DEVELOPMENT OF SUPRAMOLECULAR MULTICOMPONENT CHROMOPHORES TOWARDS PHOTOLUMINESCENCE

This thesis is submitted in partial fulfillment of the requirements for the degree of Master of Science in Chemistry. Rabah Iranti from the Department of Biology, UAEU for their enormous contributions to the success of this thesis.

Introduction

Overview
Statement of Problem
Relevant Literature

Indicator Displacement Assay (IDA)
Cucurbituril Family
Drug Sensors based on Cucurbit[n]uril Macrocycles
Hosting Attributes of CBn
Photoinduced Electron Transfer (PET)
Density Functional Theory (DFT)
PET in CB-Encapsulated Organic Dyes
pK a Shift
Carnosol Anticancer Drugs

The Objective of this Work

At the time an analyte is added to the complex in solution, the analyte displaces the dye from the binding site, which will result in a change in the fluorescence of the dye. Conversely, when the dye is displaced from the cavity due to competitive binding of an analyte, the fluorescence of the dye decreases significantly (Figure 1) [3]. The name is taken from the similarity between this molecule and a pumpkin from the group of the Cucurbitaceae family.

For example, in a recent determination of the amino acid phenylalanine [19] and the anticancer drug methotrexate [20] with the palmatine/CB7 complex, the fluorescence signal of the indicator dye palmatine was enhanced within CB7 due to prolonged host-mediated conjugation. the. These examples were putatively dependent on a number of processes, such as host-assisted conjugations between the aromatic rings of the indicator dye [19–21] and host-assisted aggregation-induced emission (AIE) [22] . In the off state, excitation of the fluorophore component of the sensor results in electron transfer from the receptor to the fluorophore as one possibility [29].

Figure 1: Schematic representation of (a) fluorescence indicator displacement assays for analyte sensing using a macrocyclic host and a fluorescent dye (indicator) and, (b) guest displacement assays (GDA)

Methods

Research Design
Experimental Reagents
Organic Synthesis of 4PBZC Probe (4-((4-(1H-benzo[d]
Proton Nuclear Magnetic Resonance ( 1 H NMR) Spectroscopy
Absorption/Steady-State Fluorescence Spectroscopy
pH Titration Studies
Consistent Binding Titration Study
Host-Guest Interaction
Competitive Displacement Studies
DFT Calculations

The inclusion complex of 4PBZC with CB7 was confirmed using proton nuclear magnetic resonance spectroscopy (1H NMR). A calculated weight of CB7 corresponding to 2 mM was added to a required volume of 30 μM 4PBZC stock solution to prepare the complex of 4PBZC-CB7. The interaction between carnosol and the complex of 4PBZC and CB7 was investigated by weighing a calculated amount of carnosol, dissolved in the stock solution of 4PBZC/CB7 to form 150 μM carnosol.

This solution was gradually added to 2.6 mL of the 4PBZC/CB7 inclusion complex in the quartz cuvette, followed by evaluation of the absorption and photoluminescence spectra of each additional solution containing CAR (in μL). The average concentration of 4PBZC (C4PBZC) and CB7 (CCB7) in the fresh solution can be estimated by the following equation. All boundaries (K, Y(CB7)24PBZC and ∈4PBZC/CB7 except Y4PBZC except Y_4PBZC were left unbounded. Examination by Levenberg-Marquardt calculation was provided by product fit of SigmaPlot 6.1, USA).

Results and Discussion

Preparation of Tert-butyl 4-(1H-benzo[d]imidazol-2-yl)

Preparation of 2-(piperazin-1-yl)-1H-benzo[d]

Preparation of 4-((4-(1H-benzo[d]imidazol-2-yl)

In addition, 4PBZC (an indicator dye) was synthesized by mixing 1.1 mmol each of both 4B7MC and 2PBZ in the presence of dry acetonitrile and 4 mmol TEA together overnight at 25oC. The extract was further purified by washing with ethyl acetate and water while the mixture was separated based on the organic and aqueous layers using a separatory funnel. The organic layer was dried over anhydrous sodium phosphate and hexane was added after concentration.

4PBZC was obtained from the precipitate formed, which was then filtered and dried in a calculated 70% yield.

Figure 13: Synthesis mechanism of indicator dye 4PBZC.

