Specific Reagent for Cr(III): Imaging Cellular Uptake of Cr(III) in Hct116

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Specific Reagent for Cr(III): Imaging Cellular Uptake of Cr(III) in Hct116 Cells and Theoretical Rationalization Firoj Ali, Sukdeb Saha, Arunava Maity, Nandaraj Taye, Mrinal Kanti Si, Eringathodi Suresh, Bishwajit Ganguly, Samit Chattopadhyay, and Amitava Das J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b07565 • Publication Date (Web): 21 Sep 2015 Downloaded from http://pubs.acs.org on September 24, 2015

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The Journal of Physical Chemistry

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Specific Reagent for Cr(III): Imaging Cellular

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Uptake of Cr(III) in Hct116 Cells and Theoretical

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Rationalization

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Firoj Ali,a Sukdeb Saha,b Arunava Maity,a Nandaraj Taye,c Mrinal Kanti Si,b E. Suresh,b

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Bishwajit Ganguly,*b Samit Chattopadhyay,*c Amitava Das*a

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a

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India, E-mail: [email protected]; Fax: +91 2025902629; Tel: +91 2025902385.

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b

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Research Institute, Bhavnagar, Gujarat: 364002, India.

Organic Chemistry Division, CSIR-National Chemical Laboratory, Pune - 411008, Maharashtra,

Analytical Science and Centralized Instrument Facility, CSIR-Central Salt & Marine Chemicals

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c

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mail: [email protected].

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RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to)

Chromatin and Disease Biology Lab, National Centre for Cell Science, Pune 411007, India, E

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KEYWORDS: Cr(III) sensor, aqueous medium, computational studies, cellular imaging reagent.

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ACS Paragon Plus Environment

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ABSTRACT:

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A new rhodamine-based reagent (L1), trapped inside the micellar structure of biologically benign

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Triton-X 100, could be used for specific recognition of Cr(III) in aqueous buffer medium having

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physiological pH. This visible light excitable reagent on selective binding to Cr(III) resulted a

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strong fluorescence turn-on response with a maximum at ~ 583 nm and tail of that luminescence

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band extended till 650 nm; an optical response that is desired for avoiding the cellular

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autofluorescence. Interference studies confirm that other metal ions do not interfere with the

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detection process of Cr(III) in aqueous buffer medium having pH 7.2. To examine the nature of

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binding of Cr(III) to L1 various spectroscopic studies are performed with the model reagent L2,

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which tend to support a Cr(III)-η2-olefin π-interactions involving two olefin bonds in molecular

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probe L1. Computational studies are also performed with another model reagent LM to examine

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the possibility of such a Cr(III)-η2-olefin π-interactions. Presumably, polar functional groups of

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the model reagent LM upon coordination to Cr(III)-centre, effectively reduce the formal charge

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on the metal ion and this is further substantiated by results of the theoretical studies. This

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assembly is found to be cell membrane permeable and shows insignificant toxicity towards live

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colon cancer cells (Hct116). Confocal laser scanning microscopic studies further revealed that

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the reagent L1 could be used as an imaging reagent for detection of cellular uptake of Cr(III) in

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pure aqueous buffer medium by Hct116 cells. Example of a specific reagent for paramagnetic

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Cr(III) with luminescence ON response is scanty in the contemporary literature. This ligand

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design helped us in achieving the turn on response by utilizing the conversion from spirolactam

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to an acyclic xanthene form on coordination to Cr(III).

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Introduction:

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Apart from its natural source, Chromium is also added to the soil through anthropogenic and

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various industrial activities.1-2 The most common form of Chromium that exist in soil, is Cr(III).

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Cr(III) is an important analyte for biological processes at cellular level.3 Cr(IV) is known to be

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reduced by intracellular reducing agents to the Cr(III), which binds DNA through guanine N7

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and the adjacent phosphate backbone.4 Studies also reveal that Cr(IV) to Cr(III) reduction takes

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place in certain wetland plants.5 It is argued that this Cr(VI) to Cr(III) reduction initially happens

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in the fine lateral roots, while the Cr(III) subsequently translocates to leaf tissues and gets

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localized there presumably in the form of oxalate salt. Cr(III) is believed to be less toxic than

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Cr(IV) for mammalians and for certain biological processes Cr(III) is used as a nutrient. Cr(III)

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containing glucose tolerance factor is believed to be an important co-factor of insulin and

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catalyses the glucose metabolism to enhance the peripheral actions of insulin. Cr(III) is also

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available in many common food stuff, vegetable and animal fats.6-7 Deficiency of Cr(III) may

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cause several chronic diseases such as diabetes, cardiovascular and nervous system disorders.8

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Though Cr(III) is known to be non-toxic, but high doses may have adverse influences on human

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physiology.9 Several researchers have expressed their reservations about the usefulness of Cr(III)

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as a long-term nutritional supplement for its possible genotoxic effects and the general notion is

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that all forms of chromium, including Cr(III), are to be considered as human carcinogens.10-12

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This is especially true as the risk to benefit ratio for usages of Cr(III) has not yet been adequately

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characterized.6-7 Solubility of Cr(III) compounds is generally less in aqueous medium and this

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accounts for its lower mobility and bioaccumulation.13 Thus, there is a pressing need to develop

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an efficient and non-cytotoxic molecular receptor that allows specific recognition and binding to

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Cr(III) with an associated optical responses.


