Trimethine Cyanine Research Articles

Atom‐Efficient Synthesis of Trimethine Cyanines Using Formaldehyde as a Single‐Carbon Source

AbstractHerein, we present an innovative and atom‐efficient synthesis of trimethine cyanines (Cy3) using formaldehyde (FA) as a single‐carbon reagent. The widespread application of Cy3 dyes in bioimaging and genomics/proteomics is often limited by synthetic routes plagued by low atom economy and substantial side‐product formation. Through systematic investigation, we have developed a practical and efficient synthetic pathway for both symmetrical and unsymmetrical Cy3 derivatives, significantly minimizing resource utilization. Notably, this approach yields water as the by‐product, in alignment with sustainable chemistry principles. Moreover, the efficient one‐pot synthesis facilitates the detection of intracellular FA levels, utilizing the fluorescence signal of Cy3 in live cells. It is also possible to detect endogenous FA in the intestinal tissues. We observed a significant decrease in FA in the small intestine of inflammatory bowel disease (IBD) mice as compared to healthy mice. This methodological advancement not only enhances the scope of fluorescent dye synthesis but also contributes to sustainable practices within chemical manufacturing, offering a significant leap forward in the development of environmentally friendly synthetic strategies.

Atom-Efficient Synthesis of Trimethine Cyanines Using Formaldehyde as a Single-Carbon Source.

Herein, we present an innovative and atom-efficient synthesis of trimethine cyanines (Cy3) using formaldehyde (FA) as a single-carbon reagent. The widespread application of Cy3 dyes in bioimaging and genomics/proteomics is often limited by synthetic routes plagued by low atom economy and substantial side-product formation. Through systematic investigation, we have developed a practical and efficient synthetic pathway for both symmetrical and unsymmetrical Cy3 derivatives, significantly minimizing resource utilization. Notably, this approach yields water as the by-product, in alignment with sustainable chemistry principles. Moreover, the efficient one-pot synthesis facilitates the detection of intracellular FA levels, utilizing the fluorescence signal of Cy3 in live cells. It is also possible to detect endogenous FA in the intestinal tissues. We observed a significant decrease in FA in the small intestine of inflammatory bowel disease (IBD) mice as compared to healthy mice. This methodological advancement not only enhances the scope of fluorescent dye synthesis but also contributes to sustainable practices within chemical manufacturing, offering a significant leap forward in the development of environmentally friendly synthetic strategies.

Thermal Truncation of Heptamethine Cyanine Dyes.

Cyanine dyes are a class of organic, usually cationic molecules containing two nitrogen centers linked through conjugated polymethine chains. The synthesis and reactivity of cyanine derivatives have been extensively investigated for decades. Unlike the recently described phototruncation process, the thermal truncation (chain shortening) reaction is a phenomenon that has rarely been reported for these important fluorophores. Here, we present a systematic investigation of the truncation of heptamethine cyanines (Cy7) to pentamethine (Cy5) and trimethine (Cy3) cyanines via homogeneous, acid-base-catalyzed nucleophilic exchange reactions. We demonstrate how different substituents at the C3' and C4' positions of the chain and different heterocyclic end groups, the presence of bases, nucleophiles, and oxygen, solvent properties, and temperature affect the truncation process. The mechanism of chain shortening, studied by various analytical and spectroscopic techniques, was verified by extensive ab initio calculation, implying the necessity to model catalytic reactions by highly correlated wave function-based methods. In this study, we provide critical insight into the reactivity of cyanine polyene chains and elucidate the truncation mechanism and methods to mitigate side processes that can occur during the synthesis of cyanine derivatives. In addition, we offer alternative routes to the preparation of symmetrical and unsymmetrical meso-substituted Cy5 derivatives.

