Aza-Crown-Based Macrocyclic Probe Design for "PET-off" Multi-Cu2+ Responsive and "CHEF-on" Multi-Zn2+ Sensor: Application in Biological Cell Imaging and Theoretical Studies.

The work represents a rare example of an aza-crown-based macrocyclic chemosensor, H2DTC (H2DTC = 1,16-dihydroxy-tetraaza-30-crown-8) for the selective detection of both Zn2+ and Cu2+ in HEPES buffer medium (pH 7.4). H2DTC exhibits a fluorescence response for both Zn2+ and Cu2+ ions. The reversibility of the chemosensor in its binding with Zn2+ and Cu2+ ions is also examined using a Na2EDTA solution. H2DTC exhibits a chelation-enhanced fluorescence (CHEF) effect in the presence of Zn2+ ions and a quenching effect (CHEQ) in the presence of paramagnetic Cu2+ ions. Furthermore, the geometry and spectral properties of H2DTC and the chemosensor bound to Zn2+ have been studied by DFT and TDDFT calculations. The limit of detection (LOD) values are 0.11 × 10-9 and 0.27 × 10-9 M for Cu2+ and Zn2+, respectively. The formation constants for the Zn2+ and Cu2+ complexes have been measured by pH-potentiometry in 0.15 M NaCl in 70:30 (v:v) water:ethanol at 298.1 K. UV-vis absorption and fluorometric spectral data and pH-potentiometric titrations indicate 1:1 and 2:1 metal:chemosensor species. In the solid state H2DTC is able to accommodate up to four metal ions, as proved by the crystal structures of the complexes [Zn4(DTC)(OH)2(NO3)4] (1) and {[Cu4(DTC)(OCH3)2(NO3)4]·H2O}n (2). H2DTC can be used as a potential chemosensor for monitoring Zn2+ and Cu2+ ions in biological and environmental media with outstanding accuracy and precision. The propensity of H2DTC to detect intracellular Cu2+ and Zn2+ ions in the triple negative human breast cancer cell line MDA-MB-468 and in HeLa cells has been determined by fluorescence cell imaging.

Fluorometric trace methanol detection in ethanol and isopropanol in a water medium for application in alcoholic beverages and hand sanitizers.

Detection of methanol (MeOH) in an ethanol (EtOH)/isopropanol ( i PrOH) medium containing water is crucial to recognize MeOH poisoning in alcoholic beverages and hand sanitizers. Although chemical sensing methods are very sensitive and easy to perform, the chemical similarities between the alcohols make MeOH detection very challenging particularly in the presence of water. Herein, the fluorometric detection of a trace amount of MeOH in EtOH/ i PrOH in the presence of water using alcohol coordinated Al(iii)-complexes of an aldehydic phenol ligand containing a dangling pyrazole unit is described. The presence of MeOH in the EtOH/ i PrOH causes a change of the complex geometry from tetrahedral (Td) to octahedral (Oh) due to the replacement of the coordinated EtOH/ i PrOH by MeOH molecules. The Td-complex exhibited fluorescence but the Oh-species did not, because of the intramolecular photo-induced electron transfer (PET). By interacting the Oh species with water, its one MeOH coordination is replaced by a water molecule followed by the proton transfer from the water to pyrazole-N which generates strong fluorescence by inhibiting the PET. In contrast, the water interaction dissociates the Td-complex to exhibit fluorescence quenching. The water induced reversal of the fluorescence response from the decrease to increase between the absence and presence of MeOH is utilized to detect MeOH in an EtOH/ i PrOH medium containing water with a sensitivity of ∼0.03-0.06% (v/v). The presence of water effected the MeOH detection and allows the estimation of the MeOH contamination in alcoholic beverages and hand sanitizers containing large amounts of water.

An aluminium fluorosensor for the early detection of micro-level alcoholate corrosion.

The detection of the dry alcoholate corrosion of aluminium is vital to design a corrosion resistive aluminium alloy for the storage and transportation of biofuel (methanol or ethanol). By synthesizing an Al3+ fluorescent probe operable in an alcoholic medium, we quantified the alcoholate corrosion in terms of the fluorometrically estimated soluble alkoxide (Al(OR)3) generation under nitrogen atmosphere. With time, a linear increase in corrosion with specific aluminium dissolution rate constants ∼2.0 and 0.9 µg per day per cm2 were estimated for aluminium and Al-7075 alloy, respectively. During open atmosphere monitoring, the adsorbed moisture converted small extent of Al(OR)3 to the insoluble Al(OH)3 at the alloy surface which retarded the alcoholate corrosion appreciably.

Porphyrin-Based Probe for Simultaneous Detection of Interface Acidity and Polarity during Lipid-Phase Transition of Vesicles.

