Solvatochromic Fluorophore Research Articles

A Systematic Analysis of Aromatic Heterocyclic Rings in Solvatochromic Fluorophores

Abstract The Suzuki–Miyaura cross-coupling was found to be effective for the modification of aromatic heterocyclic rings in solvatochromic fluorophores, thereby providing quantitative evaluation of the ring effects on photophysical properties. The effect of heteroatom, a β-substituent, and the number of rings in the aromatic moiety were investigated systematically.

Fine tuning of a solvatochromic fluorophore for selective determination of Fe 3+: a new type of benzimidazole-based anthracene-coupled receptor

We synthesized a novel benzimidazole-based, anthracene-coupled fluorescent receptor capable of recognizing and estimating the concentrations of Fe 3+ in semi-aqueous solution by ratiometric estimation. Our sensor can be made highly selective for Fe 3+ over other metal ions by changing the solvent composition.

A new solvatochromic fluorophore for exploring nonpolar environments created by biopolymers

The fluorescence of a new aminocyanonaphthalene exhibits exquisite sensitivity to its environment and responds to a solvent change from water to hexane with greater than a 100-fold increase in intensity and 100 nm shift in λ(max.em). These properties should support many applications including the detection of abasic sites within duplex DNA as illustrated below.

Solvatochromic rotaxane molecular shuttles

A strongly fluorescent bistable rotaxane is described in which the relative position of the macrocyclic ring with respect to a solvatochromic fluorophore gives a strong response in the spectral domain.

Fluorescent-Responsive Synthetic C1b Domains of Protein Kinase Cδ as Reporters of Specific High-Affinity Ligand Binding

Protein kinase C (PKC) is a critical cell signaling pathway involved in many disorders such as cancer and Alzheimer-type dementia. To date, evaluation of PKC ligand binding affinity has been performed by competitive studies against radiolabeled probes that are problematic for high-throughput screening. In the present study, we have developed a fluorescent-based binding assay system for identifying ligands that target the PKC ligand binding domain (C1 domain). An environmentally sensitive fluorescent dye (solvatochromic fluorophore), which has been used in multiple applications to assess protein-binding interactions, was inserted in proximity to the binding pocket of a novel PKCδ C1b domain. These resultant fluorescent-labeled δC1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent δC1b domain analogues can be used to evaluate ligand binding affinity.

Unusual large Stokes shift and solvatochromic fluorophore: Synthesis, spectra, and solvent effect of 6-substituted 2,3-naphthalimide

In this article, four new series of 6-substituted 2,3-naphthalimides ( 1a– d, 2a– d, 3a– d, 4a– d and 4 e– h) have been designed and synthesized through the formation of key cyclic anhydride intermediate, which was the precursor of the well known environment-sensitive fluorophore [6- N, N-dimethylaminonaphthalimides (6-DMN)] and other 6-substituted 2,3-naphthalimide series ( 2, 3, 4). Based on 6-amino-2,3-naphthalimide (6-ANP) compound, a new type of fluorophore was found to exhibit moderate to unusual large Stokes shift (297–303 nm). 6-ANP derivatives display relatively low fluorescence quantum yields in high polar protic solvents such as water ( Φ F ∼ 0.004, 571–576 nm) and a significant unusual red shift due to (1) hydrogen bonding interaction of the excited state of the molecule with the solvents, which presumably enhance the intersystem crossing process in the system to quench fluorescence, (2) this large Stokes shift was assumed to be the consequence of a substantial change of the geometric structure from the ground state (S 0) to the first excited state (S 1). Compared with the other compounds studied, the fluorescence of the nitro- and halo-derivatives was rather weak, probably due to the efficient intersystem crossing leading to a non-reactive triplet state.

Benzo[b]Fluorenone as a Quadruplex Interactive Agent (Qia): Binding Studies and Quadruplex Formation with the Human Telomeric HT4 Sequence