Spectral-fluorescent study of substituted trimethine cyanine dyes in solutions and in complexes with DNA. Effects of aggregation, moderate heating, and decreasing pH

Trimethine cyanine dyes are widely used as probes for the detection, study and quantification of biomolecules. In particular, cationic trimethine cyanines noncovalently interact with DNA with growing fluorescence. However, their use is often limited by the tendency to self-association – to the formation of aggregates. Disubstituted trimethine cyanines with hydrophobic substituents are especially prone to aggregation. In this work, we studied the interaction of a number of substituted trimethine cyanines with DNA (in aqueous buffer solutions) and showed that their aggregation strongly interfered with their use as fluorescent probes for DNA. To eliminate this drawback, preliminary heating of dye solutions with DNA to 60–70 °C was used, followed by cooling to room temperature. Compared to the experiments without heating, an increase in the dye fluorescence intensity was observed due to the partial thermal decomposition of the aggregates and the interaction of the resulting monomers with DNA. To decompose aggregates, another method was also used – protonation of the dyes with amino substituents in buffer solutions with pH 5.0, which also led to growing the dye fluorescence intensity in the presence of DNA. Complexes of the dyes with DNA were modeled using molecular docking. Effective binding constants of the dyes to DNA and detection limits when using the dyes as probes for DNA (LOD and LOQ) were determined. It is shown that dye 3 with heating in neutral buffer and dye 1 in acidic buffer may be recommended as sensitive probes for DNA. It is concluded that the method of preliminary heating may be applied to dyes prone to aggregation, for improving their properties as biomolecular probes. Another possible means to reduce the interfering effects of dye aggregates is to use easily protonated dyes (with amino substituents) in slightly acidic media.

Abstract 1654 Retro-Diels-Alder Phototruncation of Heptamethine Cyanine to Trimethine Cyanine through an Allene Hydroperoxide Intermediate

Abstract 1654 Retro-Diels-Alder Phototruncation of Heptamethine Cyanine to Trimethine Cyanine through an Allene Hydroperoxide Intermediate

Crystallographic models of ribbons and ribbon-based J-aggregate nanotubes from the geometry of tube ends.

Self-assembly into tubular structures typically proceeds by helical winding of ribbon intermediates, however, only the central parts of the tubes, that retain no information on the ribbon geometry, have received attention so far. We propose the procedure of establishing the crystal structure of ribbons and ribbon-based tubes on the basis of crystallographic analysis of the tube-end geometry, where the terminal parts of the ribbons fold and form characteristic mono/bilayer polygonal shapes. The terminal parts of flattened J-aggregate nanotubes of trimethine cyanine dye were clearly resolved in electron microscopy and atomic force microscopy images, and the original parallelogram shape of ribbons was reconstructed and interpreted as a two-dimensional [1-10]/[010] facetted crystal with inclined molecular π-stacks parallel to the long ribbon side. The back-reconstructed molecular orientations in a tube wall tend to be close to the tube normal. A two-stage "nucleation and growth" type model of the ribbon to tube transition is proposed that takes into account the established ribbon mechanical asymmetry. The model includes closure of the single ribbon loop as a nucleation event and explains, mostly observed in experiments, nearly rectangular shapes of tubes by the transition kinetics. Due to its universal character, the suggested approach can be applied to any ribbon-based tubes, regardless of their chemical composition.

Bluer Phototruncation: Retro-Diels-Alder of Heptamethine Cyanine to Trimethine Cyanine through an Allene Hydroperoxide Intermediate.

The photoconversion of heptamethine to pentamethine cyanines and of pentamethine to trimethine cyanines was recently reported. Here, we report mechanistic studies and initial experimental evidence for a previously unexplored 4-carbon truncation reaction that converts the simplest heptamethine cyanine to the corresponding trimethine cyanine. We propose a DFT-supported model describing a singlet oxygen (1O2)-mediated formation of an allene hydroperoxide intermediate and subsequent 4-carbon loss through a retro-Diels-Alder process. Fluorescence and mass spectrometry measurements provide evidence of this direct conversion process. This 4-carbon truncation reaction adds to a growing body of cyanine reactivity and may provide an optical tool leading to a substantial blue-shift (Δλem) of ∼200 nm.

Sunlight-directed fluorophore-switch in photosynthesis of cyanine subcellular organelle markers for bio-imaging