Biochemical activities at a membrane interface are affected by local pH/polarity related to membrane lipid properties including lipid dynamics. pH and polarity at the interface are two highly interdependent parameters, depending on various locations from the water-exposed outer surface to the less polar inner surface. The optical response of common pH or polarity probes is affected by both the local pH and polarity; therefore, estimation of these values using two separate probes localized at different interface depths can be erroneous. To estimate interface pH and polarity at an identical interface depth, we synthesized a glucose-pendant porphyrin (GPP) molecule for simultaneous pH and polarity detection by a single optical probe. pH-induced protonation equilibrium and polarity-dependent π-π stacking aggregation for GPP are exploited to measure pH and polarity changes at the 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG) membrane interface during DMPG phase transition. An NMR study confirmed that GPP is located at the interface Stern layer of DMPG large unilamellar vesicle (LUV). Using UV-vis absorption studies with an adapted analysis protocol, we estimated interface pH, or its deviation from the bulk phase value (ΔpH), and the interface polarity simultaneously using the same spectra for sodium dodecyl sulfate micelle and DMPG LUV. During temperature-dependent gel to liquid-crystalline phase transition of DMPG, there was ∼0.5 unit increase in ΔpH from approximately -0.6 to -1.1, with a small increase in the interface dielectric constant from ∼60 to 63. A series of spectroscopic data indicate the utility of GPP for evaluation of local pH/polarity change during lipid phase transition of vesicles.

Protonation-induced pH increase at the triblock copolymer micelle interface for transient membrane permeability at neutral pH.

Achieving controlled membrane permeability using pH-responsive block copolymers is crucial for selective intercellular uptake. We have shown that the pH at the triblock-copolymer micelle interface as compared to its bulk pH can help regulate membrane permeability. The pH-dependent acid/base equilibriums of two different interface-interacting pH probes were determined in order to measure the interfacial pH for a pH-responsive triblock copolymer (TBP) micelle under a wide range of bulk pH (4.5-9.0). According to 1H NMR studies, both pH probes provided interfacial pH at a similar interfacial depth. We revealed that the protonation of the amine moiety at the micelle interface and the subsequent formation of a positive charge caused the interface to become relatively less acidic than that of the bulk as well as an increase in the bulk-to-interfacial pH deviation (ΔpH) from ∼0.9 to 1.9 with bulk pH reducing from 8.0 to 4.5. From the ΔpH vs. interface and bulk pH plots, the apparent and intrinsic protonations or positive charge formation pKa values for the micelle were estimated to be ∼7.3 and 6.0, respectively. When the TBP micelle interacted with an anionic large unilamellar vesicle (LUV) of a binary lipid (neutral and anionic) system at the bulk pH of 7.0, fluorescence leakage studies revealed that the pH increase at the micelle interface from that of the LUV interface (pH ∼ 5.5) made the micelle interface partially protonated/cationic, thereby exhibiting transient membrane permeability. Although the increasing interface protonation causes the interface to become relatively less acidic than the bulk at any bulk pH below 6.5, the pH increase at the micelle interface may not be sufficiently large to maintain the threshold for the amine-protonated condition for effecting transient leakage and therefore, a continuous leakage was observed due to the slow disruption of the lipid bilayer.

Determination of proton concentration at cardiolipin-containing membrane interfaces and its relation with the peroxidase activity of cytochrome c.

The activities of biomolecules are affected by the proton concentrations at biological membranes. Here, we succeeded in evaluating the interface proton concentration (-log[H+] defined as pH') of cardiolipin (CL)-enriched membrane models of the inner mitochondrial membrane (IMM) using a spiro-rhodamine-glucose molecule (RHG). According to fluorescence microscopy and 1H-NMR studies, RHG interacted with the Stern layer of the membrane. The acid/base equilibrium of RHG between its protonated open form (o-RHG) and deprotonated closed spiro-form (c-RHG) at the membrane interface was monitored with UV-vis absorption and fluorescence spectra. The interface pH' of 25% cardiolipin (CL)-containing large unilamellar vesicles (LUVs), which possess similar lipid properties to those of the IMM, was estimated to be ∼3.9, when the bulk pH was similar to the mitochondrial intermembrane space pH (6.8). However, for the membranes containing mono-anionic lipids, the interface pH' was estimated to be ∼5.3 at bulk pH 6.8, indicating that the local negative charges of the lipid headgroups in the lipid membranes are responsible for the deviation of the interface pH' from the bulk pH. The peroxidase activity of cyt c increased 5-7 fold upon lowering the pH to 3.9-4.3 or adding CL-containing (10-25% of total lipids) LUVs compared to that at bulk pH 6.8, indicating that the pH' decrease at the IMM interface from the bulk pH enhances the peroxidase activity of cyt c. The peroxidase activity of cyt c at the membrane interface of tetraoleoyl CL (TOCL)-enriched (50% of total lipids) LUVs was higher than that estimated from the interface pH', while the peroxidase activity was similar to that estimated from the interface pH' for tetramyristoyl CL (TMCL)-enriched LUVs, supporting the hypothesis that when interacting with TOCL (not TMCL), cyt c opens the heme crevice to substrates. The present simple methodology allows us to estimate the interface proton concentrations of complex biological membranes.

