“Turn-on” fluorescence sensing of cytosine: Development of a chemosensor for quantification of cytosine in human cancer cells

A simple new chemosensor, pyrene-appended 5-hydroxyisophthalic acid derivative (PIA), was developed and characterized for selective detection and quantification of cytosine in different human cancer cells. PIA shows “turn-on” fluorescence in presence of cytosine through intermolecular hydrogen bonding with a detection limit of 32 nM at neutral pH.


Experimental Section Materials and Methods
1-Pyrenemethylamine hydrochloride, 5-Hydroxyisophthalic acid, Bromoacetyl chloride and cytosine were purchased from Sigma-Aldrich Pvt. Ltd. (India).Unless otherwise mentioned, materials were obtained from commercial suppliers and were used without further purification.Solvents were dried according to standard procedures.Elix Millipore water was used throughout all experiments. 1H and 13 C NMR spectra were recorded on a Bruker 400 MHz instrument.For NMR spectra, DMSO-d 6 and for NMR titration DMSO-d 6 and D 2 O were used as solvent using TMS as an internal standard.Chemical shifts are expressed in δ ppm units and 1 H-1 H and 1 H-C coupling constants in Hz.The mass spectrum (HRMS) was carried out using a micromass Q-TOF Micro TM instrument by using Methanol as a solvent.Fluorescence spectra were recorded on a Perkin Elmer Model LS 55 spectrophotometer.UV spectra were recorded on a SHIMADZU UV-3101PC spectrophotometer.Elemental analysis of the compounds was carried out on Perkin-Elmer 2400 series CHNS/O Analyzer.The following abbreviations are used to describe spin multiplicities in 1 H NMR spectra: s = singlet; d = doublet; t = triplet; m = multiplet.

Synthetic Procedures
The fluorescent receptor PIA was synthesized by four consecutive steps starting from 1-Pyrenemethylamine hydrochloride followed by the preparation of compound 1, 2 and 3 as shown in Scheme S1.Initially, 1 was synthesized according to a published procedure, 1 by the reaction of 1-Pyrenemethylamine hydrochloride and bromoacetyl chloride producing compound 1.PIA has been synthesized from 5-Hydroxyisophthalic acid in three steps via formation of compound 2 and 3.In the final step, compound 3 was hydrolysed to give the product PIA with 86% yield (Scheme S1).

Di-ester of 5-Hydroxyisophthalic acid:
To a solution of anhydrous ethanol (30ml) 5-Hydroxyisophthalic acid (1.82g, 10 mmol) was added.Catalytic amount of conc.sulfuric acid was then added to the reaction mixture.The mixture was then stirred for 24 hrs.at 80 0 C. The solution was extracted with chloroform (3×20 mL) and water (3×50 mL).Organic layer was separated and dried over by anhydrous MgSO 4 .The solvent was removed by rotary evaporation to give the di-ester product (yield 98%).
PIA: To a solution of anhydrous K 2 CO 3 (0.85g, 6 mmol) in dry acetonitrile was added di-ester of 5-Hydroxyisophthalic acid (0.36 g, 1.50 mmol).The mixture was stirred for 0.5 h.Then compound 1 (1.06 g, 2 mmol) was added to the solution and stirred for 48 h.Then, the reaction mixture was poured into water.The solution was extracted with chloroform (3×50 ml).The organic layer was separated and dried over anhydrous MgSO 4 .After removing the solvents, the residue was chromatographed on silica gel with chloroform/ Ethyl acetate = 4:1 v/v as eluent to give 0.31g (86%) of compound 3.The diester was dissolved in a mixture of 10% aqueous NaOH solution and EtOH (1:1) under reflux for 24 h.The reaction mixture was evaporated and it was acidified with conc.HCl into ice.The brown ppt was collected and dried at room temp (0.3 g, 98%).1H NMR (DMSO-d6, 400 MHz): δ (ppm)

UV-Vis and fluorescence titration studies UV-vis spectral studies:
A stock solution of PIA (1 × 10 -6 M) was prepared in water-DMSO (20:1, v/v).Cytosine solution of concentration 1 × 10 -5 M was prepared in Millipore water.All experiments were carried out in aqueous medium at neutral pH.During PIA and cytosine titration, each time a 1 × 10 -6 M solution of PIA was filled in a quartz optical cell of 1 cm optical path length and cytosine stock solution was added into the quartz optical cell gradually by using a micropipette.Spectral data were recorded at 1 min after the addition of cytosine.

Fluorescence spectral studies:
A stock solution of PIA (1 × 10 -6 M) was prepared in water-DMSO (20:1, v/v).Cytosine solution of concentration 1 × 10 -5 M was prepared in Millipore water.All experiments were carried out in aqueous medium at neutral pH.During PIA and cytosine titration, each time a 1 × 10 -6 M solution of PIA was filled in a quartz optical cell of 1 cm optical path length and cytosine stock solution was added into the quartz optical cell gradually by using a micropipette.Spectral data were recorded at 1 min after the addition of cytosine.For all fluorescence measurements, excitations were provided at 345 nm, and emissions were collected from 365 to 460 nm.