Interaction of the Indicator Dye with CB7 and

The formation of host-guest inclusion complexes between 4PBZC and 4PBZH+C and CB7 was confirmed by UV-visible and NMR titration at pH (pD) 3 and 9, respectively. The appearance of isosbestic points at 305 nm (Figure 14a) and the sigmoidal fit confirm the 2:1 binding stoichiometry for the 4PBZH+C/CB7 complex. Inclusion was also confirmed because two proton resonances, H-1 and H-2, shifted up by about 0.4−0.9 ppm with the addition of 1 equivalent of CB7, while other proton resonances H-8, H-9, H-5, and H-3 shifted slightly to higher ppm after addition of one equivalent of CB7 while H-6, H-7, H-4 and the methyl protons showed no shifts.

Furthermore, as a consequence of the addition of 2.0 molar equivalents of CB7, two resonance protons H-6, H7 and the methyl proton were shifted up, indicating the encapsulation of the coumarin unit in the cavity of CB7. However, the low binding affinity was demonstrated in the NMR data at pD 7 (Figure 25) by the shifts observed upon addition of CB7. Δδ represents the difference between the NMR peak for H-1 in the absence and presence of CB7.

Figure 15: Evolution of the UV−visible absorption spectra of 4PBZC (35 µM) and 4PBZC/CB7 (35 µM 4PBZC + 500 µM CB7) upon changing the pH in the range 3−9 in an aqueous solution at 298 K

Supramolecular Effects on Fluorescence and

The addition of CB7 (Figure 19) slightly increased emission from the neutral form, possibly due to a constraint imposed by the host cavity on their free rotation (confinement effects). The change in fluorescence intensity (Relative Int.) is the difference between the emission in the absence and presence of CB7. Encapsulation of the BZ unit (left) in the cavity of CB7 yields the lowest binding energy (-39.9 kcal/mol) compared to that of the C unit (middle) with -29.5 kcal/mol in the neutral shape of the probe (4PBZC ).

However, in the protonation of the 4PBZC by the BZ moiety, there is a decrease in the binding energies of the complex associated with the encapsulated moiety (bottom). The decrease in binding energy means that encapsulation via the BZ unit is more favored than that of the C unit and the protonated dye forms a more stable inclusion complex compared to that of the high binding energy neutral form. In addition, encapsulation of both units (BZ and C) is also possible, as confirmed by the NMR result, as the probe can get stuck by both BZ.

Figure 21: Variations in the fluorescence spectra of 5 μM of 4PBZH + C (a) and 4PBZC (b) with the addition of up to 300 μM of CB7

Supramolecular Invoked pK a Reposition

With the addition of 1 molar equivalent of CB7 to the solution of 4PBZC, two proton resonances of H-1 and H-2, corresponding to the proton of the BZ unit, were shifted to lower ppm, with approximately 0.95 ppm losing their encapsulation in the cavity of CB7. , while two resonance protons of the coumarin unit H-3 and H-4 were slightly shifted to higher ppm (from 3.69 ppm and 5.59 ppm to 3.80 ppm and 2.81 ppm, respectively).

Figure 24: The reversibility structure of neutral 4PBZC and protonated structure of 4PBZH + C (left) and UV-Visible Spectral probe 4PBZC (35 μM) from pH 2 to pH 10 in the absence of CB7 (filled circles) and the presence of 500 μM of CB7 (empty circles)

Supramolecular Effects on Fluorescence

The observed pKa* value for the complex at the excited state is smaller than the determined pKa value at the ground state, confirming the need to consider the photoacidity of the complex when estimating its pKa value [49] . Although the recovery of the true pKa* value with the addition of CB7 is beyond the focus of the present study, the plots in Figure 3 highlight the pH value at which one observes the host-induced turnover in the emission intensity of the indicator dye. can best be utilized when using the supramolecular IDA at pH around 5.8. Encouraged by the neutrality of the BZ and coumarin unit and their biological applications, pH 6 was chosen to develop an effective sensing method for CAR.

The Host-Retarded PET

At pH 6, the BZ unit is neutral, but when leached into the cavity of CB7, host-guest ion-dipole interactions protonate the ring and suppress the intramolecular PET process, restoring emission from the coumarin unit (see theoretical results: Figure 28). Highly significant changes were noted, highlighting the clear advantage of any of the detection strategies employed.