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There exist only few receptors that discuss some specificity towards Cr(III) either in organic or

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in mixed aqueous-organic medium that are predominantly organic in nature.14-22 Few literature

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reports discuss about selective binding to Cr(III) with interference either from Fe(III) or Hg(II) in

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mixed solvent medium that is predominantly aqueous.18,23-26 These reports further suggest that

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masking of Hg2+ (as HgI42¯) could be achieved in the presence of an excess of I¯ and it allows

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the specific detection of Cr(III). There are only three previous reports that describe the use of

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reagents for the detection of Cr(III) in pure aqueous solution;23,27,28 one of them describes the

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interference by Fe3+. The most recent report reveals that a rhodamine derivative within a

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polymeric matrix could be utilized for specific detection of Cr(III) in pure aqueous medium and

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the hydrophobic micro-environment generated around the binding core of the receptor induces a

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favorable influence for the detection of Cr(III).28 However, such a reagent is not appropriate for

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use as an imaging reagent.

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More recently, use of various molecular aggregates like micelles and vesicles have gained

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significance as drug carriers and cellular markers or imaging reagents, which provides such

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drug/markers a direct access to the cytoplasm; while subsequent steps are then needed for their

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release into the cytoplasm.29 Earlier studies have also established that TX100 is actually

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biologically benign and is being widely use for improving the cell membrane permeability of

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various drugs and molecular probes.30-39 We have adopted this methodology to allow the new

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receptor L1 to be self-assembled inside the micellar structure of Triton X-100 (TX100) and this

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self-assembled molecular aggregate is found to be specific towards Cr(III) in an ensemble of all

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common alkali/alkaline earth/transition/lanthanide metal ions in pure aqueous buffer medium

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having pH 7.2. Interestingly, the specific binding of the reagent L1 to Cr(III) inside the micellar

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structure resulted a sharp luminescence ON response. For unambiguous assignment and


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understanding of the nature of the binding mode of L1 with Cr(III), studies with a model reagent

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L2 under identical experimental condition were also performed. Cr(III)-η2-olefin π-interactions

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involving two olefin bonds in L1 are proposed based on the results of various spectroscopic as

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well as computational studies.

(A)

(B)

(C)

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Scheme 1: (A) Methodology followed for synthesis of L1 and L2. (B) single-crystal X-ray structure of L1

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[Hydrogen atoms are omitted for clarity]; (C) proposed molecular structure for Cr3+.L1.

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Experimental Section:

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Materials and method: Rhodamine B, Ethylenediamine, 3-bromoprop-1-ene, 1-bromopropane,

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all metal perchlorate salts (e.g LiClO4, NaClO4, KClO4, CsClO4, Mg(ClO4)2, Ca(ClO4)2,

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Ba(ClO4)2, Sr(ClO4)2, Cu(ClO4)2, Zn(ClO4)2, Co(ClO4)2, Ni(ClO4)2, Cr(ClO4)3, Fe(ClO4)2,

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Cd(ClO4)2, Hg(ClO4)2, and Pb(ClO4)2) and lanthanide ions as nitrate salts were obtained from

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Sigma-Aldrich and were used as received. Et3N, Triton X-100, Tris Buffer, NaCl was procured

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from S.D. fine chemicals, India and was used as received. Solvents such as acetonitrile,

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chloroform were also purchased from S.D. Fine Chemicals, India and were used without further

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purification unless mentioned otherwise. Silica gel 100-200 mesh was used for column


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chromatography. Analytical thin layer chromatography was performed using silica Gel GF 254.

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HPLC grade water (Merck, India) was used for experiments and all spectral studies.

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Aminoethylene rhodamine B (L) was synthesized following a standard procedure.40-46 5.0 mM

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Tris-HCl aq. buffer solution was used for maintaining solution pH, unless mentioned otherwise.

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ESI-MS measurements were performed using a Micromass QTof- Micro instrument. FT-IR

107

spectra were recorded as KBr pellets using a Perkin Elmer Spectra GX 2000 spectrometer. 1H

108

and 13C NMR spectra were recorded on Bruker 500 MHz FT NMR (model: Avance-DPX 500).

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Electronic spectra were recorded with a Varian Cary 500 Scan UV-Vis-NIR Spectrophotometer,

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Isothermal Titration Calorimetry studies were performed in Microcal iTC200, while emission

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spectra were recorded using either Edinburgh Instrument Xe-900 Spectrofluorometer or PTI. For

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all spectroscopic studies in aqueous buffer medium as well as for studies with L1 self-assembled

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inside the micellar structure of TX100 was used, unless mentioned otherwise.

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X-ray crystallography: Single crystal of suitable dimension was chosen under an optical

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microscope and mounted on a glass fibre for data collection on a Bruker SMART APEX

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diffractometer equipped with CCD area detector at 150K. Intensity data for the crystal was

117

collected using graphite-monochromated MoKα (λ=0.71073 Å) radiation. The data integration

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and reduction was performed with SAINT software.47 Data was subjected

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absorption correction using SADABS.48 The structure was solved by direct methods using

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SHELXTL49 and was refined by full matrix least square procedures based on F2 using the

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program SHELXL-97.50 All non-hydrogen atoms were refined anisotropically till convergence

122

was reached. Hydrogen atoms, attached to the organic moieties, were either located from the

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difference of Fourier map or stereochemically fixed in the complex. Details of crystallographic

124

data for compound L1 are provided in supporting information section.


to empirical

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Synthesis of L1: Amino ethyl rhodamine B (400 mg, 0.83 mmol) was dissolved in 20 mL dry

126

chloroform. To this Et3N (3 mL) was added and the resulting solution was stirred under N2

127

atmosphere for 20 minutes. Then, 3-bromoprop-1-ene (530 µL, 5.91 mmol) was added and

128

reaction mixture was refluxed for 12h with constant stirring. Progress of the reaction was

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monitored by TLC and was continued till all reactants were consumed. The reaction mixture was

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allowed to attain the room temperature and 10 mL of water was added. Then organic layer was

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collected and dried over anhydrous Na2SO4. Crude product was obtained when solvent was