Detection of Curvature-Radius-Dependent Interfacial pH/Polarity for Amphiphilic Self-Assemblies: Positive versus Negative Curvature.

It is possible that a defined curvature at the membrane interface controls its pH/polarity to exhibit specific bioactivity. By utilizing an interface-interacting spiro-rhodamine pH probe and the Schiff base polarity probe, we have shown that the pH deviation from the bulk phase to the interface (ΔpH)/interfacial dielectric constant (κ(i)) for amphiphilic self-assemblies can be regulated by the curvature geometry (positive/negative) and its radius. According to 1H NMR and fluorescence anisotropy investigations, the probes selectively interact with an anionic interfacial Stern layer. The ΔpH/κ(i) values for the Stern layer are estimated by UV-vis absorption and fluorescence studies. For the anionic sodium bis-2-ethylhexyl-sulfosuccinate (AOT) inverted micellar (IM) negative interface, the highly restricted water and proton penetration into the Stern layer owing to tight surfactant packing or a reduced water-exposed headgroup area may be responsible for the much lower ΔpH ≈ -0.45 and κ(i) ≈ 28 in comparison to ∼-2.35 and ∼44, respectively, for the anionic sodium dodecyl sulfate (SDS) micellar positive interface with a close similar Stern layer. With increasing AOT IM water-pool radius (1.7-9.5 nm) or [water]/[AOT] ratio ( w0) (8.0-43.0), the ΔpH and κ(i) increase maximally up to ∼-1.22 and ∼45, respectively, due to a greater water-exposed headgroup area. However, the unchanged ΔpH ≈ -0.65 and κ(i) ≈ 53.0 within radii ∼3.5-8.0 nm for the positive interface of a mixed Triton X-100 (TX-100)/SDS (4:1) micelle justify its packing flexibility. Interestingly, the continuously increasing ΔpH trend for IM up to its largest possible water-pool radius of ∼9.5 nm may rationalize the increase in ΔpH (∼-1.4 to -1.6) with the change in the curvature radii (∼15 to 50 nm) for sodium 1,2-dimyristoyl- sn-glycero-3-phosphorylglycerol (DMPG)/1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) (2:1) large unilamellar vesicles (LUV) owing to its negative interface. Whereas, similar to the micellar positive interface, the unchanged ΔpH at the positive LUV interface was confirmed by fluorescence microscopic studies with giant unilamellar vesicles of identical lipids composition. The present study offers a unique and simple method of monitoring the curvature-radius-dependent interfacial pH/polarity for biologically related membranes.

A ratiometric solvent polarity sensing Schiff base molecule for estimating the interfacial polarity of versatile amphiphilic self-assemblies.

A newly synthesised Schiff base molecule (PMP) existing in equilibrium between non-ionic and zwitterionic forms displays solvent polarity induced ratiometric interconversion from one form to another, such novelty being useful to detect the medium polarity. The specific interface localisation of PMP in versatile amphiphilic self-assembled systems has been exploited to monitor their interfacial polarity by evaluating such interconversion equilibrium with simple UV-Vis spectroscopy. In spite of the large differences in pH and/or viscosity between the bulk and interface, the unchanged equilibrium between the two molecular forms on varying the medium pH or viscosity provides a huge advantage for the exclusive detection of interfacial polarity.

A simple interfacial pH detection method for cationic amphiphilic self-assemblies utilizing a Schiff-base molecule.