Measurement of fluorescence quantum yields
The fluorescence quantum yield (QY) of PIA was determined relative to a reference compound of known QY. 2-Aminopyridine (solvent 0.1 M H 2 SO 4 ) was chosen as reference compound because it has emission profile between 320-480 nm similar to the receptor PIA.The quantum yield of PIA increased almost 2.5 fold upon addition of 1 equiv. of cytosine.

Evaluation of the association constants for the formation of (PIA-cytosine) complex:
By Fluorescence Method: Binding constant of the chemosensor PIA was calculated through emission method by using the following equation:

Calculation of limit of detection (LOD) of PIA with cytosine:
The detection limit of the receptor PIA for cytosine was calculated on the basis of fluorescence titration.To determine the standard deviation for the fluorescence intensity, the emission intensity of four individual receptors without cytosine was measured by 10 times and the standard deviation of blank measurements was calculated.
The limit of detection (LOD) of PIA for sensing cytosine was determined from the following equation 2-3 : LOD = K × SD/S Where K = 2 or 3 (we take 3 in this case); SD is the standard deviation of the blank receptor solution; S is the slope of the calibration curve.Computational details: Geometries have been optimized at the B3LYP/Def2SVP level of theory.5] The geometries are verified as proper minima by frequency calculations.Time-dependent density functional theory calculation has also been performed at the same level of theory.All the calculations have been carried out using ORCA software suite. 6

Cell imaging study
For confocal imaging studies, 1 × 10 4 A549 cells in 1000 μL of medium, were seeded on sterile 35 mm glass bottom culture dish (ibidi GmbH, Germany), and incubated at 37°C in a CO 2 incubator for 10 hours.Then cells were washed with 500 μL DMEM followed by incubation with PIA (1 µM) dissolved in 1000 μL DMEM at 37°C for 1 h in a CO 2 incubator and cells were washed thrice with phosphate buffered saline (PBS) (pH 7.0) to remove excess PIA observed under an Olympus IX81 microscope equipped with a FV1000 confocal system using 1003 oil immersion Plan Apo (N.A. 1.45) objectives.Images obtained through section scanning were analyzed by DIDS with excitation at 341 nm monochromatic laser beams, and emission spectra were integrated over the range 414 nm (single channel).The cells were again incubated with cytosine (10 µM) for 20 min and excess cytosine was thrice with PBS (pH 7.0) followed by observations under microscope.For all images, the confocal microscope settings, such as transmission density, and scan speed, were held constant to compare the relative intensity of intracellular fluorescence.

Cytotoxicity Assay
In vitro studies established the ability of the chemosensor PIA to detect cytosine in biological system with excellent selectivity.Human cell A549 (ATCC No CCL-185) were used as models.However, to materialize this objective, it is a prerequisite to assess the cytotoxic effect of PIA and PIA-cytosine complex on live cells.The well-established MTT assay 7 was adopted to study cytotoxicity of above mentioned complexes at varying concentrations detailed in method section.
A cytotoxicity measurement for each experiment shows that the chemosensor PIA does not have any toxicity on the tested cells and PIA-cytosine complex does not exert any significant adverse effect on cell viability at tested concentrations.

Quantification of cytosine and validation of the screening procedure
To quantify cellular level of cytosine 10 7 A549 human cancer cells were harvested by centrifugation at 3000 rpm for 5 minutes followed by washing of the cell pellet with PBS buffer.Cells were again harvested following similar centrifugation.Cell pellet were suspended with 100 µL cold deionized water in order to lyse by the osmotic shock.Lysates were further centrifuged and the supernatant has been collected.The supernatant has been added with 1µM PIA and the fluorescence signal was measured.The value of fluorescence intensity has been plotted to the standard curve in order to know the concentration of cytosine in tested sample (Fig. 5).All estimations have been done in triplicate.
The estimation of cytosine was validated using A549, HeLa and Hep-2 cancer cells.10 4 of each cell suspension were centrifuged to collect the cells.The cells were resuspended with 10 mM PBS buffer (pH 7.0) followed by centrifugation.The cell pellets were lysed by osmotic shock with 100 µL ice cold deionised water.Supernatant were added with 1 µL PIA and fluorescence signal were recorded.The fluorescence signal has been recorded for five independent sample of each cancer cell type and all experiments were done in triplicate.The signal to noise ratio were obtained and the screening procedure were validated by calculating Z' score (Table S4).

Fig. S8
Fig. S8 Linear fit curve of PIA at 377 nm with respect to cytosine concentration.

Fig. S12
Fig. S12 MTT assay to determine the cytotoxic effect of PIA and PIA-cytosine complex on A549 cells (Human cell A549, ATCC No CCL-185).

Table S2 .
Selected electronic excitation energies (eV), oscillator strengths (f), main configurations of the low-lying excited states of all the molecules and complexes.The data were calculated by TDDFT//B3LYP/Def2SVP based on the optimized ground state geometries [a] Only selected excited states were considered.The numbers in parentheses are the excitation energy in wavelength.[b] Oscillator strength.[c] H stands for HOMO and L stands for LUMO.

Table S3 .
Energies of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO)