Plumbagin as colorimetric and ratiometric sensor for arginine

Accepted Manuscript Title: Plumbagin as Colorimetric and Ratiometric Sensor for Arginine Author: Sheik Dawood Shahida Parveen Abdullah Affrose Kasi Pitchumani PII: DOI: Reference:

S0925-4005(15)00875-8 http://dx.doi.org/doi:10.1016/j.snb.2015.06.149 SNB 18742

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

18-2-2015 25-4-2015 18-6-2015

Please cite this article as: S.D.S. Parveen, A. Affrose, K. Pitchumani, Plumbagin as Colorimetric and Ratiometric Sensor for Arginine, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.06.149 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract (for review)

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Graphical abstract

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Research Highlights

Highlights of the Present Work  A simple and selective naphthoquinone based plumbagin for arginine sensing  Ratiometric and colorimetric sensor in aqueous solution

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 The 1:1 Pn/arg complex was characterized by Job’s plot and ESI-Mass spectrometry  Upon arginine binding intermolecular proton transfer from Pn to arginine occurs

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 The observed detection limit (DL) is 1.0 x 10-6 mol L−1

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*Manuscript(highlighted)

Plumbagin as Colorimetric and Ratiometric Sensor for Arginine Sheik Dawood Shahida Parveen,a Abdullah Affrose, a Kasi Pitchumania,b* a

School of Chemistry, Madurai Kamaraj University, Madurai - 625021, India b

Centre for Green Chemistry Processes, School of Chemistry,

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Madurai Kamaraj University, Madurai – 625021, India

*Corresponding author. Tel.: +91 452 2456614; fax: +91 452 2459181. E-mail address: [email protected] (K. Pitchumani) 1

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Abstract A simple and selective naphthoquinone based fluorescent probe involving

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Plumbagin (Pn =1,4-naphthoquinone) which is a naturally occurring phytochemical is reported for sensing of arginine in aqueous medium. The sensing probe Pn upon binding

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to Arg, induced a visible color change from light yellow to pink. The 1:1 Pn/Arg

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complex was characterized by Job’s plot, ESI-MS and DFT calculations. This naked eye colorimetric sensing is insensitive to other amino acids namely Asp, Glu, Thr, Lys, His,

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Cys, Met, Ser and Phe. In the absence of arginine, excited state intramolecular proton transfer (ESIPT) occurs and when arginine is added, intermolecular proton transfer from

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Pn to arginine takes place resulting in colorimetric and ratiometric response.

Keywords: Plumbagin, Colorimetric and ratiometric sensor, Arginine, Intramolecular proton transfer, DFT calculations.

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1. Introduction Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a quinonoid constituent

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isolated from the roots of Plumbago zeylanica L., is an anti-cancer agent, which is partly mediated by the inactivation of NF-kB-regulated anti-apoptotic, proliferative and

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angiogenic gene products [1]. Potential role of plumbagin, as an anti-cancer agent has

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been recognized [2-5] and its anti-cancer effects have been reported in diverse cancer models such as prostate, lung, cervical, ovarian as well as the melanoma.

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Naturally occurring α-amino acids are of special interest because of their

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biological prominence. The recognition and sensing of amino acids and their derivatives have been investigated in both metal containing and pure organic systems. Arginine plays

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an important role in cell division, the healing of wounds, removing ammonia from the

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body, immune function, and the release of hormones [6]. Arginine taken in combination

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with proanthocyanidins [7] or yohimbine [8], has also been used in the treatment for erectile dysfunction. The benefits and functions attributed to oral supplementation of L-arginine include quickening of repair time of damaged tissue and helping decrease blood pressure. Excess L-arginine can increase levels of stomach acid, particularly gastrine and some people experience anaphylaxis, or an allergic reaction, to L-arginine. Because of L-arginine's properties as a vasodilator, low blood pressure can be a side effect of supplementation. It can increase the body's production of potassium, chloride, creatinine, blood urea nitrogen, sodium and phosphate levels. Those suffering

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from kidney or liver problems are especially susceptible to changes in these chemical balances.

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The effective and selective molecular recognition or sensing of unprotected amino acids in aqueous solution is a challenging problem due to their highly hydrophilic

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character. Chemical or biological sensing via colorimetric naked eye detection is an

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inexpensive technique and is useful for amino acid recognition owing to their simplicity, high selectivity and sensitivity. And selective detection of a specific amino acid without

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interference from other amino acids is a challenging task. Inspite of its significant

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biological relevance, only few sensors are available for the detection of arginine. Some of the recent reports involve bis-rhodamine-thiourea/Al3+ complex for the fast visual

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detection of arginine [9], luminol dextran conjugate through ICT process [10], a ternary

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system based on fluorophore-surfactant assemblies-Cu2+ by electrostatic interaction

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between anionic surfactants and positively charged amino acids [11] and quercetinfunctionalized gold nanoparticles through aggregation of QCAuNPs [12]. The aim of the present study is to develop a simple system for detection of arginine in aqueous solution using naturally occurring molecules. With this goal in mind, for the first time we have used Pn, a natural naphthoquinone, as a sensing probe for the selective detection of arginine. This probe selectively showed a rapid color change from yellow to pink in presence of arginine.

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Materials and methods UV-Visible absorption spectra were recorded by using the JASCO-Spectra

temperature.

Fluorescence

measurements

were

made

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Manager (V-550) in 1 cm path length quartz cuvette with a volume of 2 mL at room on

a

Fluoromax-4

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Spectrofluorometer (HORIBA JOBIN YVON) with excitation slit set at 2.0 nm bandpass

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and emission at 4.0 nm bandpass in 1 cm x 1 cm quartz cell. Electrospray ionisation mass spectrometry (ESI-MS) analysis was performed in the positive ion mode on a liquid

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chromatography-ion trap mass spectrometer (LCQ Fleet, Thermo Fisher Instruments

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Limited, US). The samples were introduced into the ion source by in fusion method at flow rate 1µL/min. The capillary voltage of the mass spectrometer was 33V, with source

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voltage 4.98kV for the mass scale (m/z150–2000). The geometries of plumbagin and its

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complex with arginine were optimized using DFT-B3LYP 6-31G and LANL2DZ (d)

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levels respectively using Gaussian 03 package. 2.1. Isolation of plumbagin from Plumbago Zeylanica Plumbagin was isolated from the CHCl3 extract of Plumbago Zeylanica roots using Soxhlet method and characterised by NMR, FT-IR and ESI-MS techniques. (Figures S1-S4 in supporting information) 2.2.

Preparation of stock solution and UV titration

All the measurements are carried out in double distilled water which is free from ions. The stock solution of plumbagin (1 x 10-3 M) is prepared by dissolving 0.0188 g of 5

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plumbagin in 100 mL ethanol:water (v/v 1:1) mixture. Solutions (1x10-3M) of His, Lys, Met, Thr, Asp, Arg, Ser, Glu, Phe and Cys are prepared by dissolving them in water. For UV titration, plumbagin (1 mL of stock), amino acids (0.1-1.0 mL of stock) are taken in

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10 mL SMF and analysed.

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3. Results and discussion

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The UV-Vis. spectra of Pn were measured in presence of various amino acids in aqueous medium. Pn showed a characteristic UV-Vis absorbance band centered at 416

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nm. Upon addition of arginine, the band at 416 nm (ascribable to π –π* transition of Pn

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quinonoid moiety) undergoes a significant decrease in intensity, in addition a new band appears at 521 nm with a color change from yellow to pink which is attributed to the

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formation of Pn-Arg complex. (Figure 1) Among the various amino acids tested (Thr,

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His, Lys, Met, Asp, Arg, Ser, Glu, Phe and Cys), only Arg responded to probe Pn. The

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addition of other amino acids, in contrast, had no effect on the color or absorption spectrum. Thus, it is clear that the sensor probe Pn responds selectively to Arg, a response which can be attributed to its intermolecular proton transfer. (Fig. 1)

As the arginine concentration increases from 2.0 × 10-5 mol L−1 to 1.0 × 10-4

mol L−1, the absorbance at 416 nm decreases with a concomitant increase in the absorbance at 521nm (Figure 2). A ratiometric response and a clear isosbestic point was observed at 465 nm which demonstrated the existence of a well-defined stoichiometric 6

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complex. (Fig. 2) To determine whether the specificity in the detection of arginine was

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compromised or not in the presence of other amino acids, the color change of Pn with

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arginine was also studied in other binary systems involving other amino acids. The results

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indicate that the color of Pn changed from yellow to pink only when arginine was added to the mixture and did not undergo any change in the binary mixture. Thus, it is clear that

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Pn has high selectivity only for arginine. This result also indicates that the detection of arginine can be performed in solution even in a mixture of other amino acids. The

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corresponding absorption response in the selectivity of Pn (1.0 x 10-4 mol L−1) for arg in

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the presence of other amino acids (1.0 x 10-4 mol L−1) is shown in figure 3.