Density Functional Theory (DFT) Calculations

In addition, the calculations showed that 4PBZH+C binds more tightly than the corresponding neutrals, which is consistent with the additional stabilization through ion-dipole interactions and consistent with the NMR results. In the neutral form, the fluorescence of the PET is quenched, where the lone pair electrons on the heteroatoms (e.g., the amine group in BZ) have higher energy than the HOMO of the fluorophore (i.e., coumarin) (see Figure 28). Upon excitation of an electron from the HOMO to the LUMO of the fluorophore, the lone electron pair drops to the partially empty HOMO of the fluorophore, this prevents excited.

In contrast, the protonation of the amine group lowers the energy of the lone pair state below that of the fluorophore HOMO, which will therefore prevent the quenching process and restore the fluorescence. Collectively, the theoretical results confirm that the intramolecular PET takes place from the neutral BZ to the C unit, which can subsequently be suppressed by different inputs such as H+ or CB7 macromolecules. Experimentally, the suppression of the PET process by the addition of HCl (Figure 20) or CB7 at pH 6 due to the host-assisted protonation led to a significant emission enhancement (Figure 27).

Figure 28: DFT-optimized structures of CB7/4PBZC (top) and CB7/4PBZH + C (bottom), considering the complexation through the BZ (left) or the C (right) units

Interaction of CAR with CB7

CB7 and two proton resonances, H-c and H-d, are completely hidden under the CB7 peak, while three more proton resonances H-a, H-b, H-h and hydroxyl protons showed no or slight downshifts. This chemical shift explains the encapsulation of CAR in the CB7 cavity with the H-a and H-b resonance protons located outside the cavity. Indiscriminately, the DFT calculated binding energy (theoretical results) agrees with the above NMR results (Figure 31).

Figure 30: 1 H NMR spectra of carnosol with CB7 (0−1 molar equivalents) in 50%

Supramolecular Indicator Displacement Assay (IDA)

The addition of CAR has caused the expected evolution of the spectral profiles relevant to the neutral form, due to the absorption band shifting back to blue (325 nm) at about 335 nm with a concomitant decrease in fluorescence intensity (Figures 32a and 32c). These significant spectral changes confirm the complexation of CAR with CB7 (Figure 30) at the expense of the gradual displacement of the BZ unit from the cavity of CB7 with the increase in its concentration. The nonlinear fit of absorbance data (b) to a 1:1 binding model (solid line and experimental section) and the linear fit (d) of the fluorescence data are also shown.

Encapsulation of CAR and carnosic acid by macrocyclic compounds such as α, β and γ-CD was also reported [ 37 ]. This combined with the use of host-suppressed PET allowed us to extend the linearity to detection for very low concentrations of CAR. The high sensitivity of the present sensor highlights the importance of our newly designed sensor technology and its superiority compared to other reports for the detection of CAR (less than 0.7 µM) [52] and other spectroscopically silent drugs (Table 1).

Figure 32: The changes in the relative UV−visible absorbance (a) and

Conclusion

Research Implications

Future Research

Biedermann, “Rational design and performance of a cucurbit[8]uril-based indicator displacement assay for use in blood serum,” Chemical Science , vol. Hettiarachchi et al., “Toxicology and drug delivery with Cucurbit[n]uril-type molecular carriers,” PLoS ONE , vol. Shan et al., “Selective recognition and determination of phenylalanine by a cucurbit[8]uril and palmatine-based fluorescent probe,” Analytica Chimica Acta , vol.

Supramolecular Interaction of Methotrexate with Cucurbit[7]uril and Analytical Application," Spectrochimica Acta del A: Molecular and Biomolecular. Bhasikuttan, "Stimulus-Responsive Supramolecular p K a Tuning of Cucurbit[7]uril Encapsulated Coumarin 6 Dye," J. Nau , “Design of a Fluorescent Dye for Indicator Displacement from Cucurbiturils: A Macrocycle- Responsive Fluorescent Switch Operating through a p K a Skift,” Org. Fu, “A competitive strategy based on cucurbit[7]uril supramolecular interaction for simple and sensitive detection of dibucaine,” Talanta , vol.