Hemicyanine Chromophore Research Articles

Ribozyme‐Catalyzed Late‐Stage Functionalization and Fluorogenic Labeling of RNA

AbstractSite‐specific introduction of bioorthogonal handles into RNAs is in high demand for decorating RNAs with fluorophores, affinity labels or other modifications. Aldehydes represent attractive functional groups for post‐synthetic bioconjugation reactions. Here, we report a ribozyme‐based method for the synthesis of aldehyde‐functionalized RNA by directly converting a purine nucleobase. Using the methyltransferase ribozyme MTR1 as an alkyltransferase, the reaction is initiated by site‐specific N1 benzylation of purine, followed by nucleophilic ring opening and spontaneous hydrolysis under mild conditions to yield a 5‐amino‐4‐formylimidazole residue in good yields. The modified nucleotide is accessible to aldehyde‐reactive probes, as demonstrated by the conjugation of biotin or fluorescent dyes to short synthetic RNAs and tRNA transcripts. Upon fluorogenic condensation with a 2,3,3‐trimethylindole, a novel hemicyanine chromophore was generated directly on the RNA. This work expands the MTR1 ribozyme's area of application from a methyltransferase to a tool for site‐specific late‐stage functionalization of RNA.

Ribozyme-Catalyzed Late-Stage Functionalization and Fluorogenic Labeling of RNA.

Site-specific introduction of bioorthogonal handles into RNAs is in high demand for decorating RNAs with fluorophores, affinity labels or other modifications. Aldehydes represent attractive functional groups for post-synthetic bioconjugation reactions. Here, we report a ribozyme-based method for the synthesis of aldehyde-functionalized RNA by directly converting a purine nucleobase. Using the methyltransferase ribozyme MTR1 as an alkyltransferase, the reaction is initiated by site-specific N1 benzylation of purine, followed by nucleophilic ring opening and spontaneous hydrolysis under mild conditions to yield a 5-amino-4-formylimidazole residue in good yields. The modified nucleotide is accessible to aldehyde-reactive probes, as demonstrated by the conjugation of biotin or fluorescent dyes to short synthetic RNAs and full-length tRNA transcripts. Upon fluorogenic condensation with a 2,3,3-trimethylindole moiety, a novel hemicyanine chromo-phore was generated directly on the RNA. This work expands the MTR1 ribozyme's area of application from a methyltransferase to a tool for site-specific late-stage functionalization of RNA.

Impact of different auxochromes hydroxyl and methyl on two photon absorption coefficients and ultrafast dynamics of near-infrared hemicyanine dyes

There is immense interest in the design of organic nonlinear optical (NLO) chromophores for optical limiting and bioimaging applications. In this work, two near-infrared hemicyanine compounds which are named 2-(2-(3-Hydroxy-8,9-dihydro-7H-pyrido[1,2-a]quinazolin-10-yl)vinyl)-1,1-dimethyl-3-propy l-1H-benzo[e]indol-3-ium iodide (Hemicyanine1) and 1,1-Dimethyl-2-(2-(3-methyl-8,9-dihydro-7Hpyrido[1,2-a]quinazolin-10-yl)vinyl)-3-propyl-1H-benzo[e]indol-3-ium iodide (Hemicyanine2) were synthesized. The difference between Hemicyanine1 and Hemicyanine2 is that one of the auxochrome is hydroxyl and the other is methyl. The third-order nonlinear optical absorption properties of Hemicyanine1 and Hemicyanine2 were investigated using broadband Z-scan measurements at nanosecond time regime. The Z-scan results demonstrate that both chromophores have reverse-saturable absorption (RSA), while the NLO response of Hemicyanine2 is significantly stronger than Hemicyanine1. The corresponding physical mechanism were studied via femtosecond transient absorption (TA) spectroscopy. The results of femtosecond TA measurement demonstrate that the lifetime of excited state of Hemicyanine2 is considerably longer than that of Hemicyanine1. Our study indicated that the auxochrome is critically important in optimizing the NLO response and excited-state lifetime of hemicyanine chromophores for photonic applications.

Colorimetric and orange light-emitting fluorescent probe for pyrophosphate in water

A dual-mode probe based on a benzothiazolium hemicyanine chromophore was designed and synthesized for the detection of pyrophosphate (PPi) in water. The use of a fluorescent probe for colorimetric and long-wavelength fluorescence detection of PPi could be suitable for both rapid in-field and bioimaging experiments.

Photoactivatable Hemicyanine Chromophores as Fluorescent Lables

A series of photoactivatable hemicyanine chromophores containing azido group instead of the usual amino donor group has been designed and synthesized. Before photolysis, these compounds have negligible fluorescence. Upon photolysis, a molecule of nitrogen is lost from the azido group leaving behind a highly reactive nitrene moiety, which can react with and crosslink neighboring molecules to produce an amine. This fully restores the “push-pull” environmentally sensitive hemicyanine chromophore. If the molecule is in a cell membrane it will covalently crosslink lipid or protein molecules and remain relatively immobile. The photoactivity of the dyes was tested on neuroblastoma cells by selectively shining near UV or visible light on a small region of the cell and then observing the whole cell's fluorescence using a confocal microscope. This experiment shows strong fluorescence primarily in the illuminated area, which proves that we can selectively and stably stain a portion of the cell membrane using these photoactivatable hemicyanine dyes. The photoreleased dyes are fluorescent in the membrane because the amino-chromophore is similar to our voltage sensitive ASP and ANEP chromophores. These compounds are promising as targetable sensors of membrane potential. (This work is supported by Human Frontier Science Program grant RGP0027/2009 and NIH grant R01EB001963.)

Fluorinated Voltage Sensitive Dyes for SHG and Multiphoton Microscopy

Dyes based on hemicyanine chromophores have high membrane affinities and the ability to report on local membrane environment and act as sensors of membrane voltage. Laser-scanning second harmonic generation (SHG) microscopy utilizing such voltage sensitive dyes has shown considerable promise as an imaging modality, possessing several advantages over fluorescence for the optical mapping of membrane potentials. The addition of electronegative fluorine atoms to the chromophore is intended to lower both the ground and excited state energies, so as to make dyes less susceptible to photobleaching. Improved photostability will allow extension of the duration of optical recording measurements, permit the use of more intense laser excitation, and minimize photodamage to the biological sample. The effect of fluorination on photostability and dye performance has been systematically investigated for a series of newly synthesized dyes and found to depend critically on the location of the substituent within the chromophore. Voltage-clamped neuroblastoma cells stained with these dyes were imaged with 1064nm excitation, allowing sensitivities and response kinetics of SHG and two-photon fluorescence to be determined simultaneously for several fluorinated dyes. Our results suggest that voltage sensitive dyes can be developed which have large SHG signal changes, sufficient photostability, and the requisite speed for use as a practical tool for measuring electrical activity in biological systems.(Supported by NIH grant EB001963)

Improving the Photostability of Membrane Probes through Fluorination

High photostability is a desired property for fluorescent probes, especially when long recording time is necessary. It becomes more crucial for long wavelength fluorescent dyes because they generally have lower fluorescence quantum yields and are more vulnerable to chemical and photochemical attacks. We have developed a series of optical probes with high membrane affinities that are able to report on their local membrane environment and act as sensors of membrane voltage. These dyes have hemicyanine chromophores with donor-bridge-acceptor structures. Following our recent success in developing near-infrared fluorescent membrane probes, we further tried to improve the performance of these dyes through fluorination. Synthetic pathways to hemicyanines with fluorine substitution at various positions have been developed and the performances of these fluorinated dyes have been evaluated. It was found that the effect of fluorination depends critically on the site of fluorination: donor, acceptor, or bridge. Fluorination on the vinylene bridge appears to give optimal outcomes: improvement in photostability, red-shifts in absorption and emission spectra, and increase in absorption cross section. On the other hand, fluorination on the pyridinium acceptor results in an undesirable outcome: the dye is much less stable than the nonfluorinated counterpart in PBS buffer. Fluorinated membrane dyes generally show response kinetics and voltage sensitivities similar to their nonfluorinated counterparts when tested in a voltage-clamped hemispherical lipid bilayer (HLB) apparatus and in nonlinear optical imaging. (Supported by NIH grants EB001963 and U54RR022232)

The synthesis and spectroscopic investigation of dichromophoric hemicyanine dyes

Alkane α,γ-bis[4-methylpyridinium] diiodide and alkane α,γ-bis[4-methylquinolinium] diiodide were prepared and used as starting materials in the synthesis of dichromophoric cyanine dyes. Structural confirmation of the synthesized dyes was carried out by 1H NMR, MS spectroscopy and elemental analysis. The electronic absorption and the emission spectra for all the hemicyanines were measured in four organic solvents of varying polarity. All derivatives absorb in the region of about 500 nm and have molar absorptivity of about 10 4 M −1 cm −1. The absorption spectra of tested compounds are affected by their structure i.e. the structure of dialkyl(aryl)amino group, the electron acceptor part of the dye and the linker between two identical hemicyanine chromophores. The fluorescence bands' positions, in contrast to the absorption, are strongly affected by the solvent polarity.

Enhanced fluorescence in electrospun dye-doped DNA nanofibers.

Nanoscale fibers and non-woven meshes composed of DNA complexed with a cationic surfactant (cetyltrimethylammonium chloride, or CTMA) have been fabricated through electrospinning. The DNA-CTMA complex can be electrospun far more easily than DNA alone. Incorporation of a hemicyanine chromophore resulted in materials that demonstrated amplified emission as compared to thin films of identical composition. The enhanced fluorescence resulted from both the fiber morphology (5-6-fold amplification) and specific interactions (groove-binding) between the chromophore and DNA (18-21-fold amplification). The mechanical properties of freestanding electrospun non-woven fiber meshes were evaluated, and revealed stress-induced alignment of DNA strands within the DNA-CTMA fibers. These fiber-based materials are easily processable into a variety of morphologies, and have promise for applications in molecular electronics, filtration, sensors, and the medical industry.

Near-infrared photoluminescence of lanthanide complexes containing the hemicyanine chromophore

The near-infrared luminescence properties of three ( E)- N-hexadecyl- N′, N′-dimethylamino-stilbazolium tetrakis(1-phenyl-3-methyl-4-benzoyl-5-pyrazolonato) lanthanide(III) complexes are described. These three complexes, containing trivalent neodymium, erbium and ytterbium, respectively, show near-infrared luminescence in acetonitrile solution upon UV irradiation. Luminescence decay times have been measured. The complexes consist of a positively charged hemicyanine chromophore with a long alkyl chain and a tetrakis(pyrazolonato) lanthanide(III) anion. Because of the absence of an α-hydrogen atom in the pyrazolonato ligands, and because of the saturation of the coordination sphere by four bidentate ligands, the luminescence properties are enhanced when compared to, e.g. quinolinate complexes.

Hyper-Rayleigh scattering in the Fourier domain for higher precision: Correcting for multiphoton fluorescence with demodulation and phase data

An improved experimental technique for the suppression of the multiphoton fluorescence contribution in hyper-Rayleigh scattering experiments for the determination of the first hyperpolarizability of molecules in solution is presented. This improvement allows for a better correction for the fluorescence artifact, so as to eliminate any overestimation for the value of the first hyperpolarizability. The measurement of the demodulation only of the fluorescence as a function of modulation frequency [Olbrechts et al., Rev. Sci. Instrum. 69, 2233 (1998)] is now complemented by the measurement of the phase lag between the intermediate scattering and the time-delayed fluorescence. From the simultaneous data reduction of demodulation and phase shift toward the hyperpolarizability, fluorescence contribution, and fluorescence lifetime, an improvement in precision of 1 order of magnitude is demonstrated. This level of precision has been used to show the relative impact of f-orbital filling and ligands on the molecular second-order nonlinear optical response of lanthanide complexes containing a hemicyanine chromophore.

Molecular First Hyperpolarizability Data for Lanthanate Complexes Containing the Hemicyanine Chromophore

The molecular nonlinear optical polarizability, or first hyperpolarizability β, of four lanthanate complexes containing the hemicyanine 1-hexadecyl-4-{2-[4-(dimethylamino)phenyl]ethenyl}pyridinium chromophore has been determined with high precision. The experimental measurement of the phase shift and the demodulation between immediate hyper-Rayleigh scattering and time-delayed multiphoton fluorescence as a function of modulation frequency allows for the simultaneous data analysis of phase and demodulation toward precise values for fluorescence-free hyperpolarizability, multiphoton fluorescence contribution, and fluorescence lifetime. One order of magnitude improvement in precision is obtained with respect to the earlier analysis of demodulation data only. This level of precision has been used to show the relative impact of f-orbital filling and ligands on the molecular second-order nonlinear optical response of lanthanate complexes containing a hemicyanine chromophore. Implications for the earlier conclusions about better film formation for lanthanate complexes are discussed.

Ionic liquid crystals with hemicyanine chromophores

(E)-N-alkyl-4-(2-(4-dimethylaminophenyl)ethenyl) pyridinium bromides (hemicyanine bromides) with C14, C16 and C18 straight alkyl chains are described. The thermal behaviour of these compounds was investigated by differential scanning calorimetry (DSC) and by optical microscopy. The textures observed in polarised light revealed that these compounds exhibit a smectic A mesophase. A focal-conic fan texture was observed, together with regions showing homeotropic alignment. The phase behaviour of the compound with the C16 chain can be summarized as: Cr1·99·Cr2·249·SmA·268·I (dec.). The transition temperatures are rather independent of the length of the alkyl chain. The compounds have a strong tendency to supercool to a glassy mesophase. A structural model for the mesophase is proposed. The bromide anion of the hemicyanine bromides can be replaced by the bulky tetrakis(β-diketonato)lanthanide(III) anion. As the β-diketone, 2,4-pentanedione (acetylacetone, acac) and 1,3-diphenyl-1,3-propanedione (dibenzoylmethane, DBM) are chosen. None of the lanthanide complexes containing the hemicyanine chromophore shows a mesophase. The complexes either melt directly to an isotropic liquid, or decompose without melting.

Influence of hemicyanine dye structures on spectral properties of their supramolecular complexes with amylose

Hemicyanine chromophores bearing long-chain alkyl substitutents on the donor and/or acceptor ends have been prepared, and absorption and emission spectra of their supramolecular complexes with amylose have been studied in order to relate them to their supramolecular structures.

Langmuir Film-Forming and Second Harmonic Generation Properties of Lanthanide Complexes

A series of amphiphilic lanthanide complexes were designed and synthesized, in which the lanthanide complex anions act both as the counterions of hemicyanine and as the spacer within a Langmuir-Blodgett (LB) film. Studies on the surface pressure-area ({pi}-A) isotherms of these complexes show that the film-forming properties can be clearly improved if appropriate {Beta}-diketone ligands were chosen. The effects of molecular structures of the complexes, including the variation of lanthanide central ions, the structures of {Beta}-diketone ligands, and the length of alkyl chains in hemicyanines, on the film-forming properties of the materials are discussed. From second-harmonic generation experiments, the largely enhanced second-order molecular hyperpolarizability of lanthanide complexes with good film-forming properties were obtained compared with the hemicyanine iodide. This effect may be due in part to the local field effect but primarily molecular ordering and ordered segregation of hemicyanine chromophores by the bulky lanthanide complex anions. The charge separation of the hemicyanine chromophores was supported by the crystal structure of a model complex. The homogeneity of the film was verified by low angle X-ray diffraction and also by the linear relationship of the absorbance vs the number of layers of the LB films of a selected complex. 37 refs., 7more » figs., 3 tabs.« less

Efficient second-harmonic generation from all-polymeric Langmuir-Blodgett “AB” films containing up to 600 layers

Alternating Langmuir-Blodgett films were prepared for second harmonic (SH) generation from an “active” polymer containing a hemicyanine chromophore and various “passive” polymers. With the best “passive” polymer, films containing up to 300 active layers (600 layers in total with a total thickness of 1.5 μm) had the intensity of the SH signal produced from incident light of wavelength 1.064 μm proportional to the square of the number of active layers. The intensity of the SH signal was unchanged after the film had been left in the dark at 20 °C for 8 months. Over a period of 4 days the intensity of the SH signal was stable at 60 °C, but unstable at 80 °C.

Characterization of hemicyanine aggregates in air-water monolayers

Absorption spectra of a docosyl derivative of the hemicyanine chromophore were measured at the air-water interface as a function of surface concentration. At low compression, a broad band is observed corresponding to the monomer species. With increasing surface pressure two new bands grow in concurrently at the expense of the monomer band. Principal factor analysis of the spectra indicates that the two aggregate bands are produced by a single aggregate species. In terms of exciton theory, this corresponds to the aggregate geometry having two molecules per unit cell. The sensitivity of the aggregate structure to the local environment is shown by the shift in position of the absorption bands for a mixed hemicyanine-cationic diluent monolayer

Improved second-harmonic generation from Langmuir–Blodgett films of hemicyanine dyes

THE phenomenon of second-harmonic generation (SHG), whereby a material under illumination generates light at twice the incident frequency, is finding increasing use in optical signal processing. The principal requirement for SHG is a non-centrosymmetric structure; in organic materials, this can be achieved through the use of Langmuir–Blodgett films, which offer control of structure at the molecular level. SHG has been demonstrated in films composed of hemicyanine dyes of the general formula D—C6H4— CH=CH—C5H4N+— RX-, where D is an electron donor, R is usually a hydrophobic alkyl chain and X- is a counterion such as Br- or I-. The frequency-doubling properties of these films are sensitive to the choice of donor group1,2 the extent of molecular aggregation3,4and the type of packing5–7 Mixed films, in which the dye molecules are interspersed with an inert phase, have been used to reduce aggregation and consequently enhance SHG3,8, but these films have the potential disadvantage of phase separation. Here we show that the use of an amphiphilic anion as both counterion and spacer molecule gives rise to an ordered segregation of the hemicyanine chromophores, and greatly enhanced SHG.

Anisotropic second-order nonlinearities of organic monolayers.

Monolayers prepared from hemicyanine chromophores of high second-order nonlinearity (\ensuremath{\beta}=1.3\ifmmode\times\else\texttimes\fi{}${10}^{\mathrm{\ensuremath{-}}26}$ esu at resonance) have been used to study anisotropies of the concomitant ${\mathrm{\ensuremath{\chi}}}^{(2)}$-susceptibility tensor. All components of this third-rank tensor have been determined by Fourier analysis of the anisotropic coverage density ${\mathit{N}}_{\mathit{s}}$(\ensuremath{\varphi}) with respect to the angle of rotation about surface normal. Depending on the preparation both ${\mathit{C}}_{\mathit{s}}$ and ${\mathit{C}}_{2\mathit{v}}$ symmetry could be identified.

Local-field effects in Langmuir-Blodgett films of hemicyanine and behenic acid mixtures.

The effective local-field factors for a system of adsorbed molecules are measured in Langmuir-Blodgett films consisting of mixtures of the n-docosyl homologue of hemicyanine dye and behenic acid. The effect of the local-field correction factors on the interpretation of the molecular orientation of the hemicyanine chromophore, as determined by macroscopic measurements of the optical second-harmonic generation from these molecules, is reported, and the measured values of the local-field correction factors are compared with the theory of Ye and Shen. The range of the induced-dipole--induced-dipole interaction of hemicyanine chromophores in Langmuir-Blodgett films is seen to be \ensuremath{\sim}10 A\r{}.