Many previously investigated agents capable of binding to and stabilizing G-quadruplex DNA conformations possess aromatic, planar chemical structures. Here, initial investigations on the interaction of benzo[b]fluorenone (BF) with the human telomeric forming oligonucleotide HT4 using circular dichroism (CD), thermal melting and steady-state fluorescence are reported. CD studies suggest that BF exhibits binding selectivity for G-quadruplex over model double-stranded DNA structures. BF binds to hybrid-mixed type quadruplex structures in the presence of physiological concentrations of K+ ions (100mM). However, even in the absence of salt, BF can induce an anti-parallel type quadruplex conformation. Thermal melting studies on quadruplex HT4 in the presence and absence of salt show BF has little effect on the melting temperature, suggesting that BF may associate with the G-quadruplex structure through a non-intercalating mode. As BF is known to be a solvatochromic fluorophore, steady state fluorescence emission spectra were taken for BF titrated with the HT4 quadruplex formed at 100mM K+, to explore the ability of BF to act as a potential probe of the environment of its binding site. Based on the red shift of BF emission, BF/quadruplex association appears to involve a relatively polar and hydrogen-bonded environment. This work was supported by NIH SCORE Grant S06 GM 060654.

Monitoring protein interactions and dynamics with solvatochromic fluorophores.

Solvatochromic fluorophores possess emission properties that are sensitive to the nature of the local microenvironment. These dyes have been exploited in applications ranging from the study of protein structural dynamics to the detection of protein-binding interactions. Although the solvatochromic indole fluorophore of tryptophan has been utilized extensively for in vitro studies to advance our understanding of basic protein biochemistry, the emergence of new extrinsic synthetic dyes with improved properties, in conjunction with recent developments in site-selective methods to incorporate these chemical tools into proteins, now open the way for studies in more complex systems. Herein, we discuss recent technological advancements and their application in the design of powerful reporters, which serve critical roles in modern cell biology and assay development.

Synthesis and property of solvatochromic fluorophore based on D-π-A molecular system: 2-{[3-Cyano-4-(N-ethyl-N-(2-hydroxyethyl)amino)styryl]-5,5-dimethylfuran-2(5H)-ylidene}malononitrile dye

2-[[3-Cyano-4-(N-ethyl-N-(2-hydroxyethyl)amino)styryl]-5,5-dimethylfuran-2(5H)-ylidene]malononitrile styryl dye was prepared by the condensation of 4-[(2-hydroxy-ethyl)-methyl-amino]-benzaldehyde (donor moiety) with 2-cyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (acceptor moiety). The corresponding design, synthesis and solvatochromic characteristics of the intramolecular charge-transfer (ICT) dye chromophore were discussed and determined. Optical properties such as absorption and fluorescence emission spectra were monitored in several solvent media with different polarity. In this determination, the prepared dye chromophore showed positive solvatochromism effect and the resulting solvatochromic characteristics were studied with semiempirical calculations. The energy potentials of this dye chromophore such as HOMO and LUMO values were calculated by computational simulation approaches using Material Studio 4.3. Furthermore, the functions as a molecular switching sensor with pH stimulation of alkali-acid addition were determined in DMSO, which was operated by deprotonation/protonation effects based on intramolecular charge-transfer system.

Thiol-Reactive Derivatives of the Solvatochromic 4-N,N-Dimethylamino-1,8-naphthalimide Fluorophore: A Highly Sensitive Toolset for the Detection of Biomolecular Interactions

The solvatochromic fluorophore 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN) possesses extremely sensitive emission properties due largely to the low intrinsic fluorescence it exhibits in polar protic solvents such as water. This makes it well suited as a probe for the detection of a wide range of biomolecular interactions. Herein we report the development and evaluation of a new series of thiol-reactive agents derived from this fluorophore. The members of this series vary according to linker type and the electrophilic group required for the labeling of proteins and other biologically relevant molecules. Using the calcium-binding protein calmodulin as a model system, we compare the performance of the 4-DMN derivatives to that of several commercially available solvatochromic fluorophores identifying many key factors important to the successful application of such tools. This study also demonstrates the power of this new series of labeling agents by yielding a fluorescent calmodulin construct capable of producing a greater than 100-fold increase in emission intensity upon binding to calcium.

