concentration range of 5-100 µM (Inset of Figure 4c). This yields a lowest detection limit of (10 ± 1) µM, which is (168± 16) ppb of Cys-SeH on silica gel strips and hence this technology can be used for a large scale screening of biological samples routinely.

200 75 (c)

(b)

150

60

I/I0 at 540 nm

120

45

100

80 40 0 450 500 550 600 650 Wavelength (nm)

100 75 50 25 0

Intensity at 540 nm

(a)

Intensity (a.u)

160 Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30

50

15 0

0 450 500 550 600 650 Wavelength (nm)

0 2 4 6NO8 10 [Cys-SeH/

0

5 10 NO 15 [Cys-SeH/ L] 2

2

L]

20

Figure 4: (a) Fluorescence spectra of NO2L (5 µM, λex = 360 nm) obtained in the medium

of fetal bovine serum in PBS buffer upon addition of different equivalents of Cys-SeH. (b) Fluorescence spectra obtained in the titration of

NO2

L (λex = 360 nm) with different

equivalents of Cys-SeH on the silica gel strips. Inset: photograph of the corresponding samples under 365 nm UV light. (c) Plot of relative fluorescence intensity (I/Io) vs. mole ratio of [Cys-SeH]/[ NO2L]. Inset: Linear plot obtained in the concentration range 5 to 100 µM. The error bars are a result of experiments being repeated three times.

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Page 18 of 28

(c) Detection of Cys-SeH by Imaging HepG2 cells using fluorescence microscopy: In order to show the practical utility of

NO2

L in the detection of cellular selenols,

fluorescence microscopy studies were carried out using HepG2 cells.

Since the

physiological concentration of Cys-SeH is low in the cells, exogenous Cys-SeH was used in the study. The HepG2 cells were incubated in PBS buffer (pH = 7.4) containing 10 µM of the conjugate (NO2L) for 30 min at 37 ºC, and found that HepG2 cells showed no fluorescence emission at this stage when no Cys-SeH was added (Figure 5b). Upon addition of Cys-SeH to these cells exogenously, the cells exhibited effective intracellular green fluorescence emission owing to the reaction of selenols on

NO2

L (Figure 5c). In a

control experiment, even the cells incubated with excess concentrations of Cys-SH followed by Cys-SeH, did not show any interference with the detection of Cys-SeH by NO2

L (Figure 5d).

Figure 5: Fluorescence microscopy images obtained from HepG2 upon treatment with

the probe NO2L (10 µM) for 30 min in PBS buffer. (a) Control, (b) NO2L, (c) {NO2L +Cys-

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SeH (20 µM)} and (d) {NO2L +Cys-SH (30 µM)}. Panels (e - h) are the corresponding overlap images for those present in above panel (a – d).

It is known in the literature that the cells incubated with Na2SeO3 generate intracellular Cys-SeH metabolically36 and hence this was used in the present study to generate intracellular Cys-SeH. Under this, two distinct experiments were carried out, of which one is as a function of the incubation time of 20 µM Na2SeO3 (0 to 12 hrs) and the other is as a function of the concentration of the added Na2SeO3 (0–20 µM) at 12 hrs of incubation. These results showed noticeable fluorescence emission with the samples incubated with 20 µM Na2SeO3 for 3 hrs, however, significant increase in the fluorescence emission was observed after 6h and it further increases as the time of incubation increases (Figure 6a-e). Enhanced fluorescence emission was also observed at 12 hrs of incubation of cells with 5 to 20 µM of Na2SeO3 as can be seen from Figure 6ko. The merged images clearly supports that the fluorescence is exhibited through the cells.

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Figure 6:

Fluorescence microscopy images obtained from HepG2 cells upon the

following treatment. Upper panel: Initially, four samples of cells were incubated for 1, 3, 6 and 12 hrs with 20 µM Na2SeO3. At this stage, each of these is treated with probe NO2

L in PBS buffer and kept for 30 min before measuring microscopy and the resulting

pictures are given in (b), (c), (d) and (e) respectively. Panels (f to j) are the corresponding overlap images. (a) Is a control system used without the addition of Na2SeO3. Lower panel: This is same as upper panel except that in place of time it is the concentration of

Na2SeO3 (0–20 µM) that is varied and all the samples were kept for 12 hrs of incubation. The micrographs labeled as (k to l) corresponds to the concentration of Na2SeO3 of 0 (control), 5, 10, 15 and 20 µM respectively. The panels (p to t) are the corresponding overlap images.

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Therefore, the cellular studies clearly suggest that the conjugate

NO2

L exhibit good cell

permeability and shows effective intracellular fluorescence emission upon reacting with selenols present in the cells (or metabolically generated) in the cells. The result reveals that the probe can monitor the dynamic changes of Cys-SeH concentration level and is capable of detecting and imaging Cys-SeH present in the cells. HepG2 cells are viable up to 50 µM and 12 hr and were affected to only a little extent beyond this (Figure S14 in ESI) and hence NO2L is a biologically adaptable probe.