The photoconvertible fluorophore synthesis enables the light controlled imaging channels switch for accurate tracking the quantity and localization of intracellular biomolecules in chemical biology. Herein, we repurposed the photochemistry of Fischer's base and developed a sunlight-directed fluorophore-switch strategy for high-efficiency trimethine cyanine (Cy3.5/Cy3) synthesis. The unexpected sunlight-directed photoconversion of Fischer's base proceeds in conventional solvents and accelerates in chloroform via photo-oxidation and hydrogen atom transfer without using extra additives, and the heterogenous dimerization mechanism was proposed and confirmed by isolation of the reactive intermediates. The reliable strategy is employed in the photosynthesis of commercially available cytomembrane marker (DiI) and other cyanine based organelle markers with appreciable yields. Sunlight-controlled fluorophore-switch of subcellular organelle markers in living cells validated the feasibility of our strategy with cell-tolerant character. Moreover, remote control synthesis of Cy3.5 in vivo directed via sunlight further demonstrated the extended application of our strategy. Therefore, this sunlight-directed strategy will facilitate exploitation of cyanine-based probes with switched fluorescence imaging channels and further enable precise description of the dynamic variations in living cells with minimal autofluorescence and cellular disturbance.

Polymethine chain modified trimethine cyanine based fluorescent probe to selectively light up G-quadruplexes DNA in mitochondria

G-quadruplex (G4) is a noncanonical nucleic acid secondary structure that has been demonstrated to have an impact on many physiological and pathological processes. To facilitate the development of small molecule drugs targeting G4, it is critical to develop functional G4 fluorescent probes that selectively reflect the unknown physiological functions of G4 and enrich the library of G4 ligand molecules. In this study, trimethine cyanine with a p-hydroxybenzyl substituent at the α-position on the polymethine chain (α-HBCy3.5) was used as a fluorescent probe for G4. By introducing bulky benzyl substituent on the polymethine chain, α-HBCy3.5 showed excellent G4 selectivity and could identify G4s from single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) by fluorescence turn-on. Thus, red fluorescence is emitted by α-HBCy3.5 in the presence of G4s (606 nm). In addition, the ability of α-HBCy3.5 to stain DNA in mitochondria in a short time (internalization in 6 min) is expected to be developed for real-time detection and monitoring of endogenous G4 DNA in living cells.

Cy3 Cyanine Dye with Strong Fluorescence Enhancement for AGRO100 and Its Derivative.

Nucleic acids, as important substances for biological inheritance, have attracted extensive attention in the biomedical field. More and more cyanine dyes are emerging as one of the probe tools for nucleic acid detection due to their excellent photophysical properties. Here, we discovered that the insertion of the AGRO100 sequence can specifically disrupt the twisted intramolecular charge transfer (TICT) mechanism of the trimethine cyanine dye (TCy3), resulting in a clear "turn-on" response. Moreover, the fluorescence enhancement of TCy3 combined with the T-rich AGRO100 derivative is more obvious. One explanation for the interaction between dT (deoxythymidine) and positively charged TCy3 may be that its outer layer carries the most negative charge. This study provides a theoretical basis for the use of TCy3 as a DNA probe, which has promising applications in the DNA detection of biological samples. It also provides the basis for the following construction of probes with specific ability for recognition.

A trimethine cyanine dye for copper (II) detection based on the transformation between monomers and J-aggregates via G-quadruplex regulation

A trimethine cyanine dye for copper (II) detection based on the transformation between monomers and J-aggregates via G-quadruplex regulation

Spectral-fluorescent and photochemical study of 6,6′-di(benzoylamino)trimethine cyanine dyes in solutions as possible probes for DNA

Spectral-fluorescent and photochemical properties of trimethine cyanine dyes T-304, T-306, and T-307, having substituents in 6,6′-positions, in various organic solvents, in aqueous buffer solutions, in the presence of surfactants and ethanol additives, and the effect on these properties of addition of DNA have been studied. Strong aggregation of the dyes in aqueous and aqueous buffer solutions has been shown. This is due to increased hydrophobicity of the dyes, which makes it difficult to use them as spectral-fluorescent probes for DNA. In the presence of DNA, trimethine cyanines partially form highly fluorescent complexes of dye monomers with the biomolecule, with slight decomposition of the initial aggregates and the formation of aggregates on DNA molecules. The formation of different types of dye–DNA complexes, i.e., intercalation and binding in the DNA grooves, was modeled by molecular docking. Dye–DNA complexes were also studied by circular dichroism spectroscopy and by thermal dissociation of DNA. To reveal selectivity of the dyes, their interaction with human serum albumin was briefly studied. The presence of moderate concentrations of nonionic surfactants does not lead to a significant decomposition of aggregates, but leads to a biphasic dependence of the fluorescence intensity on the DNA concentration. At the same time, ethanol additives (15%) lead to a more or less linear concentration dependence of the fluorescence intensity, which makes it possible to use these dyes as fluorescent probes for DNA. The effective binding constants of the dyes to DNA and the limits of DNA detection using the dyes in the presence of 15% ethanol were estimated. Photoisomerization and generation of the triplet states of T-304, T-306, and T-307 have been also studied. Along with the fluorescence growth, complexation with DNA leads to an increase in the yield of the triplet states of the dyes. This creates a prerequisite for using the dyes in targeted PDT. In the presence of DNA, the decay kinetics of the triplet states are biexponential, which indicates different types of dye complexes with DNA. The rate constants of oxygen quenching of the triplet states of the dyes bound to DNA are significantly lower than the diffusion-controlled values (taking into account the spin-statistical factor), which is explained by the shielding effect on the triplet molecules in complexes with DNA. The data obtained show that dyes T-304, T-306 and T-307, with addition of 15% ethanol, can be used as possible fluorescent probes for DNA.