Photophysical properties, laser activity and photoreactivity of a heteroaryl chalcone: A model of solvatochromic fluorophore

The absorption and fluorescence characteristics of 3-(4′-dimethylaminophenyl)-1-(2-thienyl)prop-2-en-1-one (DMATP) have been investigated in different solvents. DMATP dye exhibits a large red shift in both absorption and emission spectra as solvent polarity increases, indicating a large change in the dipole moment of molecules upon excitation due to an intramolecular charge transfer interaction. The fluorescence quantum yield depends strongly on the properties of the solvents, which was attributed to positive and negative solvatokinetic effects. A crystalline solid of DMATP gave an excimer like emission at 570 nm due to the excitation of molecular aggregates. This is expected from the idealized crystal structure of the dye that belongs to the B-type class of Steven's classification. A dye solution ca. 10 −3 mol dm −3 in CHCl 3 gave a good laser emission in the range 480–560 nm with emission maximum at 530 nm upon pumping by nitrogen laser ( λ ex=337.1 nm). The excitation energy transfer from 7-dimethylamino-4-methyl coumarine (DMC) to DMATP has been also studied in CHCl 3 and the values of energy transfer rate constant and critical transfer distance indicate a Főrster-type mechanism. The photoreactivity and net photochemical quantum yield of DMATP in chloromethane solvents are also determined. We applied semiempirical MO calculation using AMI and ZINO/S calculation to understand the geometric and electronic structure of DMATP molecule in both ground and excited states.

Monitoring of three distinct structures of restriction enzyme complexes using characteristic fluorescence from site-selectively incorporated solvatochromic probe

The local change in the three different structures of restriction enzyme BamHI, which include DNA-free dimer and non-specific and specific complexes with DNA, were detected by the fluorescence from a site-selectively introduced solvatochromic fluorophore Nbeta-L-alanyl-5-(N,N-dimethylamino)naphthalene-1-sulfonamide (DanAla). According to the crystal structure, alpha-helices of the non-specific complex containing Ile82, Glu86 and Trp206 residues are converted into random coil by the formation of specific complex with a substrate. To understand the microenvironmental change caused by the structural transition around these positions, the DanAla probe was site-specifically introduced into the positions, and steady-state and time-resolved fluorescence was observed. The steady-state fluorescence gave us information that the rigidity of the polypeptide chains would be enhanced by the formation of the specific complex. The time-resolved fluorescence supported that the change in a water molecule-accessible space was induced by DNA-binding. We revealed that the change in rigidity and solvation around the specific positions was detected by the characteristic fluorescence using the combination of steady-state and time-resolved fluorescence techniques.

Self-quenching Mechanism: the Influence of Quencher and Spacer on Quencher-fluorescein Probes

Fluorescent chemosensors provide an extremely sensitive optical method for the real-time monitoring of molecular recognition events. An understanding of the fluorescent on/ off mechanism is essential for the rational design of fluorescent chemosensors. The majority of fluorescent chemosensors working in aqueous media operate by one of three mechanisms: (1) the suppression of photoinduced electron transfer (PET); (2) alteration of the microenvironment of a solvatochromic fluorophore; and (3) modulation of the fluorescence resonance energy transfer (FRET) between two fluorophores. The first two mechanisms are suitable for small molecule sensing (e.g., cations, small organic compounds, etc.), but FRET is very useful in the biopolymer state, for its distance information can be obtained in the 10 to 100 A region. In spite of the excellent applications of FRET for biomolecule sensing (c-AMP, insulin, etc.), the operations depend on a complicated biological pathway. Peptides with an FRET pair have been used as monitoring probes for a given enzyme activity. However, in various fluorophore pairs that have been examined, FRET peptide probes did not operate properly i.e., they were self-quenched. This self-quenching can be explained in terms of an intramolecular ground-state dimer complex between the FRET fluorophore pair in an aqueous solution. The strength of the aggregation between the two dye molecules depends on the molecular structure, solvent, temperature, and the presence or absence of electrolytes. Our aim is to systematically study the self-quenching mechanism for covalently linked fluorophore pairs. For simplicity, the factors influencing the efficiency of selfquenching are disassembled into four components (Figure 1). A FRET pair was replaced by a quencher-fluorophore pair as the reporting group. This overcomes the restriction for the FRET donor-acceptor resonance condition. From a practical viewpoint, water and fluorescein were chosen as the solvent and fluorophore, respectively; water is a crucial solvent for physiological systems; furthermore, fluorescein has a high fluorescence quantum yield in aqueous solution and its synthetic handling has been well established. As the quencher part, carbazole, methyl red and 1,8naphthalimide were selected. The effect of the spacer can be controlled by varying the alkyl chain length (-(CH2)n-, n = 5, 11, 15) and a flexible tetraethylene glycol unit (Figure 2). At pH 7.5 (50 mM HEPES buffer, I = 0.1 (NaNO3)), the fluorescence emission of C-n (n = 5, 11, 15) and C-TE ([probes] = 1 μM) was measured (Figure 3). It has been reported that there are two opposing factors determining the stability of aggregates of the derivatives containing two Rose Bengal moieties: the longer the chain, the easier it is to

