Synthesis of novel quinaldine-based squaraine dyes: effect of substituents and role of electronic factors

Condensation of squaric acid with quinaldinium salts containing electron-donating substituents gave only the semisquaraines. However, with salts possessing electronegative and electron-withdrawing groups, the squaraine dyes were isolated in quantitative yields. The semisquaraines formed undergo condensation with highly nucleophilic salts yielding the unsymmetrical squaraine dyes. These results demonstrate the role of electronic factors and provide valuable information for the design of efficient squaraine-based sensitizers that can have potential applications in photodynamic therapy

Aggregation Behavior of halogenated squaraine dyes in buffer, electrolytes, organized media, and DNA

Aggregation properties of bis(3,5-dibromo-2,4,6-trihydroxyphenyl)squaraine (1) and bis(3,5-diiodo-2,4,6-trihydroxyphenyl)squaraine (2) have been examined in buffer and in the presence of electrolytes, β-cyclodextrin, micelles and DNA. These dyes were found to form aggregates in buffer and methanol-water solutions that have absorption bands blue-shifted to those of the monomeric forms. The iodo derivative 2 forms aggregates at much lower concentrations (1.7 × 10<SUP>−6</SUP> M) compared to the bromo derivative 1 (2.35 × 10<SUP>−6</SUP> M) in 20% (vol/vol) methanol-buffer solution. Increase in methanol concentration in methanol-water solutions resulted in the disruption of the aggregates. The intermediate dimer in the monomer to aggregate conversion process can be detected under specified conditions. The entropy and the standard free energy for the dimer formation in the case of 1 are found to be -16.12 eu and 7.46 k cal mol<SUP>−1</SUP>, respectively. Addition of electrolytes (LiCl, NaCl, and KCl) and calf thymus DNA resulted in the enhancement of aggregate formation, whereas the monomer gets stabilized for several hours in the presence of microheterogeneous media such as β-cyclodextrin and cetyltrimethylammonium bromide. These results reveal that face-to-face stacking followed by hydrogen bonding interactions between the chromophoric units are the major driving force for the formation of sandwich (H-type) aggregates. These dyes exhibit favorable photophysical properties and can be applied into the tissues using carrier systems in which they do not form aggregates and hence can have potential use as sensitizers in photodynamic therapeutical applications

Site-selective interactions: squaraine dye-serum albumin complexes with enhanced fluorescence and triplet yields

We report the effect of steric factors of a few squaraine dyes, bis(2,4,6-trihydroxyphenyl)squaraine (1), bis(3,5-dibromo-2,4,6-trihydroxyphenyl)squaraine (2), and bis(3,5-diiodo-2,4,6-trihydroxyphenyl) squaraine (3), on their binding with human (HSA) and bovine (BSA) serum albumins employing photophysical, chiroptical, biophysical, and microscopic techniques. These dyes interact with serum albumins very efficiently and exhibit site selectivity, involving synergistic effects of hydrophobic, hydrogen bonding, and electrostatic interactions. The association constants of these complexes have been determined and are found to be 4.9 × 10<SUP>6</SUP> and 4.1 × 10<SUP>5</SUP> M<SUP>−1</SUP>, respectively, for the dyes 2 and 3 with BSA, while HSA showed relatively higher association constants of 6.0 × 10<SUP>6</SUP> and 9.9 × 10<SUP>5</SUP> M<SUP>−1</SUP>. Highly clear distinction in site-selective binding can be ascertained from time-resolved fluorescence, displacement cum fluorimetry, and circular dichroism (CD) studies. The increased affinity toward the major binding site (site II, domain III) over the relatively smaller binding site (site I, domain II) in the serum albumin with the increasing size of the heavy atoms present in 2 and 3 as compared to 1 indicates the importance of steric factors thereby confirming that the dye structure has a predominant role in deciding site selectivity. The distance between the energy donor and acceptor was calculated using Frster theory, which agrees well with the reported site 1 binding agent dansylamine. In contrast, no energy transfer was observed between tryptophan (Trp-214) present in domain II of the albumins and the dyes 2 and 3, indicating that these derivatives bind less efficiently at site I due to steric conatraints but preferentially bind at site II. Laser flash photolysis studies of the dyes 2 and 3 in the presence of HSA exhibited ca. 2.5-fold enhancements in the triplet lifetimes and quantum yields when compared to that obtained in buffer. The uniqueness of these dyes is that they show substituent size-dependent selectivity at site II of serum albumins and signal the event through "turn on" fluorescence intensity as well as enhanced triplet excited state lifetimes and quantum yields, thereby indicating their potential use as NIR noncovalent protein labeling and photodynamic therapeutic agents