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(Fig. 3)

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In addition to absorbance analysis, the remarkable selectivity of Pn towards arginine over other amine acids was also studied by fluorescence spectra. Here too, arginine could be discriminated from other amino acids. While other amino acids produced negligible influence on the fluorescence of Pn at a concentration of 1.0 × 10-4 mol L−1, arginine showed significant quenching in the fluorescence as shown in figure 3. The fluorescence quenching of plumbagin by arginine is ascribed to suppression of intramolecular proton transfer in the presence arginine (Scheme 1) which is otherwise significant in the absence of arginine. When arginine is added intermolecular proton transfer occurs, thus causing a decrease in the former pathway. The intensities at 612 and 7

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662 nms decreased linearly with an increase in the concentration of Arg (figure 4a & 4b), which indicate that the present probe Pn has good potential use for the quantitative determination of Arg and the observed detection limit (DL) is 1.0 x 10-6 mol L−1.

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Fluorescence response is found to be linearly proportional to the concentration of Arg in

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the range of 1.0 x 10-6 to 1.0 x 10-4 mol L-1 and the value of linearly dependent coefficient

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(Fig. 4)

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(R2) is found to be 0.9946 as shown in Figure 5.

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(Fig. 5)

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Binding between Pn and Arg is also evident from Job’s plot and ESI-MS data.

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The formation of a 1:1 complex between the Pn and Arg, is in good agreement with 1:1

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stoichiometry determined by Job’s plot from UV-Vis. absorption data (Figure 6). (Fig. 6)

The interaction of probe Pn with Arg is further evidenced by ESI mass spectrum shown in figure 7. Initially peaks at m/z 175 and 206 (Figure S3 in supporting information) values are observed for (Arg + H+) and (Pn + NH4+) adduct respectively. (Actual mass for Arg and Pn are 174 and 188). When 1.0 equiv. of arginine is added, a new peak appears at m/z 379.58 corresponding to [ Pn + Arg + NH4+]. (Fig. 7) 8

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A plausible mechanism for the sensing of arginine is given in Scheme 1. In the absence of arginine, excited state intramolecular proton transfer (ESIPT) occurs leading

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to resonating structures I and II. When arginine is added, intermolecular proton transfer

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from Pn to arginine occurs (Scheme 1) and this suppresses the excited state intramolecular proton transfer. The proposed mechanism finds strong support from the

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observation of an intense peak at m/z. 379.58 in ESI-MS (Figure 7), which corresponds

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to (Pn + Arg + NH4+).

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(Scheme 1) Influence of pH

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Absorption spectra of (Pn and Pn + Arg ) are investigated in the pH range 2-12

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(Figure 8). The absorption intensity at A521 remains constant in the pH range 2-8 and

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increases rapidly above pH 9 with the color changing from yellow to pink due to the deprotonation of phenolic -OH. This clearly shows that Pn can be used effectively for detection of arginine at a pH range 2-8. In this range, when Arg(1 X 10-4 mol L-1) is added to the probe Pn, the color changes from yellow to pink and absorption intensity at A521 remains constant, and this is attributed to intermolecular proton transfer from Pn to arginine (Scheme 1)

(Fig. 8)

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Reproducibility To determine the reproducibility of the present sensor system, each sample was measured

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three times at three different concentrations (1 X10-6 -1 X 10-4 mol L-1) which are shown in Table 1. For each sample, the change in fluorescence intensity is very less (≤ ± 1.0 CPS/Micro

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Amps) indicating that the reproducibility of the present sensor system is excellent.

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Response time and the stability of the sensor

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(Table 1)

Pn shows an instant color change from yellow to pink upon addition of 1.0×10-4

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mol L-1 of Arg solution, Dependence of fluorescence intensity (Fig. 3d) of Pn upon addition of arginine is shown in (Figure. 9) as a function of time which shows that the

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intensity decreases rapidly and remains constant above 10s. This indicates clearly that this probe not only provides good response for the rapid measurement of arginine and

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also the sensor system is stable for a long time. (Fig. 9)

Theoretical calculations

The geometries of plumbagin and its complex with arginine were optimized using DFT-B3LYP 6-31G and LANL2DZ (d) level respectively using Gaussian 03 package. As shown in Figure 10, optimized geometry of the sensor probe plumbagin shows effective binding sites to form a 1:1 complex with arginine and this supports the experimental finding obtained from Job’s plot and ESI-MS analysis of the complex. This is also 10

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evident from a comparison of the oscillator strength (F) of the various transitions given in Table 2.

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(Fig. 10)

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(Table 2)

In free Pn the contribution of HOMO to LUMO is higher (Table 1, entry 1) and

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other transitions are absent (Table 1, entry 2 and 3). After binding of arginine to Pn the

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magnitude of HOMO to LUMO transition has decreased significantly from 0.0751 to 0.0002, 0.0006 (Table 1, entries 1 and 4) indicating clearly that suppression of excited which can account readily the fluorescence

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state intramolecular proton transfer

quenching by intermolecular proton transfer from Pn to arginine.

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To get an insight into the electronic behavior in the presence and absence of

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arginine with plumbagin, TD-DFT calculations were carried out using the same level.

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Plotting of HOMO and LUMO of plumbagin (Figure 11) shows that phenyl group behaves as a HOMO, whereas quinonoid group behaves as a LUMO. After the binding of arginine in plumbagin (Pn + Arg), phenyl group has the HOMO character, but the whole π-moiety is represented as LUMO which is shown in figure 10. It clearly shows the relatively well-separated charge distribution between HOMO and LUMO indicating substantial charge transfer from the phenyl group to the quinonoid ring when the molecule is excited. (Fig. 11)

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The significant advantages in term of sensitivity, detection limit of the developed sensor over other reported approaches are shown in the table 3. Table 3 Comparison of various probes employed for arginine sensing.

Rhodamine-thiourea/Al3+ complex

2 3

Luminol Dextran Conjugate

Assemblies-Cu

Lys, Arg

te

and His

Lys, Arg

modified gold nanoparticles

and His

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p-Sulfonatocalix[4]arene thiol

Calix[4]arene Crown Ether

7

Calix[4]arenes modified with carboxylic acid groups

8

C1- or C2-symmetrical host molecules based on a spirobisindane skeleton

9

Fluorescence

d

Quercetin-functionalized gold nanoparticles

6

Arg

Crown ether carboxylic acids

Lys and Arg

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[11]

UV-Vis.

2.5 X10-6 M

[12]

UV-Vis.

1 X10-6 M

[13]

1 X10-7 M

[14]

1 X10-3 M

[15]

NMR

1 X10-3 M

[16]

Luminescence

----

[17]

Surface Plasmon Resonance

Arg

al

Arg

[9]

1.7 X10-5 M

Fluorescence

Electrochemic

Arg

1.2 X10-6 M

[10]

Lys and

Lys and

Reference

5 X10-6 M

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2+

5

Fluorescence

Arg

Limit for Arg

Arg

Ternary System Based on Fluorophore-Surfactant

4

Method

for

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1

Sensing Probe

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No

Detection

Sensor

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S.

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10

Zn(II)-terpyridine complex

Arg

Fluorescence

2.06 X10-6 M

Arg

Fluorescence

1 X10-6 M

11

Present work

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Plumbagin

[18]

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4. Conclusion

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We report herein Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) Pn, as a selective colorimetric and ratiometric sensing probe for arginine in aqueous solution.

