The sensing behaviour of S1 was evaluated in following two different media; 1. In acetonitrile: 2. In aqueous solution:

Transcription

1 The union of merely hundred plus elements lead to billions of molecules with varieties of properties. This union takes place at the level of electrons between two bonding entities i.e. atoms. This union of elements provides the basic building blocks of life. The next level of association between molecules leading to varieties of selfassemblies of molecules like cell membrane, chromosomes, the higher order structures of enzymes and proteins came into picture in decades of 40 s of last century. From the decade of 40 s of last century a proper understanding of these molecular associations started pouring in, which got riper in the decade of 60 s in the form of ion-ion, ion-dipole, dipole-dipole, dipole-induced dipole etc. Pedersen, Cram and Lehn were awarded Nobel Prize in They started a new vista in chemistry in the name of supramolecular chemistry where weak interactions were at the heart of molecular association for forming supramolecules with varieties of properties. The molecular recognition is very important field of supramolecular chemistry and is attracting the Scientists from all disciplines of Science viz. Chemistry, Physics, Biology, Biochemistry, Genetics etc. The chemical/biochemical reactions involve a no. of ionic/molecular drivers which drive the reactions in a particular direction for achieving a particular physiological task. A large no. of metal ions viz. Na +, K +, Ca 2+, Mg 2+, Cr 3+ /Cr 6+, Mn 2+, Fe 2+ /Fe 3+, Co 2+, Ni 2+, Cu 2+, Zn 2+, Cd 2+, Hg 2+ play crucial roles in a variety of reactions occurring in living beings. Similarly a large no. of anions viz. OH, SO 2 4 /SO 2 3, NO 3 /NO 2, HSO 4 /HSO 3, F, Cl, Br, I, C 2 O 2 4, CH 3 COO etc. play important roles in various processes. Besides these a no. of neutral molecules like amino acids, alcohol, ammonia etc. also play important roles. The working concentration of the above type of substances in living cells are generally very small of the order of micro, nano or picomolar etc. At these concentrations the routine chemical/analytical methods do not work. Although a wide variety of instrumental methods are available but their cost and operation are not user friendly many times. Hence for dealing with such situation the chemists particularly supramolecular chemists have started a new field of developing substance specific sensors particularly optical sensors. An optical sensor is a programmed molecule of abiotic origin which undergoes characteristic colour change upon its interaction with a particular analyte. A huge no. of optical sensors have been devised and are in practice since then. The Page 150

2 biggest bottleneck with these sensors is their cost, tedious synthesis, water intolerance, non-specific and poor reversibility. Present dissertation concentrates upon the design, synthesis and evaluation of some cost effective optical sensors having good water compatibility and fair reversibility. Some simple but smart Schiff bases having potential to identify ionic/neutral analytes of biological importance like Cu 2+, Zn 2+, F, HSO 4, cysteine and methanol have been synthesized and characterized under present study. The unravelling of mechanistic aspects of these sensors is yet another important objective of present work. The comparison of optical activities of these molecular sensors with the truth table of a particular logic gate in terms of Boolean algebra opens a new dimension for these optical sensors. This new dimension is molecular machine and a no. of pioneer workers are involved in the same for last 2-3 decades. Present thesis involved construction of optical sensors particularly the fluorescent ones which are truly intramolecular charge transfer (ICT) probes on the skeleton of coumarin. Their evaluation against a particular specific analyte from the matrix of a large no. similar types of analytes is yet another important objective of this work. The entire work has been presented in five chapters besides Introduction and general experimental procedure. The chapter 1 describes synthesis, characterization and evaluation of a coumarin containing Schiff base S1 as an anion sensor. The same was fully characterized through various spectroscopic techniques like IR, 1 H & 13 C NMR along with mass determination. The structure of S1 was finally established through single crystal X-ray diffraction studies (figure 1). Figure 1: Ortep diagram of S1 with 50% elipsoid The sensing behaviour of S1 was evaluated in following two different media; 1. In acetonitrile: 2. In aqueous solution: Page 151