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removed under vacuum. It was finally purified by column chromatography using silica gel

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column and ethyl acetate-hexane (1:9, v/v) as eluent. This enabled us to isolate L1 in pure form

134

with 50% yield. 1H NMR (500 MHz, CDCl3, SiMe4, J (Hz), δ ppm): 7.8 (1H, d, J = 4.6, H18),

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7.32 (2H, m, H16, H17), 6.99 (1H, d, J = 4.4, H15), 6.35 (4H, t, J = 10, H4, H5, H9, H10), 6.16 (2H,

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d, J = 8.8, H2, H12), 5.56 (2H, td, J = 16.5, 6.5, H27, H24), 4.92 (4H, d, J = 18.5, H25, H28), 3.24

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(8H, q, J = 6.8, H29, H31, H33, H35), 3.18 (2H, t, J = 6.8, H22), 2.85 (4H, d, J = 6.2, H23, H26), 2.1

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(2H, t, J = 6.8, H21), 1.07 (12H, t, J = 6.8, H30, H32, H36, H34).

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SiMe4, δ ppm): 167.70, 153.38, 148.70, 135.13, 132.22, 131.54, 128.98, 127.97, 123.78, 122.63,

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117.53, 108.03, 105.58, 97.73, 64.85, 56.60, 50.46, 44.37, 37.75, 29.69, 12.60. IR (KBr):

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νmax/cm-1 = 1684, 1617. ESI-MS (+ve mode, m/z): 565.93 (M + H+), Calc. for C36H44N4O2 is

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564.76.

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L2 synthesis: Amino ethyl rhodamine B (200 mg, 0.41 mmol) was dissolved in 15 mL dry

144

chloroform. To this Et3N (500 µL) was added and the resulting solution was stirred for 20

145

minutes under N2 atmosphere. Then 1-bromopropane (120 µL, 1.35 mmol) was added and the

146

resulting reaction mixture was refluxed for 24h until all the starting materials were consumed

147

and this was monitored by TLC. After this reaction mixture was allowed to attain the room


13

C NMR (125 MHz, CDCl3,

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temperature, 10 mL of water was added. The organic layer, after drying over anhydrous Na2SO4,

149

was collected and followed by the removal of chloroform under vacuum to yield the crude

150

product. Column chromatography was performed using silica gel as stationary phase and 10%

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ethyl acetate in hexane as mobile phase for isolating L2 in pure form with 40% yield. 1H NMR

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(500 MHz, CDCl3, SiMe4, J (Hz), δ ppm): δ 7.81 (dd, 1H, J = 5.9, 2.6 , H18), 7.37 (dd, 2H, J =

153

5.6, 3.0, H16, H17), 7.05 – 7.00 (m, 1H, H15), 6.35 (s, 1H, H12), 6.33 (s, 1H, H2), 6.31 (d, 2H, J =

154

2.5, H5, H9), 6.19 (dd, 2H, J = 8.9, 2.6, H4, H10), 3.26 (q, 8H, J = 7.0, H29, H31, H33, H35), 3.09 (d,

155

2H, J = 5.8, H21), 2.15 (s, 6H, H22, H23, H26), 1.18 (d, 4H, J = 6.7, H24, H27), 1.09 (t, 12H, J = 7.0,

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H30, H32, H36, H34), 0.68 (t, 6H, J =6.8, H25, H28).13C NMR (125 MHz, CDCl3, SiMe4, δ ppm):

157

167.75, 153.77, 148.72, 148.25, 132.25, 129.18, 127.78, 123.93, 122.59, 108.48, 105.63, 97.94,

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64.95, 56.78, 50.96, 44.34, 37.79, 37.47, 20.27, 12.54, 11.82. IR (KBr): νmax/cm-1 = 1680. ESI-

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MS (+ve mode, m/z): 569.29 (M + H+), Calc. for C36H44N4O2 is 568.79. Elemental Analysis:

160

Calculated C 76.02, H 8.51, N 9.85; experimentally obtained C 76.20, H 8.50, N 9.88.

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General experimental procedure for UV-Vis and Fluorescence studies:

162

A solution of the perchlorate salts of the respective ion (Li+, Na+, K+, Cs+, Ca2+, Mg2+, Ba2+, Sr2+,

163

Fe2+, Ni2+, Co2+, Cu2+, Mn2+, Cd2+, Pb2+, Ba2+, Zn2+, Sr2+, Hg2+ and Cr3+) and nitrate salts of

164

lanthanides ions (Tb3+, Ho3+, Ce3+, Sm3+, Rb+, Pr3+, Eu3+, Gd3+, Nd3+, Dy3+, Tm3+, Er3+, Yb3+) in

165

pure aqueous buffer medium having pH 7.2 were used for all studies. The effective final

166

concentrations of all metal salts were maintained at 1.62 x 10-4 M. A stock solution of the

167

receptor L1 (6.9 x 10-4 M) was prepared in acetonitrile medium and 57 µL of this stock solution

168

was added to 2.5 ml of 0.4 mM TX100 in Tris-HCl aqueous buffer medium having solution pH

169

7.2 to make the effective ligand concentration of 1.59 x 10-5 M. The solution was used for all the

170

photophysical studies. Cr3+ stock solution (4.75 x 10-3 M) was prepared in pure aqueous medium


8

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171

and was used for all studies. Emission titrations were also performed as a function of [TX100]

172

(0.1 mM, 0.23 mM, 0.32 mM, 0.4 mM, 0.6 mM) in Tris-HCl buffer medium of pH 7.2 by

173

monitoring the increase in emission intensity (using λExt = 530 nm, λMon = 583 nm and slit width

174

2/2 nm) on binding of L1 to Cr3+ for optimizing the maximum enhancement of the emission

175

intensity. The relative fluorescence quantum yields (Φf) were estimated using equation 1 for

176

different concentration of TX100 (0.1 mM, 0.23 mM, 0.32 mM, 0.4 mM, 0.6 mM) in Tris-HCl

177

buffer medium (having solution pH of 7.2) and by using the Rhodamine B (Φf = 0.3 in aqueous

178

medium at RT) as a reference.