PRODAN-Conjugated DNA: Synthesis and Photochemical Properties

A solvatochromic fluorophore, PRODAN, has been used as a microenvironment-sensitive reporter. Based on the chemistry of PRODAN, we designed and synthesized four novel fluorescent nucleosides, PDNX (X = U, C, A, and G), to which a PRODAN fluorophore was attached at pyrimidine C5 or purine C8. The fluorescent nucleosides sensitively varied the Stokes shift values depending on the orientational polarizability of the solvent. The PDNX incorporated into DNA also changed the Stokes shift values depending on the DNA structure. In particular, the excitation spectrum of the PDNX-containing duplex shifted to a longer wavelength and gave a smaller Stokes shift value when the base opposite PDNX could form a Watson-Crick base pair with PDNX. A lower energy excitation of PDNX-containing DNA resulted in a strong fluorescence emission selective to the Watson-Crick pairing base. This unique photochemical character was applicable to the efficient typing of single-nucleotide polymorphisms of genes.

Detection of the Local Structural Changes in the Dimer Interface of BamHI Initiated by DNA Binding and Dissociation Using a Solvatochromic Fluorophore

To detect the local structural change in an interface between proteins induced by the substrate binding and dissociation, a solvatochromic fluorescent N(beta)-L-alanyl-5-(N,N-dimethylamino)-naphthalene-1-sulfonamide (DanAla) was introduced into 132 position of the dimer interface in BamHI. Before addition of the substrate, the fluorescence from the normal planer excited state of DanAla moiety was observed as a main emission, and thereby the DanAla in the dimer interface is located in the hydrophobic microenvironment. The incubation with the substrate for 20 min induced the gradual increase in fluorescence intensity around 430 nm. The fact reflects that the polarity is reduced by the slight structural change initiated by the formation of the complex with the substrate. Furthermore, the incubation for more than 20 min caused the slight decrease in fluorescence around 430 nm and an appearance of fluorescence (560 nm) due to twisted intramolecular charge transfer (TICT) excited state. Therefore, the DanAla is exposed to comparative polar environment after the dissociation of the substrate. The fluorescence lifetime as a minor component, which is attributed to the TICT excited state, was reduced by addition of the substrate. The results provide that the hydrophobicity in the dimer interface is increased by the substrate binding. Interestingly, we found that the structure of an initial form is different from that of a refolded form after the dissociation of the substrate using a spectral subtraction technique. We have achieved detection of the changing structure induced by the substrate binding and dissociation using a steady-state and time-resolved fluorescence.

Patterned poly(N-isopropylacrylamide) brushes on silica surfaces by microcontact printing followed by surface-initiated polymerization.

Patterned poly(N-isopropylacrylamide) (PNIPAAm) brushes were fabricated on oxidized silicon wafers by surface-initiated atom transfer radical polymerization of N-isopropylacrylamide from a micropatterned initiator. The patterned surface initiator was prepared by microcontact-printing octadecyltrichlorosilane and backfilling with 3-(aminopropyl)triethoxysilane followed by amidization with 2-bromo-2-methylpropionic acid. XPS and FTIR confirmed the chemical structure of the surface initiator and the PNIPAAm brushes. Surface analysis techniques, including ellipsometry, contact angle goniometry, and X-ray reflectometry (XRR), were used to characterize the thickness, roughness, hydrophilicity, and density of the polymer brushes. Tapping-mode AFM imaging confirmed the successful patterning of the PNIPAAm brushes on the oxidized silicon substrates. Variable temperature ellipsometry indicated that the lower critical solution temperature of the hydrated PNIPAAm brush was broad, occurring over the range of 20-35 degrees C. A solvatochromic fluorophore, 6-propionyl-2-dimethylaminonaphthalene (Prodan), in the PNIPAAm brush layers yielded a very similar emission to that in DMF, which can be attributed to the similarity of their chemical structures. Fluorescence microscopy further proved the successful patterning of the polymer brushes and suggested that the Prodan is localized in the patterned PNIPAAm brushes and excluded from the surrounding octadecyltrichlorosilane regions.

Preface: Special Issue on Solvatochromic Fluorophores

Preface: Special Issue on Solvatochromic Fluorophores