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This naked eye colorimetric sensing is insensitive to other amino acids namely Asp, Glu,

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Thr, Lys, His, Cys, Met, Ser and Phe. In the absence of arginine, excited state intramolecular proton transfer (ESIPT) occurs leading to resonating structures I and II.

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When arginine is added, intermolecular proton transfer from Pn to arginine takes place

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(Scheme 1) and this suppresses the excited state intramolecular proton transfer. The

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proposed mechanism finds strong support from the observation of an intense peak at m/z. 379.58 in ESI-MS which corresponds to (Pn + Arg + NH4+), Job’s plot and also from DFT calculations.

Acknowledgements

S. S. P gratefully acknowledges the financial assistance from UGC, New Delhi for UPE project fellowship, A. A gratefully acknowledges the financial assistance from USRF University Stipendiary Research Fellowship (USRF), MKU, Tamil Nadu, India. K. P thanks DST, New Delhi for financial support. 13

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References [1] S. K. Sandur, H. Ichikawa, G. Sethi, K. S. Ahn, B. B. Aggarwal, Plumbagin (5hydroxy-2-methyl-1,4-naphthoquinone) suppresses NF-kappaB activation and NF-

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kappaB-regulated gene products through modulation of p65 and IkappaBalpha kinase activation, leading to potentiation of apoptosis induced by cytokine and

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chemotherapeutic agents, J. Biol. Chem., 281 (2006) 17023-33.

[2] R. Parimala, P. Sachdanandam, Effect of Plumbagin on some glucose matabolising

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enzymes studied in rats in experimental hepatoma, Mol. Cell Biochem., 125 (1993) 59-63.

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[3] R. A. Naresh, N. Udupa, P. U. Devi, Niosomal plumbagin with reduced toxicity and improved anticancer activity in BALB/C mice, J. Pharm. Pharmacol., 48 (1996)

M

1128-32.

[4] S. Sugie, K. Okamoto, K. M. Rahman, T. Tanaka, K. Kawai, J. Yamahara, H. Mori,

d

Inhibitory effects of plumbagin and juglone on azoxymethane-induced intestinal

te

carcinogenesis in rats, Cancer Lett., 27 (1998) 177-83. [5] B. Hazra, R. Sarkar, S. Bhattacharyya, P. K. Ghosh, G. Chel, B. Dinda, Synthesis of

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plumbagin derivatives and their inhibitory activities against Ehrlich ascites carcinoma in vivo and Leishmania donovani Promastigotes in vitro, Phytother. Res., 16 (2002) 133-7. [6]

H. Tapiero, G. Mathe, P. Couvreur, K. D. Tew, I. Arginine,Biomedicine and Pharmacotherapy, 56 (2002) 439-45.

[7] R. Stanislavov, V. Nikolova, Treatment of erectile dysfunction with pycnogenol and L-arginine, Journal of Sex and Marital Therapy, 29 (2003) 207-13.

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[8] T. Lebret, J. M. Hervea, P. Gornyb, M. Worcelc, H. Botto, Efficacy and safety of a novel combination of L-arginine glutamate and yohimbine hydrochloride: a new oral therapy for erectile dysfunction, European Urology, 41 (2002) 608-13. L. He, V. L. L. So, J. H. Xin, A new rhodamine-thiourea/Al3+ complex

ip t

[9]

cr

sensor for the fast visual detection of arginine in aqueous media, Sensors and

us

Actuators B, 192 (2014) 496–502.

[10] W. Nasomphan, P. Tangboriboonrat, S. Smanmoo, Selective Sensing of L-

an

Arginine Employing Luminol Dextran Conjugate, Macromolecular Research, 20 (2012) 344-346.

M

[11] J. Cao, L. Ding, W. Hu, X. Chen, X. Chen, Y. Fang, Ternary System Based on Fluorophore-Surfactant Assemblies-Cu2+ for Highly Sensitive and Selective

te

d

Detection of Arginine in Aqueous Solution, Langmuir, 30 (2014) 15364−15372. [12] K. A. Rawat, S. K. Kailasa, Visual detection of arginine, histidine and lysine

Ac ce p

using quercetin-functionalized gold nanoparticles, Microchim Acta, 181 (2014) 1917–1929.

[13] G. Patel and S. Menon, Recognition of lysine, arginine and histidine by novel psulfonatocalix[4]arene thiol functionalized gold nanoparticles in aqueous solution, Chem. Commun., (2009) 3563-65. [14] H. Chen, L. Gu, Y. Yin, K. Koh, J. Lee, Molecular Recognition of Arginine by Supramolecular Complexation with Calixarene Crown Ether Based on Surface Plasmon Resonance, Int. J. Mol. Sci., 12 (2011) 2315-24. 15

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[15] W. M. Hassen, C. Martelet, F. Davis, S. P.J. Higson, A. Abdelghani, S. Helali, N. J. Renault, Calix[4]arene based molecules for amino-acid detection,

Sensors and

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Actuators B, 124 (2007) 38-45.

for Arginine and Lysine, Org. Lett., 5 (2000) 605-08.

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[16] M. Wehner, T. Schrader , P. Finocchiaro , S. Failla , G. Consiglio, A Chiral Sensor

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[17] L. Jiang, K. Tang, X. Ding, Q. Wang, Z. Zhou, R. Xiao, A Chiral Sensor for Arginine and Lysine, Materials Science and Engineering: C, 33 (2013) 5090-94.

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[18] X. Zhou, X. Jin, D. Li and X. Wu, Selective detection of zwitterionic arginine with a

M

new Zn(II)-terpyridine complex: potential application in protein labeling and

Ac ce p

te

d

determination, Chem. Commun., 47 (2011) 3921–3923.

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Biographies Sheik Dawood Shahida Parveen (1979) received her B.Sc. (Chemistry) degree from Fatima

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College (Madurai Kamaraj University), Madurai in 1999, M.Sc. (Chemistry) from Madura College (Madurai Kamaraj University), Madurai, India in 2001. At present she is a research

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scholar in Madurai Kamaraj University, Madurai, India. Her research interests are isolation of

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phytochemicals, nanoformulation, DNA binding and cleavage studies and sensors.

Abdullah Affrose (1985) received her B.Sc., (Chemistry) degree from M. S. S. Wakf board

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college (Madurai Kamaraj University), Madurai in 2006, M.Sc., (Chemistry) from Saraswathi Narayanan College (Madurai Kamaraj University), Madurai, India in 2008, M. Phil., (Chemistry)

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from Madurai Kamaraj University, Madurai, India in 2009 and doing Ph.D., in the same

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university. Her research interests are studies on naturally occurring secondary metabolites.

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Kasi Pitchumani (1954) received his MSc (Chemistry) from Madurai Kamaraj University,

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Madurai, India. He has received Ph. D., and D. Sc., degrees from the same university in 1981 and was appointed as Professor in Organic Chemistry from 1996 to till present. He did his postdoctoral research with Prof. V. Ramamurthy, University of Miami, USA and Prof. Akihiko Ueno, Tokyo Institute of Technology, Japan. He has 32 year of teaching experience in Organic Chemistry and published 172 research articles in peer reviewed journals. His research interests are supramolecular photochemistry and chemistry in confined media like clays, zeolites, hydrotalcites and cyclodextrins. He is also involved in synthesis of modified cyclodextrins, isolation of natural products and newer nanomaterials for developing sensor applications.

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Figure 1 Effect of addition of different amino acids, namely Thr, His, Lys, Met, Asp, Arg, Ser, Glu, Phe and Cys on the UV-Vis. spectra of probe Pn (λmax= 416 nm)(1.0 x 10-4

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mol L−1). Probe Pn+arg (λmax= 521nm).

Figure 2 (a) Ratiometric response of Pn, 1 x 10-4 mol L−1 at different concentrations of

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Arg in aqueous medium (2 x 10-5 mol L−1 to 1 x 10-4 mol L−1). b) Plot of absorption intensity variation (in triplicate) of Pn with the change in [Pn + Arg] (2 x 10-5 mol L−1 to

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1 x 10-4 mol L−1). Inset: Absorption titration curve in [Pn + Arg] (4 x 10-5 mol L−1 to 1 x

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10-4 mol L−1)

Figure 3 Bar chart illustrating absorption response in the selectivity of Pn (1.0 x 10-4

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mol L−1) for arg in the presence of other amino acids (1.0 x 10-4 mol L−1). The violet bars represent the absorption intensity of Pn in the presence of one equivalent of the other amino acids. The brown bars represent the change in absorption intensity that

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and the other amino acids.