179

Φf

180

where, Φf′ was the absolute quantum yield for the rhodamine B and was used as reference; Isample

181

and Istd are the integrated emission intensities; Asample and Astd are the absorbances at the

182

excitation wavelength, and ηsample and ηstd are the respective refractive indices.

183

In order to optimize the optical responses of the reagent L1, self-assembled within the micellar

184

structure of TX100 in the aq. buffer medium (pH 7.2), emission intensities of L1 (1.59 x 10-5 M)

185

in presence of 5 mole equivalent excess of Cr(III) at 583 nm were recorded with varying

186

[TX100] (SI). Results clearly revealed that maximum response is achieved for [TX100] of 0.4

187

mM. This surfactant concentration was maintained for all our studies.

188

Computational Methodology:

189

The interaction of olefinic π-bonds in the model compound with the chromium ion was examined

190

with DFT M06-2X functional level of theory in water using polarizable continuum (PCM)

191

solvent model.51,52 The Cr3+ ion was treated with LANL2DZ53,54 basis set and the rest of the

192

atoms were treated with 6-31G* basis set.55 All structures were optimized in Gaussian 09

193

version.56 This interaction was modeled with the core unit around the Cr3+ ion. The optimized

= Φf′ (Isample/Istd)(Astd/Asample)( η2sample/η2std)


Eq. 1

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194

geometry shows that the Cr3+ ion is coordinated with two nitrogen atoms, one oxygen atom, two

195

olefinic π-bonds and a water molecule (Figure 4C(i)). The “atoms in molecules” AIM analysis

196

performed with the optimized geometry showed significant interaction of Cr3+ ion with these

197

ligand units.57 The AIM calculations were performed by using multiwfn software.57,58-60

198

Cell studies:

199

Materials: Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen), Fetal Bovine Serum

200

(FBS) (Invitrogen), Penicillin Streptomycin antibiotics (Invitrogen), Hct116 cell line (National

201

Centre

202

diphenyltetrazolium bromide) (Sigma), Iso-propanol (Fischer Scientific), Triton X 100 (USB

203

Corporation), and Paraformaldehyde (Sigma) were used as received.

204

Cell culture & Confocal laser scanning microscopic Imaging:

205

Hct116 cells were seeded on cover slips placed in 6 well plates using DMEM medium and

206

supplemented with 10% FBS and Penicillin Streptomycin antibiotics. After 24 hours of

207

incubation at 37ºC, cells were treated with L1 (15.9 µM) for 30 minutes. Cells were then washed

208

thrice with Phosphate Buffer Saline (1X PBS). The L1-stained colon cancer cells Hct116

209

incubated with Cr3+ (10 µM) for 30 min. Then these cultures were washed thoroughly and fixed

210

with 4% PFA for 20 minutes. After washing, cover slips were mounted onto glass slides using

211

Fluoroshield with DAPI (Sigma) mounting medium. Nail paint was used to seal the cover slips

212

mounted on the glass slides. Images were acquired in Olympus Fluoview Microscope (λExt/λEm =

213

530 nm/573 nm). 0.4 mM TX100 in aqueous Tris buffer solution (25 mM; pH 7.2) were used for

214

confocal imaging studies and this allowed reagent L1 to be trapped inside the micellar structure

215

of TX100 and this allowed us to perform all recognition and binding studies in pure aqueous

216

medium having a physiologically relevant pH of 7.2.

For

Cell

Science),

trypsin-EDTA,

MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-


10

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217

Results and Discussion:

218

Reported literature procedure was adopted for synthesis of the intermediate compound L.40-46

219

Methodologies used for synthesis of the receptor L1 and the model compound L2 are described in

220

scheme 1. Desired compound L1 and L2 were isolated in pure form after necessary workup and

221

were characterized by using various analytical/spectroscopic techniques. Analytical and

222

spectroscopic data confirmed the desired purity and these compounds were utilized for further

223

studies. Molecular structure for the receptor L1 was also confirmed by X-ray single crystal

224

structural analysis (Scheme 1B). Receptor L1 and L2, being present in cyclic spirolactam form,

225

did not show any absorption band beyond 380 nm in acetonitrile medium.40-46,61-62

226

Limited solubility of L1 in water restricted us from studying the preferential binding of this

227

reagent towards different metal ions in water. Further, the deleterious and high solvation

228

enthalpy of the target cation could also impose an effective energetic barrier to inhibit binding in

229

aqueous medium. More recently, it has been demonstrated that the micelles obtained from

230

appropriate surfactant not only provide the suitable hydrophobic environment for solubilizing

231

such lypophobic reagents in water, but also provides a favourable local hydrophobic

232

environment for an efficient receptor-cation binding.63-67 TX100 (CMC = 0.23 mM) was used as

233

a neutral surfactant for solubilizing the receptor L1 in aq. medium. Reagent L1 was found to form

234

water soluble self-assembled micellar structure with TX100 having average diameter of 8.2 nm

235

(SI). It is also reported that the micellar structure is expected to improve the cell membrane

236

permeability of such lypophobic reagent.67, 68 Typically, aqueous solution of Tris buffer (5 mM,

237

25 mM NaCl; pH 7.2) was used as solvent for the studies that contained 0.26 gL-1 of TX100 (0.4

238

mmol dm-3). This concentration for TX100 was optimized based on the results of the

239

fluorescence studies and is discussed latter.


11


240

Electronic and steady state luminescence spectra for L1 were recorded in the absence and

241

presence of various metal ions in aq. buffer medium of pH 7.2 (Figure 1). This clearly revealed

242

that spectra recorded for L1 and L1 + Mn+ (when Mn+ was all other metal ions, except for Cr(III))

243

did not show any change in absorption or emission spectra of L1 (ΦL1 = 0.003 with λExt = 530

244

nm). The complete absence of any absorption or emission band in the visible region of the

245

spectrum accounted for the colourless nature of its aqueous solution under the specific

246

experimental conditions.

248 249

(A)

1.5x105

Cr3+

(B)

Cr

Intensity

0.10

247 Absorbance

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Page 12 of 28

3+

1.0x105

0.05

L1, L1 + Mn+

L1, L1 + Mn+

5.0x104

250 251

0.0

0.00 525

252

600

675

Wavelength (nm)

550

600 650 Wavelength (nm)

700

253 254 255 256

Figure 1. Changes in (A) absorption and (B) emission spectra (λExt of 530 nm) of the receptor L1 (1.59 x10-5 M) in absence and presence of different metal ions (Mn+: 1.62 x 10-4M: Li+, Na+, K+, Cs+, Ca2+, Mg2+, Sr2+, Ba2+, Cr3+, Fe2+, Co3+, Ni2+, Cu2+, Zn2+, Hg2+, Cd2+ and Pb2+); All studies were performed in aq. solution of 0.4 mM TX100 and Tris buffer (5 mM, 25 mM NaCl; pH 7.2).

257

This also confirmed that L1 remained exclusively in the spirolactam form at pH 7.2. However,

258

sharp changes in the electronic and emission spectra were observed when respective spectrum

259

was recorded in presence of Cr(III). Figure 1 clearly reveals that among all these cations new

260

absorption and emission spectral bands at 562 nm and 583 nm (ΦCr3+.L1 = 0.022 with λExt = 530

261

nm), respectively, are evident only in the presence of Cr(III). This spirolactam structure for L1

262

was also confirmed by characteristic signal at 64.85 ppm for the tertiary C-atom in the 13C NMR

263

spectrum recorded in CDCl3 medium (SI).40-46,61,62 Emission spectra recorded for the receptor L1

264

in aq. medium at various pH revealed that the spirolactam form was stable within the pH range

265

5-12 (SI). Thus, the observed absorption and emission spectral responses in presence of Cr(III)


12

Page 13 of 28

were solely due to a specific binding of Cr(III) to L1, which accounted for the conversion of the

267

spirolactam form to an acyclic xanthene form of the rhodamine derivative.40-46,61,62 Further,

268

interference studies in presence of 10 mole excess of all other metal ions (SI) ensured that the

269

reagent L1 was specific towards Cr(III) even in an ensemble of all other common competing

270

cationic analytes.

0.06

60 40

(B) 5

8.0x10

20 3

3

3

4x10 5x10 6x10 7x10 3+

3

1/[Cr ]

0.04

5

4.0x10

1.23 x10-3M [Cr3+] 0.0 M

6.0x10

-4

4.0x10

-4

2.0x10

-4

0.0

Intensity

1.84 x10-3M 0.08 [Cr3+] 0.0 M

1/(A-A0)

(A)

1/(I-I0)

266

Absorbance

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


3x10

3

3

4x10

5x10 3+

3

6x10

3

1/[Cr ]

0.02 0.00 500

0.0

550 600 Wavelength (nm)

550

600 650 Wavelength(nm)

271 272 273 274 275

Figure 2. Change in (A) Uv-Vis and (B) emission (λExt of 530 nm) spectral pattern for L1 (1.59 x 10-5 M) in presence of varying [Cr3+] (0 - 1.84x10-3 M for Uv-Vis spectral titration and 0 - 1.23x10-3 M for emission spectral titration studies). Inset: Benesi-Hildebrand (B-H) plots of spectral titration. All studies were performed in aq. solution of 0.4 mM TX100 and Tris buffer (5 mM, 25 mM NaCl; pH 7.2).

276

Apart from the specific affinity of the reagent L1 towards Cr(III), higher solvation enthalpy of

277

the other probable competing cations could have also imposed an unfavourable energy barrier for

278

inhibiting the binding process. These influences eventually had contributed to the observed

279

specificity towards Cr(III). Results of the Job plot as well as the B-H plot confirmed a 1:1

280

binding stoichiometry (SI). Formation of Cr3+.L1 was also confirmed from the result of FAB Ms

281

study (SI). Affinity of L1 towards Cr(III) and the associated binding constant for the formation of

282

Cr3+.L1 in aq. buffer medium (pH = 7.2) was evaluated from the data obtained from B-H plots of

283

the systematic absorption (KaAbs = (3.2 ± 0.2).103 M-1) and emission (KaEms = (3.3 ± 0.2).103 M-1)

284

spectral titrations (Figure 2). Analogous absorbance and emission titrations performed with

285

Cr(ClO4)3 and L1 ([Cr(III)] = (0 - 1.93) x 10-4 M; [L1] = 1.59 x 10-5 M; without using TX 100) in


13


286

acetonitrile medium helped us in evaluating formation constant for Cr3+.L1 in acetonitrile: KaAbs

287

= (1.1 ± 0.03).106 M-1, KaEms = (1.0 ± 0.02).106 M-1 (using λExt: 530 nm and λMon: 583 nm for

288

emission titration). The binding affinity of L1 towards Cr3+ was also evaluated as KaITC = (1.6 ±

289

0.02).106 M-1 in acetonitrile medium at 25°C using ITC experiments (Figure 3). Comparison of

290

the binding constants evaluated in pure aq. buffer medium and in acetonitrile clearly revealed the

291

unfavourable energy barrier imposed by the deleterious solvation of Cr3+ in aqueous medium.