Site-selective binding and dual mode recognition of serum albumin by a squaraine dye

With the objective of developing small molecule based probes for proteins, interactions of polyhydroxyl-substituted squaraine dye (SQ) with bovine serum albumin (BSA) have been investigated by absorption, steady-state and time-resolved fluorescence, circular dichroism (CD), cyclic voltammetry (CV), 1H NMR, scanning electron, and tapping mode atomic force microscopic techniques. Increase in addition of BSA resulted in increase in absorbance and fluorescence quantum yields (80-fold) of SQ, along with significant bathochromic shifts in the absorption and fluorescence maxima. Half-reciprocal analysis of the absorption data gave a 1:1 stoichiometry for the complex between BSA and SQ with high association (Kass) constant of (1.4 ± 0.1) × 10<SUP>6</SUP> M<SUP>-1</SUP> and change in free energy of -35 kJ/mol. The complex formation was further confirmed by observation of induced CD signal corresponding to the SQ chromophore at 610 nm, upfield shift (about Δδ 0.1 ppm) of aromatic protons of SQ in <SUP>1</SUP>H NMR spectra, and decrease in current intensity (CV) of SQ when bound to BSA. The picosecond time-resolved fluorescence studies indicated that the BSA-SQ complex exhibits biexponential decay with significantly enhanced lifetimes of 0.5 and 1.5 ns when compared to the lifetime of SQ (τ = 121 ps) in the absence of BSA. Employing displacement cum fluorimetry using site-specific binding ligands, such as dansylproline and dansylamide, indicated that SQ binds with protein selectively at site II involving hydrophobic, hydrogen bonding, and electrostatic interactions. The uniqueness of this molecular system is that it interacts with BSA selectively at site II and signals the binding event through dual mode recognition of "visual color" change and "turn on" fluorescence mechanism

β-Cyclodextrin as a photosensitizer carrier: effect on photophysical properties and chemical reactivity of squaraine dyes

With the objective of understanding the utility of β-cyclodextrin (β-CD) as a carrier system, we have investigated its interactions with a few near-infrared absorbing squaraine dyes (i.e., 1a,b and 2a,b) through absorption and steady-state and time-resolved fluorescence techniques. The addition of β-CD to the phloroglucinol dyes 1a,b resulted in a significant bathochromic shift in absorption, together with a ca. 1.5-2.5-fold enhancement in fluorescence intensity, whereas for the aniline-based dyes 2a,b, a hypsochromic shift in the absorption and a ca. 5-12-fold fluorescence enhancement were observed in a 10% (v/v) ethanol/water mixture. Benesi-Hildebrand analysis showed that both the dyes 1a,b and 2a,b form 2:1 stoichiometric complexes with β-CD. The complex formation was confirmed by competitive binding analysis employing adamantyl-1-carboxylic acid (ACA) and adamantyl-1-ammonium chloride (ADAC). The displacement of the dyes 1a,b and 2a,b from the [dye-β-CD] complex by ADAC and ACA unambiguously establishes the encapsulation of these dyes in the hydrophobic nanocavity of β-CD. Uniquely, the formation of the inclusion complexes with β-CD provides unusual protection from nucleophilic attack by aminothiols such as cysteine and glutathione for dyes 1a,b, whereas negligible protection was observed for dyes 2a,b. These results demonstrate the substituent-dependent encapsulation of potentially useful squaraine dyes in β-CD, thereby indicating its potential as a carrier system for the squaraine dyes 1a,b useful in photodynamic therapy

Aggregation properties of heavy atom substituted squaraine dyes: evidence for the formation of J-type dimer aggregates in aprotic solvents

The squaraine dye bis(2,4,6-trihydroxyphenyl)squaraine (SqH) was earlier reported to form J-type dimer aggregates in acetonitrile solutions at higher concentrations. Subsequent studies have suggested that concentration-dependent changes in the absorption spectrum of SqH in acetonitrile could be attributed to shifts in the acid-base equilibrium due to the presence of water as an impurity. In this work, we describe our studies on the effect of varying acid and dye concentration on the absorption spectra of the bromo and iodo substituted dyes, bis(3,5-dibromo-2,4,6-trihydroxyphenyl)squaraine (SqBr) and bis(3,5-diiodo-2,4,6-trihydroxyphenyl)squaraine (SqI). Analysis of the changes in the absorption spectra as a function of dye concentration and the nature of the solvent composition confirmed the formation of J-type dimer aggregates in aprotic solvents in this class of dyes. Further confirmation for the formation of the J-type dimer aggregates could be obtained by comparing the differences in the triplet excited state properties of the neutral and aggregated forms of the dyes using time-resolved spectroscopy