A simple pH-sensing method for cationic micelle and vesicle interfaces is introduced, utilizing a Schiff-base molecule, 2-((4H-1,2,4-triazol-4-ylimino)methyl)-6-(hydroxymethyl)-4-methylphenol (AH). AH containing a phenolic moiety was obtained by the reaction between 4-amino-4H-1,2,4-triazole containing polar O- and N-centres with opposite polarity to the cationic interface and 2-hydroxy-3-(hydroxymethyl)-5-methylbenzaldehyde. The acid/base equilibrium of AH was investigated at the interfaces of cetrimonium bromide (CTAB) micelles, tri-block-copolymeric micelles (TBPs) and large unilamellar vesicles (LUVs) of different lipid compositions using steady state UV-Vis absorption spectroscopy. AH interacted strongly with the micelle and vesicle interfaces, according to the binding studies with LUV. A larger amount of AH proton dissociation was observed when localized at the interface of micelles and vesicles compared to that in the bulk phase, indicating that the pH values at the cationic interfaces are higher than in the bulk phase. The pH values were about 2.2 and 1.6 units higher at the CTAB and TBP micelle interfaces, respectively, than the bulk pH. The pH variation decreased from 2.4 to 1.5 units by increasing the neutral 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid content from 0 to 50% in the cationic dimethyldioctadecylammonium (DDAB) LUV, indicating that the interfacial positive charges are responsible for the higher interfacial pH. Detailed structural and absorption characteristics of neutral AH and its anionic A(-) forms were investigated by fluorescence spectroscopic measurements and DFT based theoretical calculations. The present simple pH detection method may be applied to various biological micelle and vesicle interfaces.

Formation of domain-swapped oligomer of cytochrome C from its molten globule state oligomer.

Many proteins, including cytochrome c (cyt c), have been shown to form domain-swapped oligomers, but the factors governing the oligomerization process remain unrevealed. We obtained oligomers of cyt c by refolding cyt c from its acid molten globule state to neutral pH state under high protein and ion concentrations. The amount of oligomeric cyt c obtained depended on the nature of the anion (chaotropic or kosmotropic) in the solution: ClO4(-) (oligomers, 11% ± 2% (heme unit)), SCN(-) (10% ± 2%), I(-) (6% ± 2%), NO3(-) (3% ± 1%), Br(-) (2% ± 1%), Cl(-) (2% ± 1%), and SO4(2-) (3% ± 1%) for refolding of 2 mM cyt c (anion concentration 125 mM). Dimeric cyt c obtained by refolding from the molten globule state exhibited a domain-swapped structure, in which the C-terminal α-helices were exchanged between protomers. According to small-angle X-ray scattering measurements, approximately 25% of the cyt c molecules were dimerized in the molten globule state containing 125 mM ClO4(-). These results indicate that a certain amount of molten globule state oligomers of cyt c convert to domain-swapped oligomers during refolding and that the intermolecular interactions necessary for domain swapping are present in the molten globule state.

Formation of oligomeric cytochrome c during folding by intermolecular hydrophobic interaction between N- and C-terminal α-helices.

We have previously shown that horse cytochrome c (cyt c) forms oligomers by domain swapping its C-terminal α-helix when interacting with ethanol. Although folding of cyt c has been studied extensively, formation of domain-swapped oligomers of cyt c during folding has never been reported. We found that domain-swapped oligomeric cyt c is produced during refolding from its guanidinium ion-induced unfolded state at high protein concentrations and low temperatures. The obtained dimer exhibited a domain-swapped structure exchanging the C-terminal α-helical region between molecules. The extent of dimer formation decreased significantly for the folding of C-terminal cyt c mutants with reduced hydrophobicity achieved by replacement of hydrophobic residues with Gly in the C-terminal region, whereas a large amount of heterodimers was generated for the folding of a mixture of N- and C-terminal mutants. These results show that cyt c oligomers are formed through intermolecular hydrophobic interaction between the N- and C-terminal α-helices during folding. A slow phase (4-5 s) was observed in addition to a 400-500 ms phase during folding of a high concentration of cyt c in the presence of 1.17 M guanidine hydrochloride. The fast phase is attributed to the intramolecular ligand exchange process, and we attribute the slow phase to the ligand exchange process in oligomers. These results show that it is important to consider formation of domain-swapped oligomeric proteins when folding at high protein concentrations.

Fluorescence correlation spectroscopy at single molecule level on the Tat-TAR complex and its inhibitors.

The TAR element of HIV and the viral protein Tat form a molecular switch regulating transcriptional efficiency in HIV. We show that fluorescence correlation spectroscopy at the single molecule level is a powerful method to study the association between a Tat-derived peptide and TAR fragments. We also investigated the inhibition of the peptide-RNA complex by different ligands. Utilizing cross correlation measurements, the dissociation constants (K(D)) were determined. To demonstrate the important role of the bulge for the binding of Tat, we compared wt-TAR with three RNA mutants, mainly differing in the bulge region. For the TAR mutants studied at equimolar concentration of RNA and peptide (25 nM), the K(D) values are 15-35 times larger than that of wt-TAR. This gives evidence that the bulge region is the most crucial part of the TAR RNA for specific Tat binding. The IC(50) values for different inhibitors of the Tat/TAR complex both with wt-TAR and mutants have been determined. Neamine conjugate proved to be the best inhibitor of the complex formation. Our results are in agreement with earlier published data on this system using alternative biophysical and biochemical methods, respectively.