292

Thermodynamic parameters were obtained from ITC studies (∆G = -(8.48 ± 0.02) kcal mol-1, ∆H

293

= -(20.8 ± 0.4) kcal mol-1 and ∆S = -(41.4 ± 0.05) cal mol-1; all symbols are used following

294

standard terminology).

295

Time (min) 0

296

10

20

30

40

µcal/sec

0.00

297 298

-5.00

-10.00 0.0

299

kcal mol-1 of injectant

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

Page 14 of 28

300 301

-8.0 -16.0

302 0.0

303

0.5

1.0

Molar Ratio

304 305 306

Figure 3. Isothermal Titration Calorimetry (ITC) titration profile for the binding of Cr3+ to receptor L1 at 25°C in acetonitrile; Top plot: raw data for the sequential 2 µL injection of Cr3+ (1.2 x 10-3 M) into solution of L1 (2.0 x 10-4 M) and bottom plot of the heat evolved (kcal mol-1) of Cr3+ added.

307

The observed plot of the data from ITC experiment (Figure 3) also supported the 1:1 binding

308

stoichiometry.69,70 The higher -∆H value revealed that binding was exclusively driven by

309

enthalpy change; while small but negative entropy of binding supported the formation of the

310

adduct Cr3+.L1.


14

Page 15 of 28

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


311

Reversible binding of Cr(III) to receptor L1 was also established with the restoration of the

312

original absorption or emission spectra for L1 on treating the solution of Cr3+.L1 with excess

313

Na2EDTA (SI). EDTA2- is known to have a much higher affinity (pKa = 23.6 in aq. medium)

314

towards Cr(III) than L1 and thus leads to generation of free L1, which eventually underwent the

315

cyclization reaction restoring the original receptor L1 in its spirolactam form. This was evident

316

from the restoration of the spectra of L1 in aq. solution of 0.4 mM TX100 and Tris buffer (5 mM,

317

25 mM NaCl; pH 7.2) in the absence of any Cr(III) (SI).

318

Further, to examine the role of structural variation in L1 and L2 on the binding affinity towards

319

Cr(III), emission spectra for L2 were also recorded in absence and presence of various metal ions

320

(SI) that were used for scanning responses of the reagent L1 (Figure 1).

321

Spectroscopic studies under identical experimental condition (i.e solvent medium having 0.4 mM

322

Triton X-100 in Tris buffer, pH 7.2; SI) with the model reagent (L2; Scheme 1) and different

323

cations, like Hg2+, Fe3+, Cr3+, Pb2+, Cd2+, Ni2+, Zn2+, Cu2+, Ca2+, Na+, and K+ were carried out.

324

Results of such studies revealed that on substituting olefin group (of L1) with –C2H5 group (in

325

L2) (Scheme 1), the observed specificity towards Cr(III) was lost when L2 was used for studies

326

(SI). L2 was found to bind all metal ions like Hg(II), Fe(III), Cr(III), Pb(II) with associated

327

increase in absorbance and emission band intensity at 561 nm and 585 nm, respectively in 0.4

328

mM TX100 and Tris buffer medium (pH 7.2) (SI). Results of such studies revealed that the extent of

329

absorption and emission changes as well as the binding efficiency for L2 towards Cr(III) was

330

found to be much lower than that compared to L1. Respective binding constants for these ions

331

were evaluated: KaHg

332

KaPb

333

suggest that the appended olefin functionality/ies in the rhodamine derivative (L1) has a distinct

2+.L2

2+.L2

3+.L2

= 1.08 x 103 M-1; KaFe

3+.L2

= 5.80 x 102 M-1; KaCr

= 5.20 x 102 M-1;

= 2.10 x 102 M-1. A comparison of these results with those for reagent L1, tends to


15


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

Page 16 of 28

334

role in achieving the desired specificity for Cr(III). Our results clearly revealed that in absence of

335

appended olefin groups, seemingly analogous reagent L2 showed much lower binding affinity

336

towards Cr(III). Despite efforts, we failed to get suitable single crystal of Cr3+.L1 for structural

337

analysis.

338

FTIR spectra recorded for L1 and Cr3+.L1, revealed a distinct shift from 1617 cm-1 to 1587 cm-1

339

(∆Ѵ = 30 cm-1) for C=C stretching frequency (SI). Blue shift in absorption of vinyl group in the

340

FTIR spectrum on coordination to metal ion is reported earlier.71,72 This supported the

341

involvement of olefin functionality(ies) in coordination to Cr(III)-centre. A recent report reveals

342

that Cr(III) could form a high yield air stable isolable coordination complex with soft donor

343

ligand bis(2-picolyl)phenyl phosphine.73 This also support the possibility of Cr(III)-olefin

344

interaction in Cr3+.L1 complex for achieving the observed specificity of the reagent L1 towards

345

Cr(III).

346

To examine the feasibility of the proposed complex formation between Cr(III) and L1 (Scheme

347

IC), DFT calculations and “atoms in molecules” AIM analyses with a model compound (LM)

348

(Figure 4B(i) and 4C(i)) was performed. These model compounds were used to reduce the

349

computational time, however, the active site of the reagent L1 for binding to Cr(III) was retained.