3 In acetonitrile solution; S1 showed selective naked eye colour change from olive green to pink for F while other anions did not produce any categorical change (figure 2). S1 F - AcO BzO I Br Cl BF 4 PF 6 HPO 4 2 H 2 PO 4 HSO 4 ClO 4 Figure 2: Visible colour change of S1 on addition of 1.0 equiv. different anions in acetonitrile at 10μM. The UV-vis. titration study was performed by the concomitant additions of TBA salt of F (0-10 equivalents) to the 10μM acetonitrile solution of S1. The two isosbestic points at ~455 and ~316 nm were observed during the above titration indicating a chemical interaction of F with S1. The binding constant determinations as well as Job s plot indicated 1:1 stoichiometry. The 1 H NMR studies clearly indicated the hydrogen bonding between Ar-OH and F at lower equivalent while at higher equiv. it was deprotonated (figure 3). Figure 3: 1 HNMR titration spectrum of S1 with F In aqueous solution, the olive green colour of S1 was bleached by the HSO4 selectively from the matrix of a large no. of anions (figure 4). The UV-vis. titration studies between S1 and HSO 4 showed two well-defined isosbestic points centred at ~395 and ~316 nm indicated an interaction between S1 and HSO 4. S1 F AcO BzO I Br Cl BF 4 PF 6 HSO 4 HPO 4 2 H 2 PO 4 ClO 4 Figure 4: Visible colour change of S1 with different anions in CH 3 CN-H 2 O (1:1, v/v) solution at 50μM. Page 152

4 The S1 (3μM) also gave intense blue fluorescent response with TBA salt of HSO 4 only in CH 3 CN-H 2 O (1:1, v/v) solution (figure 5 & 6a). The fluorescence titration between S1 and HSO 4 showed linear enhancement in fluorescent intensity (figure 6b). S1 F AcO BzO I Br Cl BF 4 PF 6 HSO 4 HPO 4 2 H 2 PO 4 ClO 4 Figure 5: Fluorescence image of S1 under UV lamp (365 nm) with different anions in CH 3 CN-H 2 O (1:1, v/v) solution. 4x10 6 HSO nm 3x Intensity, a.u. 2x10 6 S1, F, Aco, Bzo, H 2 PO 4 Cl, Br, I, BF 4, ClO 4, 2 PF 6, HPO 4 Intensity, a.u x Wavelength, nm Wavelength, nm Figure 6: (a) Fluorescence spectrum of S1 with different anions and (b) Fluorescence titration spectrum of S1 with HSO 4 at 3.0μM in CH 3 CN-H 2 O (1:1, v/v) solution. The sensing mechanism of S1 towards HSO 4 was revealed from the 1 H NMR studies which clearly proved the hydrolysis of S1 in the presence of HSO 4 (figure 7). The hydrolysis of S1 with HSO 4 leading to its rupture into its constituents was also supported by the mass spectral studies. Further to justify the above results of hydrolysis we isolated the hydrolytic product of S1 in the presence of HSO 4 in aqueous medium and characterized it through 1 H NMR study which again supported the above observation. Hence in this chapter, we successfully studied the mechanistic aspects of optical sensing of HSO 4 in acetonitrile and in aqueous media. In this chapter we were successful in establishing operation of two different mechanistic aspects of Schiff bases viz. Hydrogen bonding/hydrolysis towards the optical sensing of HSO 4 in nonaqueous (Acetonitrile) and in aqueous media respectively (Scheme1). We also studied Page 153

5 the colorimetric naked eye responses of F with S1. However, the same involved hydrogen bonding (Scheme 2). TBA- HSO 4 Ar-H & -NH 2 -OH -CH=N Ar-H Figure 7: 1 H NMR spectrum of S1 and with HSO 4 in CD 3 CN-D 2 O Scheme 1: Proposed Correct sensing mechanism of Schiff base S1 towards HSO 4 in water medium Page 154