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occurs upon subsequent addition of one equivalent of arg to the solution containing Pn

Figure 4 a) Emission spectra of Pn (1.0 x 10-4 mol L−1) in the presence of various amino acids (1.0 x 10-4 mol L−1= His, Leu, Met, Thr, Asp, Ser, Glu, Phe and Cys). b) Fluorescence response of Pn (1.0 x 10-4 mol L-1) upon addition of arg (1.0 x 10-6 mol L-1 to 1.0 x 10-4 mol L-1) in aqueous medium; b) Fluorescence emission spectra of Pn (1.0 x 10-4 mol L-1) upon addition of Arg (1.0 x 10-6 mol L-1 to 1.0 x 10-4 mol L-1.) in aqueous medium (λex = 430 nm, λem = 615 and 662 nm, slit width: 5 nm/5 nm).

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Figure 5 Linear plot indicating the fluorescence intensity change (in triplicate) of probe Pn with concentration of arg(1.0 x 10-6 to 1.0 x 10-4 mol L-1).

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and arg was kept constant at arg 1.0 equiv in aqueous medium.

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Figure 6 Job’s plot of the complexation between Pn and arg, total concentration of Pn

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Scheme 1 Mechanism of sensing of arginine by Pn

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Figure 7 ESI-Mass spectrum of Pn with arg (Pn + Arg + NH4+).

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Figure 8 Plot of absorption intensity of Pn and Pn+Arg with variation of pH (from 2 to

d

12)

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Table 1 Reproducibility of the sensor probe Pn.

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Figure 9 Time-dependent fluorescence intensity of Pn with arginine in aqueous solution (λex = 290 nm, λem = 471 nm).

Figure 10 Optimized geometry of sensor probe plumbagin and its 1:1 complexes with arginine

Table 2 Oscillator strength values of Pn and Pn / arg complex calculated from Gaussian software.

Figure 11 Frontier molecular orbitals optimized at the B3LYP/LANL2DZ (d) level of theory. 19

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Lys

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Thr

Met

Asp

Arg

Ser

Glu

Phe

Cys

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te

d

Pn

Figure 1 Effect of addition of different amino acids, namely Thr, His, Lys, Met, Asp, Arg, Ser, Glu, Phe and Cys on the UV-Vis. spectra of probe Pn (λmax= 416 nm)(1.0 x 10-4 mol L−1). Probe Pn+arg (λmax= 521nm).

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cr

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a)

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d

b)

Figure 2 (a) Ratiometric response of Pn, 1 x 10-4 mol L−1 at different concentrations of Arg in aqueous medium (2 x 10-5 mol L−1 to 1 x 10-4 mol L−1). b) Plot of absorption intensity variation (in triplicate) of Pn with the change in [Pn + Arg] (2 x 10-5 mol L−1 to 1 x 10-4 mol L−1). Inset: Absorption titration curve in [Pn + Arg] (4 x 10-5 mol L−1 to 1 x 10-4 mol L−1) 21

Page 23 of 69

ip t cr us an

Figure 3 Bar chart illustrating absorption response in the selectivity of Pn (1.0 x 10-4

M

mol L−1) for Arg in the presence of other amino acids (1.0 x 10-4 mol L−1). The violet bars represent the absorption intensity of Pn in the presence of one equivalent of the

d

other amino acids. The brown bars represent the change in absorption intensity that

Ac ce p

and the other amino acids.

te

occurs upon subsequent addition of one equivalent of Arg to the solution containing Pn

22

Page 24 of 69

a

an

us

cr

ip t

b

Figure 4 a) Emission spectra of Pn (1.0 x 10-4 mol L−1) in the presence of various amino

M

acids (1.0 x 10-4 mol L−1= His, Leu, Met, Thr, Asp, Ser, Glu, Phe and Cys). b) Fluorescence response of Pn (1.0 x 10-4 mol L-1) upon addition of Arg (1.0 x 10-6 mol L-1

d

to 1.0 x 10-4 mol L-1) in aqueous medium; b) Fluorescence emission spectra of Pn (1.0 x 10-4 mol L-1) upon addition of Arg (1.0 x 10-6 mol L-1 to 1.0 x 10-4 mol L-1.) in aqueous

Ac ce p

te

medium (λex = 430 nm, λem = 615 and 662 nm, slit width: 5 nm/5 nm).

23

Page 25 of 69

y = -95.73x + 0.9707 2

an

us

cr

ip t

R = 0.9946

M

Figure 5 Linear plot indicating the fluorescence intensity change (in triplicate) of probe

Ac ce p

te

d

Pn with concentration of Arg(1.0 x 10-6 to 1.0 x 10-4 mol L-1).

24

Page 26 of 69

ip t cr us an

Figure 6 Job’s plot of the complexation between Pn and Arg, total concentration of Pn

Ac ce p

te

d

M

and Arg was kept constant at arg 1.0 equiv in aqueous medium.

25

Page 27 of 69

an

us

cr

ip t

(Pn + Arg + NH4+)

Ac ce p

te

d

M

Figure 7 ESI-Mass spectrum of Pn with arg (Pn + Arg + NH4+).

26

Page 28 of 69

ip t cr us

Ac ce p

te

d

M

an

Scheme 1 Mechanism of sensing of arginine by Pn

27

Page 29 of 69

ip t cr us an M d te Ac ce p

Figure 8 Plot of absorption intensity of Pn and Pn+Arg with variation of pH (from 2 to 12)

28

Page 30 of 69

ip t cr us an M d te

Ac ce p

Figure 9 Time-dependent fluorescence intensity of Pn with arginine in aqueous solution (λex = 290 nm, λem = 471 nm).

29

Page 31 of 69

[Arg] mol L-1

Fluorescence intensity S1c /

Average

Reproducibility

≤ ± 1.0

97865, 97866, 97864

97865.67

1 X 10-5

84227, 84227, 84229

84227.67

1 X 10-4

65432, 65433, 65432

65432.67

≤ ± 1.0

cr

1 X 10-6

ip t

R1(CPS/MicroAmps)

us

Ac ce p

te

d

M

an

Table 1 Reproducibility of the sensor probe Pn

≤ ± 0.5

30

Page 32 of 69

ip t cr

Plumbagin

us

Plumbagin + Arg

Ac ce p

te

d

M

an

Figure 10 Optimized geometry of sensor probe plumbagin and its 1:1 complexes with arginine

31

Page 33 of 69

Transition

Oscillator strength(F)

1

H

0.0751

H-1

L

an

3

L

H

M

4 Pn / Arg complex

H

0.0000 0.0000

L

0.0002, 0.0006

L+2

0.0009

te

d

5

cr

H-2

us

Free Pn

2

L

ip t

Entry

Table 2 Oscillator strength values of Pn and Pn / Arg complex calculated from Gaussian

Ac ce p

software.

32

Page 34 of 69

us

cr

ip t

Plumbagin

an

Plumbagin + Arg

LUMO

M

HOMO

Figure 11 Frontier molecular orbitals optimized at the B3LYP/LANL2DZ (d) level of

Ac ce p

te

d

theory

33

Page 35 of 69

*Manuscript Click here to view linked References

Plumbagin as Colorimetric and Ratiometric Sensor for Arginine Sheik Dawood Shahida Parveen,a Abdullah Affrose, a Kasi Pitchumania,b* a

School of Chemistry, Madurai Kamaraj University, Madurai - 625021, India b

Centre for Green Chemistry Processes, School of Chemistry,

Ac ce p

te

d

M

an

us

cr

ip t

Madurai Kamaraj University, Madurai – 625021, India

*Corresponding author. Tel.: +91 452 2456614; fax: +91 452 2459181. E-mail address: [email protected] (K. Pitchumani) 1

Page 36 of 69

Abstract A simple and selective naphthoquinone based fluorescent probe involving

ip t

Plumbagin (Pn =1,4-naphthoquinone) which is a naturally occurring phytochemical is reported for sensing of arginine in aqueous medium. The sensing probe Pn upon binding

cr

to Arg, induced a visible color change from light yellow to pink. The 1:1 Pn/Arg

us

complex was characterized by Job’s plot, ESI-MS and DFT calculations. This naked eye colorimetric sensing is insensitive to other amino acids namely Asp, Glu, Thr, Lys, His,

an

Cys, Met, Ser and Phe. In the absence of arginine, excited state intramolecular proton transfer (ESIPT) occurs and when arginine is added, intermolecular proton transfer from

Ac ce p

te

d

M

Pn to arginine takes place resulting in colorimetric and ratiometric response.