350

Results of these studies revealed that the proposed Cr(III) interactions with olefinic π-bonds

351

could exist in such systems in aqueous medium. We have optimized two model geometries as

352

shown in Figure 4B(i) and 4C(i), which represent the interaction of Cr(III) with one (Figure

353

4B(i)) and two (Figure 4C(i)) olefin bond(s), respectively. The optimized geometry of 4B(i)

354

suggests that the Cr3+ ion is coordinated with two nitrogen atoms, one oxygen atom, one olefinic

355

π-bond of the ligand and a water molecule. The optimized geometry of 4C(i) showed the

356

participation of the two olefinic π-bonds. The “atoms in molecules” AIM analysis performed


16

Page 17 of 28

357

with the optimized geometry showed significant interaction of Cr3+ ion with probable

358

coordinating sites in both cases.58 (ii)

(B) (i)

(A)

(ii)

(C) (i)

(iii)

(H2O) 2.02

2.38

1.510

2.04

2.0

LM

2.12

3.61

359 360 361 362 363 364 365 366 367 368 369 370 371

Figure 4. (A) Molecular structure of the model compound (LM) that was used for computational studies; B(i) optimized structure with the possible binding sites of the model reagent to Cr(III) where one olefinic double bond is interacted; B(ii) contour plots of the Laplacian distributions 2ρ(r) in the plane containing the atom Cr and two carbon atoms in olefinic double bonds in model compound B(i); solid and dotted lines designate regions of local charge concentration and depletion, respectively; the bond paths are indicated by solid black lines, bond critical points are marked with a blue sphere; C(i) optimized structure with the possible binding sites of the model reagent to Cr(III) where two olefinic double bond is interacted; C(ii) and (iii) contour plots of the Laplacian distributions 2ρ(r) in the plane containing the atom Cr and two carbon atoms in olefinic double bonds showing bond critical path and bond critical point. C(ii) and C(iii) represents the interaction of two different π-bonds with Cr3+ ion in model compound C(i) [H- atoms are omitted for clarity; Green: Cr(III), Gray: C; Blue: N; Red: O; distances are shown in Å).

372

The NBO charge calculated for the Cr(III) centre in the Cr(III) complex with the LM (Figure

373

4B(i)) is (+1.295), which was much smaller than the bare Cr(III) ion. The polar functional groups

374

of the model ligand LM upon coordination with Cr(III) is expected to reduce the formal charge

375

on the metal ion, which is further exhibited with 1.110 on the metal center of the complex shown

376

in Figure 4C(i).

377 378

Table-1: Topological parameters calculated of B(ii), C(ii) and C(iii) (in au): electron density at C=C bond…Cr(III) bond critical point (BCP) and Laplacian, 2 (r) are given.

∆

∆

∆

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

Interacted bond

ρ(r)

B(ii)

0.03758

0.12930

C(ii)

0.03213

0.11784

C(iii)

0.02119

0.08227


∇

ρ(r)

17


379

The Laplacian is obtained using this equation of ¼ [∇2ρ(r)] = 2G(r)+V(r).59 The interaction

380

between C=C π-bond and Cr(III) ion is a closed shell interaction as the Laplacian calculated to

381

be positive in these cases. Laplacian value of B(ii) is relatively larger than C(ii) and C(iii),

382

however, the cumulative effect in the later case override the stability than the former case.

383

The calculated results with DFT M06-2X functional level of theory using basis set 6-31G* (C,

384

N, H, O) and LANL2DZ (Cr) in aqueous phase suggest that the structure shown in 4C(i) is

385

energetically more stable than 4B(i) by 4.9 kcal mol-1. The experimental results corroborate the

386

calculated results. The AIM calculations were performed by using Multiwfn software, a

387

multifunctional program for wave function analysis.58-60 The AIM analysis indicates the T-

388

shaped paths of maximum electron density, the so called bond paths, which connect the Cr(III)-

389

ion with the bond critical point of C=C bonds. The contour plots of the Laplacian distributions

390

2

391

Imaging Studies:

∆

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

Page 18 of 28

ρ(r) also reveals the interaction of olefinic π-bonds with Cr(III) ion (Table 1).60

392

After ensuring the fact that reagent L1 binds specifically to Cr(III) in an ensemble of several

393

other interfering metal ions under physiological condition, possibility of using this molecule as

394

an imaging reagent for detection of cellular uptake of Cr(III) by human colon cancer cells

395

(Hct116) was examined. However, prior to such an experiment, toxicity of this reagent towards

396

live Hct116 cells was tested using MTT assay technique. MTT assay confirmed insignificant

397

toxicity of the reagent L1 towards Hct116 cells (ESI). This reagent L1 was further used for the

398

detection of Cr3+ in Hct116 cells by confocal laser scanning microscopic (CLSM) studies using

399

laser excitation source of 530 nm. Hct116 cells were incubated with L1 (15.9 µM) for 30 min at

400

37 οC.


18

Page 19 of 28

(B)

(C)

(D)

(E)

(F)

(G)

(H)

(ii) (A)

(B)

(C)

(D)

Intensity

(i) (A)

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


401 402 403 404 405 406 407 408 409

Figure 5. (i) CLSM image of Hct116 cells treated with probe L1 (15.9 µM) for 30 min at 37 °C: (A) the bright field image, (B) red channel image, (c) overlay of (A) and (B); CLSM Images of the cells upon treatment with probe L1 (15.9 µM) and then Cr3+ (10 µM) for 30 min at 37 °C: (E) bright field image, (F) red channel image, (G) overlay images of (E) and (F); (D) 3D representation of CLSM images of L1 (15.9 µM), (H) 3D representation of CLSM images of L1+10 µM of Cr3+, (λExt/λEm = 530/573 nm). (ii) CLSM images of Hct116 cells (pretreated with 10 µM of Cr3+) stained with 15.9 µM of L1, ER tracker green and DAPI: CLSM image (A) with DAPI, (J) with ER-Tracker green, (K) with L1+10 µM of Cr3+ and (D) overlay image of image I, J and C.