CONCLUSIONS AND CORRELATIONS

An NO2 derivatized benzene sulfonyl possessing triazole linked glucopyranosyl conjugate (NO2L) was synthesized and characterized and was demonstrated for its selective recognition of Cys-SeH over the simple Cys-SH in PBS buffer owing to its water solubility. The NO2L is a selective reactive probe for Cys-SeH over the other amino acids, thiol molecules (viz., MPA, TAA, Hcy, DTTred and GSHred,) and selenium compounds (viz., SeO2, Na2SeO3, Cys-Se-Se-Cys, and PhCH2Se-SeCH2Ph) by displacing the p-NBS group from the probe conjugate and thereby releasing L4 and L5, of which L4 is fluorescent. Thus all these thio- and seleno- compounds, except Cys-SeH, exhibited no change in the fluorescence emission intensity, implying no reaction of these on

NO2

L.

This reaction can be monitored by different spectroscopy techniques including that of fluorescence and was indeed studied by emission, absorption, 1H NMR and ESI MS, and the thermodynamics of the reaction were monitored by ITC. All the spectral studies

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Page 22 of 28

clearly supported the nucleophilic attack of Cys-SeH on

NO2

L and the peaks

corresponding to L4 and L5 were observed both in 1H NMR and ESI MS. The release of fluorescent L4 only in case of Cys-SeH (but not with other thiol molecules) was further supported by its colour when shined using UV light. Thus,

NO2

L exhibited a minimum

detection limit of 25±2 ppb for Cys-SeH in aqueous PBS buffer as revealed by fluorescence measurements.

The importance of NO2L in the selective detection of Cys-SeH over other conjugates, viz., 2NO2

L,

5F

L,

2F

L

1F

L and

fluorinated conjugate

2F

0F

L has been supported by fluorescence study. The partially

L is less reactive by exhibiting only 20 fold enhancement, while

the 1FL and 0FL derivatives exhibit no fluorescence enhancement supporting that the CysSeH does not react with the last two derivatives, though marginal reactivity was observed with

2F

L.

Further, the

2NO2

L and

5F

L are non-selective for Cys-SeH because these

respond to Cys-SH and CN– respectively as reported by us recently.33,34 All these results (Scheme 2) suggests that the NO2L selectively responds to Cys-SeH with more than ~210 fold increase in fluorescence emission intensity in aqueous PBS buffer at pH 7.4.

The feasibility of the reaction with 2NO2L has been supported in both the cases, viz., CysSeH and Cys-SH by releasing energy with ∆H values of -506 and -425 kcal/mol respectively as obtained from ITC and this suggests that the di-nitro derivative cannot differentiate these two analytes. However, the mono-nitro derivative,

NO2

L can clearly

distinguish these two analytes by releasing higher energy in case of Cys-SeH (-382 kcal/mol) as compared to Cys-SH (-45 kcal/mol) and hence

22


NO2

L reacts selectively with

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Cys-SeH. Further the reaction of Cys-SeH or Cys-SH with 1FLOH and 0FLOH showed no significant heat changes, implying that these two analytes does not exhibit any reactivity on these two conjugates.

Scheme 2. Schematic representation of different features noticed in sensing Cys-SeH.

The products of the reaction of NO2L with Cys-SeH in PBS buffer solution, in serum and on silica gel surface exhibits a fluorescence enhancement of 210±20, 175±15 and 75±5 fold respectively and this is further reflected in their minimum detection limits, viz., 25±2, 80±5 and 168±16 ppb respectively. Though the sensitivity of the detection follows a trend, viz., PBS buffer solution > in serum > on silica surface, the Cys-SeH can be very well detected when 10 µL of a 10 µM of the receptor NO2L is loaded on a silica gel strip and or when the detection is carried out in biological medium using serum. The receptor, NO2

L clearly crosses the membrane to get into HepG2 cells where in the Cys-SeH

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produced inside the cells reacts with the receptor to result in fluorescent L4 and provides fluorescence imaging. All these results (Scheme 2) clearly suggest the compatibility of NO2

L to extend its utility to biological applications.

ASSOCIATED CONTENT

The supporting information includes the experimental

Supporting information:

methods, synthesis and characterization, spectral data, fluorescence and absorption data and the ITC data. This material is available free of charge via the Internet at http:// pubs.acs.org.

AUTHOR INFORMATION

Corresponding author: To whom correspondence should be addressed. Phone: 91 22 2576 7162. Fax: 91 22 2572 3480. Email: [email protected] #

Dedication: We dedicate this paper to Professor Harkesh B. Singh on his 60th birthday.

ACKNOWLEDGEMENTS

CPR acknowledges the financial support from DST (SERB & nano mission), CSIR and DAE-BRNS, chair professorship from IIT Bombay and JC Bose National Fellowship from DST. AS acknowledges UGC for fellowship. We thank CRNTS of IIT Bombay for the cell imaging facility.

We thank D. S. Yarramala for helping with the ITC

measurements.

REFERENCES 1. Stadtman, T. C. Annu. Rev. Biochem. 1996, 65, 83-100.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2. Johansson, L.; Gafvelin, G.; Arner, E. S. Biochim. Biophys. Acta, 2005, 1726, 113. 3. Rayman, M. P. Lancet, 2000, 356, 233– 241. 4. G. N. Schrauzer, Cell Mol Life Sci., 2000, 57, 1864-1873. 5. Duffield-Lillico, A. J.; Reid, M. E.; Turnbull, B. W.; Combs Jr, G. F.; Slate, E. H.; Fischbach, L. A.; Marshall, J. R.; Clark, L. C. Cancer Epidemiol., Biomarkers Prev. 2002, 11, 630–639. 6. Zhang, S. Y.; Ong, C.-N.; Shen, H.-M. Cancer Lett., 2004, 208, 143–153. 7. Dalton, T. P.; Shertzer, H. G.; Puga, A. Annu. Rev. Pharmacol. Toxicol. 1999, 39, 67–101. 8. Pastore, A.; Piemonte, F.; Locatelli, M.; Russo, A. L.; Gaeta, L. M.; Tozzi, G.; Federici, G. Clin. Chem. 2001, 47, 1467–1469. 9. Shahrokhian, S. Anal. Chem. 2001, 73, 5972–5978. 10. Chen, J.; Berry, M. J. J. Neurochem. 2003, 86, 1–12. 11. Schomburg, L.; Schweizer, U.; Kohrle, J. Cell. Mol. Life Sci. 2004, 61, 1988 – 1995. 12. Tapiero, H.; Townsend, D.; Tew, K. Biomed. Pharmacother. 2003, 57, 134–144. 13. Kleinman, W. A.; Richie, J. P. Biochem. Pharmacol. 2000, 60, 19–29. 14. Brenneisen, P.; Steinbrenner, H.; Sies, H. Mol. Aspects Med. 2005, 26, 256–267. 15. Arteel, G. E.; Sies, H. Toxicol. Pharmacol. 2001, 10, 153– 158. 16. Reeves, M. A.; Hoffmann, P. R. Cell. Mol. Life Sci. 2009, 66, 2457–2478. 17. Park, S.; Imlay, J. A. J. Bacteriol. 2003, 185, 1942-1950. 18. Schroederm, H. A.; Frosr, D. V.; Balass, J. J. J. Chron. Dis., 1970, 23, 227-243

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

19. Huber, R. E.; Criddle, R. S. Arch. Biochem. Biophys. 1967, 122, 164-173. 20. Arner, E. S. J. Experrimental cell research, 2010, 316, 1296-1303. 21. Rocher, C.; Lalanne, J. L.; Chaudiere, J. Eur. J. Biochem. 1992, 205, 955-960. 22. Baoxin, Z.; Chunpo, G.; Juan, Y.; Yaping, L.; Huichen, X.; Jianguo, F., J. Am. Chem. Soc., 2015, 137, 757–769. 23. Fanpeng, K.; Bo, H.; Yan, G.; Kehua, X.; Xiaohong, P.; Fang, H.; Qiuling, Z.; Hao, C.; Bo, T., Chem. Commun. 2015, 51, 3102-3105. 24. Chen, H.; Dong, B.; Tang, Y.; Lin, W. Chem. Eur. J., 2015, 21, 11696–11700. 25. Yang, L.; Sturgeon, R.E.; McSheehy, S.; Mester, Z. J. Chromatogr. A. 2004, 1055, 177-184. 26. Encinar, J. R.; Schaumloffel, D.; Ogra, Y.; Lobinski, R. Anal. Chem. 2004, 76, 6635 – 6642. 27. Iscioglu, B.; Henden, E. Anal. Chim. Acta. 2004, 505, 101 – 106. 28. Maeda, H.; Katayama, K.; Matsuno, H.; Uno, T. Angew. Chem., Int. Ed. Engl. 2006, 45, 1810-1813.

29. Bouffard, J.; Kim, Y.; Swager, T. M.; Weissleder, R.; Hilderbrand, S. A. Org. Lett. 2008, 10, 37-40. 30. Shao, J.; Sun, H.; Guo, H.; Ji, S.; Zhao, J.; Wu, W.; Yuan, X.; Zhang, C.; James, T. D. Chem. Sci. 2012, 3, 1049–1061. 31. Maity, D.; Govindaraju, T. Org. Biomol. Chem. 2013, 11, 2098-2104. 32. Murale, D. P.; Kim, H.; Choi, W. S.; Churchill, D. G. Org. Lett. 2013, 15, 3630– 3633.

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33. Areti, S.; Bandaru, S.; Yarramala, D. S.; Rao, C. P. Anal. Chem. 2015, 87, 2396−12403. 34. Areti, S.; Teotia, R.; Rao, C. P. Analyst, 2015, 140, 7391-7398 35. Wallenberg, M.; Olm, E.; Hebert, C.; Bj¨ornstedt, M.; Fer-nandes, A. P. Biochem. J., 2010, 429, 85–93. 36. Kryukov, G. V.; Castellano, S.; Novoselov, S. V.; Lobanov, A. V.; Zehtab, O.; Guigo, R.; Gladyshev, V. N. Science, 2003, 300, 1439-1443.

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Table of Contents (Graphical TOC)

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