6 Scheme 2: Proposed sensing mechanism of Schiff base S1 towards F in acetonitrile The Chapter 2 describes synthesis, characterization and evaluation of S2 which showed enhanced fluorescence in the presence of Zn 2+ in semi aqueous media (EtOH H 2 O (1:1, v/v)) with nanomolar level of detection (figure 8). The single crystals of S2 showed beautiful ladder like structure incorporating non-classical hydrogen bonding while the resulting species between sensor and Zn 2+ exhibited 1D coordination polymeric framework in its single crystal XRD pattern (figure 9 & 10). S2 Na + K + Mg 2+ Ca 2+ Al 3+ Cr 3+ Mn 2+ Fe 3+ Co 2+ Ni 2+ Cu 2+ Zn 2+ Cd 2+ Hg 2+ Ag + Pb 2+ Figure 8: Fluorescence image of S2 with different cations under UV lamp in EtOH H 2 O (1:1, v/v) solution Figure 9: Formation of ladder-like supramolecular architecture of S2 due to weak nonclassical hydrogen bonding Figure 10: The 1D chain coordination polymer framework of [Zn.NO 3.S2)] n Page 155

7 The chapter 3 describes a simple coumarin based Schiff base S3 which is the minor modification of S2 (chapter 2).The same involves hydrogen instead of CH 3 across the >C=N. The S3 and S3-Zn 2+ complex were fully characterized through various spectroscopic techniques including single crystal XRD (figure 11 &12). The S3-Zn 2+ complex exhibited 1D coordination polymeric framework in its single crystal XRD pattern (figure 12). Figure 11: Crystal structure of S3: thermal ellipsoids are drawn at the 50% probability level and Intermolecular Hydrogen bond interaction in S3 Figure 12: A supramolecular architecture of [Zn.Cl.S3] n having non-classical hydrogen bond interaction with DMF molecule The S3 showed remarkable fluorescence turn on response for Zn 2+ in aqueous solution with nanomolar detection limits (figure 13). It is noteworthy that the minor modification of S2 was justified by enhanced water compatibility of S3 towards sensing of Zn 2+. The turn on response of S3 was understood in terms of strong chelation-enhanced fluorescence (CHEF) upon addition of Zn 2+ to S3 that weakens the intramolecular charge transfer (ICT) causing blue shift and inhibits the >C=N isomerization as well as PET processes (figure 14). Page 156

8 S3 Na + K + Mg 2+ Ca 2+ Ba 2+ Al 3+ Cr 3+ Mn 2+ Fe 3+ Co 2+ Ni 2+ Cu 2+ Zn 2+ Cd 2+ Hg 2+ Pb 2+ Figure 13: Fluorescence image of S3 towards different cations under UV lamp (excited at 365 nm) ICT strong, PET and >C=N isomerization allowed, No CHEF Zn 2+ Weak Fluorescence Strong Fluorescence ICT weak, PET and >C=N inhibited, CHEF allowed Figure 14: proposed sensing behaviour of S3 towards Zn 2+ The fluorescent response of the S3-Zn 2+ ensemble was further examined in the presence of different cations/anions in aqueous solution (EtOH: H 2 O, 1:9, v/v). The same study indicated selective fluorescence turn off response of S3-Zn 2+ ensemble towards Cu 2+ /HSO 4 (figure 15 & 16). S3-Zn 2+ Na + K + Mg 2+ Ca 2+ Ba 2+ Al 3+ Cr 3+ Mn 2+ Fe 3+ Co 2+ Ni 2+ Cu 2+ Cd 2+ Hg 2+ Pb 2+ S3-Zn 2+ F - AcO BzO I Br Cl BF 4 PF 6 HPO 4 2 H 2 PO 4 HSO 4 ClO 4 PPi Figure 15: Fluorescence image of S3-Zn 2+ ensemble towards different cations and anions under UV lamp (excited at 365 nm) in aqueous solution Page 157