410

After necessary washing, no intracellular fluorescence was observed in the confocal images of

411

these cells and these were used as control. Further, pre-treated Hct116 cells (with 15.9 µM

412

reagent L1) were washed thoroughly and subjected to a follow-up treatment with Cr3+ (10 µM)

413

showed strong intracellular fluorescence when viewed through the red channel of the confocal

414

microscope (Figure 5). Images shown in Figure 5 also reveal that intracellular fluorescence is

415

preferentially localized in the lipid dense region of the cells. This was further confirmed from

416

CLSM images recorded after colocalization studies with ER-tracker green (ER-specific reagent)

417

and DAPI (It stains nuclei specifically, with little or no cytoplasmic labeling) (Figure 5(ii)),

418

which confirmed that the reagent L1 was preferentially localized in the lipid dense region of

419

Hct116 cells. This was further substantiated by the high value for the overlap coefficient (0.931)

420

of L1 (15.9 µM) in presence of 10 µM of Cr3+ and ER-tracker green. Thus, our studies confirmed

421

that the biologically benign reagent L1 could permeate through the cell membrane and was


19


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

Page 20 of 28

422

preferentially localized in the lipid dense region of the cells. Further, this reagent could detect the

423

cellular uptake of Cr3+ in Hct116 cells.

424

Conclusion:

425

In brief, we had designed and synthesized a new molecular probe L1, which was found to be

426

self-assembled inside the micellar structure of TX100 in aqueous medium. This self-assembled

427

molecular aggregate could be used as a reagent for specific detection of Cr(III) in pure aqueous

428

buffer medium (pH 7.2) with an associated fluorescence turn-on response even in presence of

429

several other competing cations. Interestingly, this reagent can be easily up-taken by cells via

430

permeation across cell membrane of human colon cancer cells (Hct116). Further, results of the

431

MTT assay studies confirmed that the reagent L1 showed insignificant toxicity towards human

432

colon cancer cells (Hct116). This reagent could detect the Cr(III) ion uptake in Hct116 cells from

433

aqueous environment and showed a luminescence ON response on binding to Cr(III) ions. Use of

434

the rhodamine based reagent with conversion from non-luminescent spirolactam form to the

435

strongly luminescent acyclic form on binding to Cr(III) had helped us in achieving the

436

luminescence ON response. Most importantly the reagent L1 could be utilized as an imaging

437

reagent for the detection of Cr3+ in Hct116 colon cancer cells. To the best of our knowledge, this

438

is a rare example of a chemosensor that is completely specific towards Cr(III) and works is pure

439

aqueous medium with an associated fluorescence turn on response. Example of the luminescence

440

ON based receptor for Cr(III) is not common due to the paramagnetic nature of Cr(III), which is

441

known to efficiently quench the luminescence of the organic fluorophore to which it is bound.

442

ASSOCIATED CONTENT

443

AUTHOR INFORMATION


20

Page 21 of 28

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444

Corresponding Author

445

* E-mail: [email protected]; [email protected]; [email protected].

446

Acknowledgement: A.D. acknowledges SERB (India) Grant (SB/S1/IC-23/2013) and CSIR-

447

National Chemical Laboratory Grant MLP 028226 for funding. Authors (AD & BG) also

448

acknowledge financial support through project MSM, CSC-0129. F.A., A.M., and N.T

449

acknowledge CSIR for their research fellowships. M.K.S acknowledges UGC research grant

450

funding.

451

Supporting Information Available (SI): Characterization data of L1 and L2; spectral details

452

Crystal structure has been deposited at the Cambridge Crystallographic Data Centre and

453

allocated the deposition number: CCDC 961378.This material is available free of charge via the

454

Internet at http:// pubs.acs.org.

455

References:

456 457

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458 459

2 Nriagu, J. O.; Nieboer, E. Chromium in the Natural and Human Environments; Wiley, New York,

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3 Latva, S.; Jokiniemi, J.; Peraniemi, S.; Ahlgren, M. Separation of Picogram Quantities of Cr(III) and

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4 Arakawa, H.; Ahmad, R.; Naoui, M.; Ali, H.; Riahi, T. A Comparative Study of Calf Thymus DNA

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6 Sundaram, S; Raghavan, P. S. Chromium-VI Reagents: Synthetic Applications; Springer: Berlin, 2011.

Environ. Int. 2005, 31, 739−753.

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Binding to Cr(III) and Cr(VI) Ions. J. Biol. Chem. 2000, 275, 10150–10153. Lytle, C. M.; Lytle, F. W.; Yang, N.; Qian, J.-H.; Hansen, D.; Zayed, A.; Terry, N. Reduction of Cr(VI) to Cr(III) by Wetland Plants: Potential for In Situ Heavy Metal Detoxification. Environ. Sci. Technol. 1998, 32, 3087-3093.


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12 DM, S.; JJ, B.; KE, W. A Prediction of Chromium(III) Accumulation in Humans from Chromium

481 482

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27


Table of Contents: Specific Reagent for Cr(III): Imaging Cellular Uptake of Cr(III) in Hct116 Cells and Theoretical Rationalization Firoj Ali, Sukdeb Saha, Arunava Maity, Nandaraj Taye, Mrinal Kanti Si, E. Suresh, Bishwajit Ganguly, Samit Chattopadhyay and Amitava Das

H2O

H2O

H2O

H2O

H2O (H2O)

H2O

2.02

2.04

H2O

H2O

H2O

H2O

1.510

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

2.0 2.12

Specific molecular probe for recognition of Cr(III) in aqueous buffer with a luminescence ON response and its usage as an imaging reagent.


Page 28 of 28

Specific Reagent for Cr(III): Imaging Cellular Uptake of Cr(III) in Hct116

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