Keywords: Plumbagin, Colorimetric and ratiometric sensor, Arginine, Intramolecular proton transfer, DFT calculations.

2

Page 37 of 69

1. Introduction Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a quinonoid constituent

ip t

isolated from the roots of Plumbago zeylanica L., is an anti-cancer agent, which is partly mediated by the inactivation of NF-kB-regulated anti-apoptotic, proliferative and

cr

angiogenic gene products [1]. Potential role of plumbagin, as an anti-cancer agent has

us

been recognized [2-5] and its anti-cancer effects have been reported in diverse cancer models such as prostate, lung, cervical, ovarian as well as the melanoma.

an

Naturally occurring α-amino acids are of special interest because of their

M

biological prominence. The recognition and sensing of amino acids and their derivatives have been investigated in both metal containing and pure organic systems. Arginine plays

d

an important role in cell division, the healing of wounds, removing ammonia from the

te

body, immune function, and the release of hormones [6]. Arginine taken in combination

Ac ce p

with proanthocyanidins [7] or yohimbine [8], has also been used in the treatment for erectile dysfunction. The benefits and functions attributed to oral supplementation of L-arginine include quickening of repair time of damaged tissue and helping decrease blood pressure. Excess L-arginine can increase levels of stomach acid, particularly gastrine and some people experience anaphylaxis, or an allergic reaction, to L-arginine. Because of L-arginine's properties as a vasodilator, low blood pressure can be a side effect of supplementation. It can increase the body's production of potassium, chloride, creatinine, blood urea nitrogen, sodium and phosphate levels. Those suffering

3

Page 38 of 69

from kidney or liver problems are especially susceptible to changes in these chemical balances.

ip t

The effective and selective molecular recognition or sensing of unprotected amino acids in aqueous solution is a challenging problem due to their highly hydrophilic

cr

character. Chemical or biological sensing via colorimetric naked eye detection is an

us

inexpensive technique and is useful for amino acid recognition owing to their simplicity, high selectivity and sensitivity. And selective detection of a specific amino acid without

an

interference from other amino acids is a challenging task. Inspite of its significant

M

biological relevance, only few sensors are available for the detection of arginine. Some of the recent reports involve bis-rhodamine-thiourea/Al3+ complex for the fast visual

d

detection of arginine [9], luminol dextran conjugate through ICT process [10], a ternary

te

system based on fluorophore-surfactant assemblies-Cu2+ by electrostatic interaction

Ac ce p

between anionic surfactants and positively charged amino acids [11] and quercetinfunctionalized gold nanoparticles through aggregation of QCAuNPs [12]. The aim of the present study is to develop a simple system for detection of arginine in aqueous solution using naturally occurring molecules. With this goal in mind, for the first time we have used Pn, a natural naphthoquinone, as a sensing probe for the selective detection of arginine. This probe selectively showed a rapid color change from yellow to pink in presence of arginine.

4

Page 39 of 69

Materials and methods UV-Visible absorption spectra were recorded by using the JASCO-Spectra

temperature.

Fluorescence

measurements

were

made

ip t

Manager (V-550) in 1 cm path length quartz cuvette with a volume of 2 mL at room on

a

Fluoromax-4

cr

Spectrofluorometer (HORIBA JOBIN YVON) with excitation slit set at 2.0 nm bandpass

us

and emission at 4.0 nm bandpass in 1 cm x 1 cm quartz cell. Electrospray ionisation mass spectrometry (ESI-MS) analysis was performed in the positive ion mode on a liquid

an

chromatography-ion trap mass spectrometer (LCQ Fleet, Thermo Fisher Instruments

M

Limited, US). The samples were introduced into the ion source by in fusion method at flow rate 1µL/min. The capillary voltage of the mass spectrometer was 33V, with source

d

voltage 4.98kV for the mass scale (m/z150–2000). The geometries of plumbagin and its

te

complex with arginine were optimized using DFT-B3LYP 6-31G and LANL2DZ (d)

Ac ce p

levels respectively using Gaussian 03 package. 2.1. Isolation of plumbagin from Plumbago Zeylanica Plumbagin was isolated from the CHCl3 extract of Plumbago Zeylanica roots using Soxhlet method and characterised by NMR, FT-IR and ESI-MS techniques. (Figures S1-S4 in supporting information) 2.2.

Preparation of stock solution and UV titration

All the measurements are carried out in double distilled water which is free from ions. The stock solution of plumbagin (1 x 10-3 M) is prepared by dissolving 0.0188 g of 5

Page 40 of 69

plumbagin in 100 mL ethanol:water (v/v 1:1) mixture. Solutions (1x10-3M) of His, Lys, Met, Thr, Asp, Arg, Ser, Glu, Phe and Cys are prepared by dissolving them in water. For UV titration, plumbagin (1 mL of stock), amino acids (0.1-1.0 mL of stock) are taken in

ip t

10 mL SMF and analysed.

cr

3. Results and discussion

us

The UV-Vis. spectra of Pn were measured in presence of various amino acids in aqueous medium. Pn showed a characteristic UV-Vis absorbance band centered at 416

an

nm. Upon addition of arginine, the band at 416 nm (ascribable to π –π* transition of Pn

M

quinonoid moiety) undergoes a significant decrease in intensity, in addition a new band appears at 521 nm with a color change from yellow to pink which is attributed to the

d

formation of Pn-Arg complex. (Figure 1) Among the various amino acids tested (Thr,

te

His, Lys, Met, Asp, Arg, Ser, Glu, Phe and Cys), only Arg responded to probe Pn. The

Ac ce p

addition of other amino acids, in contrast, had no effect on the color or absorption spectrum. Thus, it is clear that the sensor probe Pn responds selectively to Arg, a response which can be attributed to its intermolecular proton transfer. (Fig. 1)

As the arginine concentration increases from 2.0 × 10-5 mol L−1 to 1.0 × 10-4

mol L−1, the absorbance at 416 nm decreases with a concomitant increase in the absorbance at 521nm (Figure 2). A ratiometric response and a clear isosbestic point was observed at 465 nm which demonstrated the existence of a well-defined stoichiometric 6

Page 41 of 69

complex. (Fig. 2) To determine whether the specificity in the detection of arginine was

ip t

compromised or not in the presence of other amino acids, the color change of Pn with

cr

arginine was also studied in other binary systems involving other amino acids. The results

us

indicate that the color of Pn changed from yellow to pink only when arginine was added to the mixture and did not undergo any change in the binary mixture. Thus, it is clear that

an

Pn has high selectivity only for arginine. This result also indicates that the detection of arginine can be performed in solution even in a mixture of other amino acids. The

M

corresponding absorption response in the selectivity of Pn (1.0 x 10-4 mol L−1) for arg in

d

the presence of other amino acids (1.0 x 10-4 mol L−1) is shown in figure 3.

te

(Fig. 3)

Ac ce p

In addition to absorbance analysis, the remarkable selectivity of Pn towards arginine over other amine acids was also studied by fluorescence spectra. Here too, arginine could be discriminated from other amino acids. While other amino acids produced negligible influence on the fluorescence of Pn at a concentration of 1.0 × 10-4 mol L−1, arginine showed significant quenching in the fluorescence as shown in figure 3. The fluorescence quenching of plumbagin by arginine is ascribed to suppression of intramolecular proton transfer in the presence arginine (Scheme 1) which is otherwise significant in the absence of arginine. When arginine is added intermolecular proton transfer occurs, thus causing a decrease in the former pathway. The intensities at 612 and 7

Page 42 of 69

662 nms decreased linearly with an increase in the concentration of Arg (figure 4a & 4b), which indicate that the present probe Pn has good potential use for the quantitative determination of Arg and the observed detection limit (DL) is 1.0 x 10-6 mol L−1.

ip t

Fluorescence response is found to be linearly proportional to the concentration of Arg in

cr

the range of 1.0 x 10-6 to 1.0 x 10-4 mol L-1 and the value of linearly dependent coefficient

an

(Fig. 4)

us

(R2) is found to be 0.9946 as shown in Figure 5.

M

(Fig. 5)

d

Binding between Pn and Arg is also evident from Job’s plot and ESI-MS data.

te

The formation of a 1:1 complex between the Pn and Arg, is in good agreement with 1:1

Ac ce p

stoichiometry determined by Job’s plot from UV-Vis. absorption data (Figure 6). (Fig. 6)

The interaction of probe Pn with Arg is further evidenced by ESI mass spectrum shown in figure 7. Initially peaks at m/z 175 and 206 (Figure S3 in supporting information) values are observed for (Arg + H+) and (Pn + NH4+) adduct respectively. (Actual mass for Arg and Pn are 174 and 188). When 1.0 equiv. of arginine is added, a new peak appears at m/z 379.58 corresponding to [ Pn + Arg + NH4+]. (Fig. 7) 8

Page 43 of 69

A plausible mechanism for the sensing of arginine is given in Scheme 1. In the absence of arginine, excited state intramolecular proton transfer (ESIPT) occurs leading

ip t

to resonating structures I and II. When arginine is added, intermolecular proton transfer

cr

from Pn to arginine occurs (Scheme 1) and this suppresses the excited state intramolecular proton transfer. The proposed mechanism finds strong support from the

us

observation of an intense peak at m/z. 379.58 in ESI-MS (Figure 7), which corresponds

an

to (Pn + Arg + NH4+).

M

(Scheme 1) Influence of pH

d

Absorption spectra of (Pn and Pn + Arg ) are investigated in the pH range 2-12

te

(Figure 8). The absorption intensity at A521 remains constant in the pH range 2-8 and

Ac ce p

increases rapidly above pH 9 with the color changing from yellow to pink due to the deprotonation of phenolic -OH. This clearly shows that Pn can be used effectively for detection of arginine at a pH range 2-8. In this range, when Arg(1 X 10-4 mol L-1) is added to the probe Pn, the color changes from yellow to pink and absorption intensity at A521 remains constant, and this is attributed to intermolecular proton transfer from Pn to arginine (Scheme 1)

(Fig. 8)

9

Page 44 of 69

Reproducibility To determine the reproducibility of the present sensor system, each sample was measured

ip t

three times at three different concentrations (1 X10-6 -1 X 10-4 mol L-1) which are shown in Table 1. For each sample, the change in fluorescence intensity is very less (≤ ± 1.0 CPS/Micro

cr

Amps) indicating that the reproducibility of the present sensor system is excellent.

an

Response time and the stability of the sensor

us

(Table 1)

Pn shows an instant color change from yellow to pink upon addition of 1.0×10-4

M

mol L-1 of Arg solution, Dependence of fluorescence intensity (Fig. 3d) of Pn upon addition of arginine is shown in (Figure. 9) as a function of time which shows that the

te

d

intensity decreases rapidly and remains constant above 10s. This indicates clearly that this probe not only provides good response for the rapid measurement of arginine and

Ac ce p

also the sensor system is stable for a long time. (Fig. 9)

Theoretical calculations

The geometries of plumbagin and its complex with arginine were optimized using DFT-B3LYP 6-31G and LANL2DZ (d) level respectively using Gaussian 03 package. As shown in Figure 10, optimized geometry of the sensor probe plumbagin shows effective binding sites to form a 1:1 complex with arginine and this supports the experimental finding obtained from Job’s plot and ESI-MS analysis of the complex. This is also 10

Page 45 of 69

evident from a comparison of the oscillator strength (F) of the various transitions given in Table 2.

ip t

(Fig. 10)

cr

(Table 2)

In free Pn the contribution of HOMO to LUMO is higher (Table 1, entry 1) and

us

other transitions are absent (Table 1, entry 2 and 3). After binding of arginine to Pn the

an

magnitude of HOMO to LUMO transition has decreased significantly from 0.0751 to 0.0002, 0.0006 (Table 1, entries 1 and 4) indicating clearly that suppression of excited which can account readily the fluorescence

M

state intramolecular proton transfer

quenching by intermolecular proton transfer from Pn to arginine.

d

To get an insight into the electronic behavior in the presence and absence of

te

arginine with plumbagin, TD-DFT calculations were carried out using the same level.

Ac ce p

Plotting of HOMO and LUMO of plumbagin (Figure 11) shows that phenyl group behaves as a HOMO, whereas quinonoid group behaves as a LUMO. After the binding of arginine in plumbagin (Pn + Arg), phenyl group has the HOMO character, but the whole π-moiety is represented as LUMO which is shown in figure 10. It clearly shows the relatively well-separated charge distribution between HOMO and LUMO indicating substantial charge transfer from the phenyl group to the quinonoid ring when the molecule is excited. (Fig. 11)

11

Page 46 of 69

The significant advantages in term of sensitivity, detection limit of the developed sensor over other reported approaches are shown in the table 3. Table 3 Comparison of various probes employed for arginine sensing.

Rhodamine-thiourea/Al3+ complex

2 3

Luminol Dextran Conjugate

Assemblies-Cu

Lys, Arg

te

and His

Lys, Arg

modified gold nanoparticles

and His

Ac ce p

p-Sulfonatocalix[4]arene thiol

Calix[4]arene Crown Ether

7

Calix[4]arenes modified with carboxylic acid groups

8

C1- or C2-symmetrical host molecules based on a spirobisindane skeleton

9

Fluorescence

d

Quercetin-functionalized gold nanoparticles

6

Arg

Crown ether carboxylic acids

Lys and Arg

ip t

[11]

UV-Vis.

2.5 X10-6 M

[12]

UV-Vis.

1 X10-6 M

[13]

1 X10-7 M

[14]

1 X10-3 M

[15]

NMR

1 X10-3 M

[16]

Luminescence

----

[17]

Surface Plasmon Resonance

Arg

al

Arg

[9]

1.7 X10-5 M

Fluorescence

Electrochemic

Arg

1.2 X10-6 M

[10]

Lys and

Lys and

Reference

5 X10-6 M

M

2+

5

Fluorescence

Arg

Limit for Arg

Arg

Ternary System Based on Fluorophore-Surfactant

4

Method

for

us

1

Sensing Probe

an

No

Detection

Sensor

cr

S.

12

Page 47 of 69

10

Zn(II)-terpyridine complex

Arg

Fluorescence

2.06 X10-6 M

Arg

Fluorescence

1 X10-6 M

11

Present work

ip t

Plumbagin

[18]

cr

4. Conclusion

us

We report herein Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) Pn, as a selective colorimetric and ratiometric sensing probe for arginine in aqueous solution.

an

This naked eye colorimetric sensing is insensitive to other amino acids namely Asp, Glu,

M

Thr, Lys, His, Cys, Met, Ser and Phe. In the absence of arginine, excited state intramolecular proton transfer (ESIPT) occurs leading to resonating structures I and II.

d

When arginine is added, intermolecular proton transfer from Pn to arginine takes place

te

(Scheme 1) and this suppresses the excited state intramolecular proton transfer. The

Ac ce p

proposed mechanism finds strong support from the observation of an intense peak at m/z. 379.58 in ESI-MS which corresponds to (Pn + Arg + NH4+), Job’s plot and also from DFT calculations.

Acknowledgements

S. S. P gratefully acknowledges the financial assistance from UGC, New Delhi for UPE project fellowship, A. A gratefully acknowledges the financial assistance from USRF University Stipendiary Research Fellowship (USRF), MKU, Tamil Nadu, India. K. P thanks DST, New Delhi for financial support. 13

Page 48 of 69

References [1] S. K. Sandur, H. Ichikawa, G. Sethi, K. S. Ahn, B. B. Aggarwal, Plumbagin (5hydroxy-2-methyl-1,4-naphthoquinone) suppresses NF-kappaB activation and NF-

ip t

kappaB-regulated gene products through modulation of p65 and IkappaBalpha kinase activation, leading to potentiation of apoptosis induced by cytokine and

cr

chemotherapeutic agents, J. Biol. Chem., 281 (2006) 17023-33.

[2] R. Parimala, P. Sachdanandam, Effect of Plumbagin on some glucose matabolising

us

enzymes studied in rats in experimental hepatoma, Mol. Cell Biochem., 125 (1993) 59-63.

an

[3] R. A. Naresh, N. Udupa, P. U. Devi, Niosomal plumbagin with reduced toxicity and improved anticancer activity in BALB/C mice, J. Pharm. Pharmacol., 48 (1996)

M

1128-32.

[4] S. Sugie, K. Okamoto, K. M. Rahman, T. Tanaka, K. Kawai, J. Yamahara, H. Mori,

d

Inhibitory effects of plumbagin and juglone on azoxymethane-induced intestinal

te

carcinogenesis in rats, Cancer Lett., 27 (1998) 177-83. [5] B. Hazra, R. Sarkar, S. Bhattacharyya, P. K. Ghosh, G. Chel, B. Dinda, Synthesis of

Ac ce p

plumbagin derivatives and their inhibitory activities against Ehrlich ascites carcinoma in vivo and Leishmania donovani Promastigotes in vitro, Phytother. Res., 16 (2002) 133-7. [6]

H. Tapiero, G. Mathe, P. Couvreur, K. D. Tew, I. Arginine,Biomedicine and Pharmacotherapy, 56 (2002) 439-45.

[7] R. Stanislavov, V. Nikolova, Treatment of erectile dysfunction with pycnogenol and L-arginine, Journal of Sex and Marital Therapy, 29 (2003) 207-13.

14

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[8] T. Lebret, J. M. Hervea, P. Gornyb, M. Worcelc, H. Botto, Efficacy and safety of a novel combination of L-arginine glutamate and yohimbine hydrochloride: a new oral therapy for erectile dysfunction, European Urology, 41 (2002) 608-13. L. He, V. L. L. So, J. H. Xin, A new rhodamine-thiourea/Al3+ complex

ip t

[9]

cr

sensor for the fast visual detection of arginine in aqueous media, Sensors and

us

Actuators B, 192 (2014) 496–502.

[10] W. Nasomphan, P. Tangboriboonrat, S. Smanmoo, Selective Sensing of L-

an

Arginine Employing Luminol Dextran Conjugate, Macromolecular Research, 20 (2012) 344-346.

M

[11] J. Cao, L. Ding, W. Hu, X. Chen, X. Chen, Y. Fang, Ternary System Based on Fluorophore-Surfactant Assemblies-Cu2+ for Highly Sensitive and Selective

te

d

Detection of Arginine in Aqueous Solution, Langmuir, 30 (2014) 15364−15372. [12] K. A. Rawat, S. K. Kailasa, Visual detection of arginine, histidine and lysine

Ac ce p

using quercetin-functionalized gold nanoparticles, Microchim Acta, 181 (2014) 1917–1929.

[13] G. Patel and S. Menon, Recognition of lysine, arginine and histidine by novel psulfonatocalix[4]arene thiol functionalized gold nanoparticles in aqueous solution, Chem. Commun., (2009) 3563-65. [14] H. Chen, L. Gu, Y. Yin, K. Koh, J. Lee, Molecular Recognition of Arginine by Supramolecular Complexation with Calixarene Crown Ether Based on Surface Plasmon Resonance, Int. J. Mol. Sci., 12 (2011) 2315-24. 15

Page 50 of 69

[15] W. M. Hassen, C. Martelet, F. Davis, S. P.J. Higson, A. Abdelghani, S. Helali, N. J. Renault, Calix[4]arene based molecules for amino-acid detection,

Sensors and

ip t

Actuators B, 124 (2007) 38-45.

for Arginine and Lysine, Org. Lett., 5 (2000) 605-08.

cr

[16] M. Wehner, T. Schrader , P. Finocchiaro , S. Failla , G. Consiglio, A Chiral Sensor

us

[17] L. Jiang, K. Tang, X. Ding, Q. Wang, Z. Zhou, R. Xiao, A Chiral Sensor for Arginine and Lysine, Materials Science and Engineering: C, 33 (2013) 5090-94.

an

[18] X. Zhou, X. Jin, D. Li and X. Wu, Selective detection of zwitterionic arginine with a

M

new Zn(II)-terpyridine complex: potential application in protein labeling and

Ac ce p

te

d

determination, Chem. Commun., 47 (2011) 3921–3923.

16

Page 51 of 69

Biographies Sheik Dawood Shahida Parveen (1979) received her B.Sc. (Chemistry) degree from Fatima

ip t

College (Madurai Kamaraj University), Madurai in 1999, M.Sc. (Chemistry) from Madura College (Madurai Kamaraj University), Madurai, India in 2001. At present she is a research

cr

scholar in Madurai Kamaraj University, Madurai, India. Her research interests are isolation of

us

phytochemicals, nanoformulation, DNA binding and cleavage studies and sensors.

Abdullah Affrose (1985) received her B.Sc., (Chemistry) degree from M. S. S. Wakf board

an

college (Madurai Kamaraj University), Madurai in 2006, M.Sc., (Chemistry) from Saraswathi Narayanan College (Madurai Kamaraj University), Madurai, India in 2008, M. Phil., (Chemistry)

M

from Madurai Kamaraj University, Madurai, India in 2009 and doing Ph.D., in the same

d

university. Her research interests are studies on naturally occurring secondary metabolites.

te

Kasi Pitchumani (1954) received his MSc (Chemistry) from Madurai Kamaraj University,

Ac ce p

Madurai, India. He has received Ph. D., and D. Sc., degrees from the same university in 1981 and was appointed as Professor in Organic Chemistry from 1996 to till present. He did his postdoctoral research with Prof. V. Ramamurthy, University of Miami, USA and Prof. Akihiko Ueno, Tokyo Institute of Technology, Japan. He has 32 year of teaching experience in Organic Chemistry and published 172 research articles in peer reviewed journals. His research interests are supramolecular photochemistry and chemistry in confined media like clays, zeolites, hydrotalcites and cyclodextrins. He is also involved in synthesis of modified cyclodextrins, isolation of natural products and newer nanomaterials for developing sensor applications.

17

Page 52 of 69

Figure 1 Effect of addition of different amino acids, namely Thr, His, Lys, Met, Asp, Arg, Ser, Glu, Phe and Cys on the UV-Vis. spectra of probe Pn (λmax= 416 nm)(1.0 x 10-4

ip t

mol L−1). Probe Pn+arg (λmax= 521nm).

Figure 2 (a) Ratiometric response of Pn, 1 x 10-4 mol L−1 at different concentrations of

cr

Arg in aqueous medium (2 x 10-5 mol L−1 to 1 x 10-4 mol L−1). b) Plot of absorption intensity variation (in triplicate) of Pn with the change in [Pn + Arg] (2 x 10-5 mol L−1 to

us

1 x 10-4 mol L−1). Inset: Absorption titration curve in [Pn + Arg] (4 x 10-5 mol L−1 to 1 x

an

10-4 mol L−1)

Figure 3 Bar chart illustrating absorption response in the selectivity of Pn (1.0 x 10-4

M

mol L−1) for arg in the presence of other amino acids (1.0 x 10-4 mol L−1). The violet bars represent the absorption intensity of Pn in the presence of one equivalent of the other amino acids. The brown bars represent the change in absorption intensity that

te

Ac ce p

and the other amino acids.

d

occurs upon subsequent addition of one equivalent of arg to the solution containing Pn

Figure 4 a) Emission spectra of Pn (1.0 x 10-4 mol L−1) in the presence of various amino acids (1.0 x 10-4 mol L−1= His, Leu, Met, Thr, Asp, Ser, Glu, Phe and Cys). b) Fluorescence response of Pn (1.0 x 10-4 mol L-1) upon addition of arg (1.0 x 10-6 mol L-1 to 1.0 x 10-4 mol L-1) in aqueous medium; b) Fluorescence emission spectra of Pn (1.0 x 10-4 mol L-1) upon addition of Arg (1.0 x 10-6 mol L-1 to 1.0 x 10-4 mol L-1.) in aqueous medium (λex = 430 nm, λem = 615 and 662 nm, slit width: 5 nm/5 nm).