9 The addition of Cu 2+ to the S3-Zn 2+ leads to transmetallation (S3-Cu 2+ ) causing fluorescence quenching via electron-transfer process. On the other hand, the addition of HSO 4 to the S3-Zn 2+ leads to stripping of Zn 2+ from S3-Zn 2+ hence producing S3 in solution which is non-fluorescent due to operative PET and >C=N isomerization. (a) (b) Figure 16: (a) Fluorescence spectrum of S3-Zn 2+ ensemble with different cations and (b) different anions in aqueous solution (EtOH: H 2 O, 1:9, v/v) at 0.5μM This sensing behavior of S3 with Zn 2+ and Cu 2+ /HSO 4 strikingly mimics the INHIBT logic gate and can be represented as off-on-off switching (figure 17). The S3 was also utilized to image intracellular Zn 2+ and Cu 2+ /HSO 4 ions in E. coli cells with a good performance. Input A Input B Output (Zn 2+ ) (Cu 2+ /HSO 4 ) Figure 17: Representation of a two-state molecular switch based on INHIBIT logic function and truth table Page 158

10 In chapter 4, a simple coumarin derived Schiff base (S4) incorporating rhodanine as a smart example of fluorescent probe for selective detection and differentiation of methanol from water/matrix of basic alcohols (C1C4) was synthesized and studied. The same is altogether a new approach as it involves suppression of PET/>C=N isomerization through methanol activated ring opening of the cyclic control unit of S4 leading to formation of a fluorescent moiety S5 (figure 18). Methanol Figure 18: Methanol mediated chemical transformation of S4 to S5 The photo physical behaviour of S4 (0.5μM) was studied in a number of solvents having different polarities showed that the methanolic solution of S4 was highly emissive at ~520 nm while other solutions of the same were either non/very feebly emissive (figure 19 & 20). Solvent such as EtOH and other basic (C2-C4) alcohols which have almost same characteristic feature as MeOH were unable to cause any observable change in photophysical properties of S4 (figure 19). ACN MeOH EtOH PrOH BuOH CHCl 3 DCM THF Tolu EA Acetone DMF DMSO H 2 O Figure 19: Selective fluorescence color changes of S4 in various solvents; from left to right: ACN, MeOH, EtOH, PrOH, BuOH, Acetone, Ethylacetate, Toluene, DCM, CHCl 3, THF, DMF, DMSO and H 2 O. Page 159

11 2.5x nm 2.0x10 6 Intensity, a.u. 1.5x x10 6 Methanol Other Solvents 5.0x Wavelength, nm Figure 20: Characteristic effect of methanol solvent over the emission spectra of S4 A comparison of 1 H NMR spectrum of S4 before and after addition of methanol supports its chemical transformation to S5 (figure 21). -NH -HC=N -OCH 3 S5 -HC=N S4 Figure 21: Partial 1 H NMR spectra of S4 and S5 and corresponding comparison of their peak positions The single crystal XRD of S5 confirmed our above speculation that the S4 undergoes ring opening at rhodanine leading to the formation of S5 which was highly fluorescent (figure 22). Page 160

12 Figure 22: ORTEP view (thermal ellipsoid are shown at 50% level) of the S5 with the partial atom numbering and Showing intermolecular hydrogen bonding in S5 The cell imaging studies demonstrated that S4 had potential to detect intracellular methanol also (figure 23). Figure 23: Cell imaging of Methanol through S4 In chapter 5, the S5 which is the open chain version of S4 reported in chapter 4 th was explored for its optical detection towards a variety of metal ions. The same showed selective fluorescent quenching with Cu 2+ followed by its fluorescent turn on response with cysteine without any interference even with glutathione (GSH) and homocysteine (figure 24). The binding constant determinations as well as Job s plot indicated 1:1 stoichiometry. The single crystal X-ray diffraction of S5 with Cu 2+ further confirms the same (figure 25). 6x10 6 5x10 6 Intensity, a.u. 5x10 6 4x10 6 3x10 6 2x10 6 S5, Na +, K +, Ca 2+,Ba 2+, Mg 2+, Al 3+, Cr 3+, Mn 2+, Fe 3+, Ni 2+, Co 2+, Zn 2+,Cd 2+, Hg 2+, Pb 2+ Cu 2+ Intensity, a.u. 4x10 6 3x10 6 2x10 6 Cys S5-Cu 2+, His, Hcy, GSH, Val, Gly, Arg, Ala, Luc, Phe, Tyr, Lys, Trp, Glu, Gln 1x10 6 1x Wavelength, nm Figure 24: (a) Fluorescence spectrum of S5 with different cation and (b) Fluorescence spectrum of S5-Cu 2+ with different amino acids in H 2 O: MeOH (99:1, v/v) solution at 0.5 μm 0 Wavelength, nm Page 161

13 Figure 25: ORTEP plot of the S5 Cu 2+ with the atom numbering scheme. The S5 was further exploited for successful bio-imaging of Cu 2+ and Cys through fluorescence microscopy in E. coli cells (figure 26). Figure 26: Fluorescent imaging of Cu 2+ and Cys in E.Coli cells with S5 The on-off-on sensing behaviour of S5 in the presence of Cu 2+ and Cys was successfully translated in the form of IMP molecular logic gate (figure 27). Input A [Cu 2+ ] Input B [Cys] Output Fluorescence [high] [low] [high] [high] Figure 27: IMP Logic gate function and Truth Table Page 162

14 IN NUTSHELL THE ENTIRE WORK CAN BE SUMMARIZED UNDER FOLLOWING POINTS The optical sensors particularly the fluorescent ones viz. S1, S2, S3, S4 and S5 of ICT type on coumarin platform were successfully synthesized, characterized and evaluated for their sensing properties against a variety of ionic/neutral analytes. The S1 having 7-amino-4-trifluoromethylcoumarin was utilized for the detection of F and HSO 4 in acetonitrile and in aqueous medium respectively. Here for the first time, we uncovered the true mechanism of sensing for the HSO 4 in aqueous medium by the family of Schiff base sensors. The S2 having 7-N, N-diethyl amino-3-acetylcoumarin was utilized for the nanomolar detection of Zn 2+ in aqueous medium. The single crystal of S2 and S2 - Zn 2+ portrayed a beautiful supramolecular ladder-like and 1D chain coordination polymeric framework respectively. The S3 which is a minor modification of S2 showed increased water compatibility and was employed for the selective fluorescent sensing of Zn 2+ ion. The S3-Zn 2+ ensemble was further utilized for its nanomolar sensing against Cu 2+ / HSO 4 ion with a fluorescent turn-off response. The S3 was also utilized to image intracellular Zn 2+ and Cu 2+ /HSO 4 ions in E. coli cells with a good performance. The S4 incorporating rhodanine as a smart example of fluorescent probe was synthesized and utilized for selective detection and differentiation of methanol from water/matrix of basic alcohols (C1-C4). The present approach is altogether a new approach as the same involves suppression of PET/C=N isomerization through methanol activated ring opening of the cyclic control unit of S4 leading to formation of a fluorescent moiety S5. The same was also utilized for the bioimaging of methanol in living cells. The S5 which is the open chain version of S4 at rhodanine, served as highly efficient fluorescent sensor for Cu 2+. The S-Cu 2+ ensemble further served as an effective and selective fluorescent sensor for Cys amino acid. The same was consequently used as input to build up an implication (IMP) logic gate by monitoring the output signal at ~520 nm. The S5 was further exploited for successful bio-imaging of Cu 2+ and Cys through fluorescence microscopy in E. coli cells. Page 163