18

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Figure 5 Linear plot indicating the fluorescence intensity change (in triplicate) of probe Pn with concentration of arg(1.0 x 10-6 to 1.0 x 10-4 mol L-1).

cr

and arg was kept constant at arg 1.0 equiv in aqueous medium.

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Figure 6 Job’s plot of the complexation between Pn and arg, total concentration of Pn

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Scheme 1 Mechanism of sensing of arginine by Pn

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Figure 7 ESI-Mass spectrum of Pn with arg (Pn + Arg + NH4+).

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Figure 8 Plot of absorption intensity of Pn and Pn+Arg with variation of pH (from 2 to

d

12)

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Table 1 Reproducibility of the sensor probe Pn.

Ac ce p

Figure 9 Time-dependent fluorescence intensity of Pn with arginine in aqueous solution (λex = 290 nm, λem = 471 nm).

Figure 10 Optimized geometry of sensor probe plumbagin and its 1:1 complexes with arginine

Table 2 Oscillator strength values of Pn and Pn / arg complex calculated from Gaussian software.

Figure 11 Frontier molecular orbitals optimized at the B3LYP/LANL2DZ (d) level of theory. 19

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ip t cr us an His

Lys

M

Thr

Met

Asp

Arg

Ser

Glu

Phe

Cys

Ac ce p

te

d

Pn

Figure 1 Effect of addition of different amino acids, namely Thr, His, Lys, Met, Asp, Arg, Ser, Glu, Phe and Cys on the UV-Vis. spectra of probe Pn (λmax= 416 nm)(1.0 x 10-4 mol L−1). Probe Pn+arg (λmax= 521nm).

20

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M

an

us

cr

ip t

a)

Ac ce p

te

d

b)

Figure 2 (a) Ratiometric response of Pn, 1 x 10-4 mol L−1 at different concentrations of Arg in aqueous medium (2 x 10-5 mol L−1 to 1 x 10-4 mol L−1). b) Plot of absorption intensity variation (in triplicate) of Pn with the change in [Pn + Arg] (2 x 10-5 mol L−1 to 1 x 10-4 mol L−1). Inset: Absorption titration curve in [Pn + Arg] (4 x 10-5 mol L−1 to 1 x 10-4 mol L−1) 21

Page 56 of 69

ip t cr us an

Figure 3 Bar chart illustrating absorption response in the selectivity of Pn (1.0 x 10-4

M

mol L−1) for Arg in the presence of other amino acids (1.0 x 10-4 mol L−1). The violet bars represent the absorption intensity of Pn in the presence of one equivalent of the

d

other amino acids. The brown bars represent the change in absorption intensity that

Ac ce p

and the other amino acids.

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occurs upon subsequent addition of one equivalent of Arg to the solution containing Pn

22

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a

an

us

cr

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b

Figure 4 a) Emission spectra of Pn (1.0 x 10-4 mol L−1) in the presence of various amino

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acids (1.0 x 10-4 mol L−1= His, Leu, Met, Thr, Asp, Ser, Glu, Phe and Cys). b) Fluorescence response of Pn (1.0 x 10-4 mol L-1) upon addition of Arg (1.0 x 10-6 mol L-1

d

to 1.0 x 10-4 mol L-1) in aqueous medium; b) Fluorescence emission spectra of Pn (1.0 x 10-4 mol L-1) upon addition of Arg (1.0 x 10-6 mol L-1 to 1.0 x 10-4 mol L-1.) in aqueous

Ac ce p

te

medium (λex = 430 nm, λem = 615 and 662 nm, slit width: 5 nm/5 nm).

23

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y = -95.73x + 0.9707 2

an

us

cr

ip t

R = 0.9946

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Figure 5 Linear plot indicating the fluorescence intensity change (in triplicate) of probe

Ac ce p

te

d

Pn with concentration of Arg(1.0 x 10-6 to 1.0 x 10-4 mol L-1).

24

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ip t cr us an

Figure 6 Job’s plot of the complexation between Pn and Arg, total concentration of Pn

Ac ce p

te

d

M

and Arg was kept constant at arg 1.0 equiv in aqueous medium.

25

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an

us

cr

ip t

(Pn + Arg + NH4+)

Ac ce p

te

d

M

Figure 7 ESI-Mass spectrum of Pn with arg (Pn + Arg + NH4+).

26

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ip t cr us

Ac ce p

te

d

M

an

Scheme 1 Mechanism of sensing of arginine by Pn

27

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ip t cr us an M d te Ac ce p

Figure 8 Plot of absorption intensity of Pn and Pn+Arg with variation of pH (from 2 to 12)

28

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ip t cr us an M d te

Ac ce p

Figure 9 Time-dependent fluorescence intensity of Pn with arginine in aqueous solution (λex = 290 nm, λem = 471 nm).

29

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[Arg] mol L-1

Fluorescence intensity S1c /

Average

Reproducibility

≤ ± 1.0

97865, 97866, 97864

97865.67

1 X 10-5

84227, 84227, 84229

84227.67

1 X 10-4

65432, 65433, 65432

65432.67

≤ ± 1.0

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1 X 10-6

ip t

R1(CPS/MicroAmps)

us

Ac ce p

te

d

M

an

Table 1 Reproducibility of the sensor probe Pn

≤ ± 0.5

30

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ip t cr

Plumbagin

us

Plumbagin + Arg

Ac ce p

te

d

M

an

Figure 10 Optimized geometry of sensor probe plumbagin and its 1:1 complexes with arginine

31

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Transition

Oscillator strength(F)

1

H

0.0751

H-1

L

an

3

L

H

M

4 Pn / Arg complex

H

0.0000 0.0000

L

0.0002, 0.0006

L+2

0.0009

te

d

5

cr

H-2

us

Free Pn

2

L

ip t

Entry

Table 2 Oscillator strength values of Pn and Pn / Arg complex calculated from Gaussian

Ac ce p

software.

32

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us

cr

ip t

Plumbagin

an

Plumbagin + Arg

LUMO

M

HOMO

Figure 11 Frontier molecular orbitals optimized at the B3LYP/LANL2DZ (d) level of

Ac ce p

te

d

theory

33

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Author Biographies

Biographies Sheik Dawood Shahida Parveen (1979) received her B.Sc. (Chemistry) degree from Fatima College (Madurai Kamaraj University), Madurai in 1999, M.Sc. (Chemistry) from Madura College (Madurai Kamaraj University), Madurai, India in 2001. At present she is a research

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scholar in Madurai Kamaraj University, Madurai, India. Her research interests are isolation of

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phytochemicals, nanoformulation, DNA binding and cleavage studies and sensors.

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Abdullah Affrose (1985) received her B.Sc., (Chemistry) degree from M. S. S. Wakf board college (Madurai Kamaraj University), Madurai in 2006, M.Sc., (Chemistry) from Saraswathi

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Narayanan College (Madurai Kamaraj University), Madurai, India in 2008, M. Phil., (Chemistry) from Madurai Kamaraj University, Madurai, India in 2009 and doing Ph.D., in

M

the same university. Her research interests are studies on naturally occurring secondary

d

metabolites.

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Kasi Pitchumani (1954) received his MSc (Chemistry) from Madurai Kamaraj University, Madurai, India. He has received Ph. D., from the same university in 1981 and was appointed

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as Professor in Organic Chemistry to till present. He did his postdoctoral research with Prof. V. Ramamurthy, University of Miami, USA and Prof. Akihiko Ueno, Tokyo Institute of

Ac

Technology, Japan. He has 32 year of teaching experience in Organic Chemistry and published 172 research articles in peer reviewed journals.

His research interests are

supramolecular photochemistry and chemistry in confined media like clays, zeolites, hydrotalcites and cyclodextrins. He is also involved in synthesis of modified cyclodextrins, isolation of natural products and newer nanomaterials for developing sensor applications.

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