Oxygen‐Insensitive Delayed Fluorescence Based on Singlet Manifold

AbstractDelayed fluorescence molecules have important applications in time‐resolved luminescence imaging (including lifetime imaging) systems. However, existing approaches of generating delayed fluorescence in organic molecules rely on triplets as long‐lived intermediate states. As such, this triplet‐based delayed fluorescence can be severely plagued by its oxygen sensitivity. As the presence of oxygen or ambient air can hardly be avoided in living biosystems, oxygen‐insensitive delayed fluorescence is highly desirable. Herein, a new pathway is reported for oxygen‐insensitive delayed fluorescence which is produced only within the singlet manifold. Such an example is found in a cyto‐compatible and water‐soluble trimethine cyanine dye C3T‐H, which bears a very high molar extinction coefficient and a narrow‐band red emission. It is demonstrated that this dye exhibits notable delayed fluorescence which cannot be diminished by the presence of oxygen or ambient air. Time‐resolved imaging of living cells in the presence of oxygen is successfully achieved. Through quantum chemical calculations, a nonemissive dark‐state structure in the S1 state is found to be thermodynamically viable (energy barrier ≈8 kcal mol−1) through the molecular twisting around the trimethine bridge of C3T‐H, which serves as a “reservoir” of the excitation energy to enable the oxygen‐insensitive delayed fluorescence.

Trimethine Cyanine Dyes as NA-Sensitive Probes for Visualization of Cell Compartments in Fluorescence Microscopy.

We propose symmetrical cationic trimethine cyanine dyes with β-substituents in the polymethine chain based on modified benzothiazole and benzoxazole heterocycles as probes for the detection and visualization of live and fixed cells by fluorescence microscopy. The spectral-luminescent properties of trimethine cyanines have been characterized for free dyes and in the presence of nucleic acids (NA) and globular proteins. The studied cyanines are low to moderate fluorescent when free, but in the presence of NA, they show an increase in emission intensity up to 111 times; the most pronounced emission increase was observed for the dyes T-2 in the presence of dsDNA and T-1 with RNA. Spectral methods showed the binding of all dyes to nucleic acids, and different interaction mechanisms have been proposed. The ability to visualize cell components of the studied dyes has been evaluated using different human cell lines (MCF-7, A2780, HeLa, and Hs27). We have shown that all dyes are cell-permeant staining nucleus components, probably RNA-rich nucleoli with background fluorescence in the cytoplasm, except for the dye T-5. The dye T-5 selectively stains some structures in the cytoplasm of MCF-7 and A2780 cells associated with mitochondria or lysosomes. This effect has also been confirmed for the normal type of cell line-human foreskin fibroblasts (Hs27). The costaining of dye T-5 with MitoTracker CMXRos Red demonstrates specificity to mitochondria at a concentration of 0.1 μM. Colocalization analysis has shown signals overlapping of dye T-5 and MitoTracker CMXRos Red (Pearson's Coefficient value = 0.92 ± 0.04). The photostability study shows benzoxazole dyes to be up to ∼7 times more photostable than benzothiazole ones. Moreover, studied benzoxazoles are less cytotoxic at working concentrations than benzothiazoles (67% of cell viability for T-4, T-5 compared to 12% for T-1, and ∼30% for T-2, T-3 after 24 h). Therefore, the benzoxazole T-4 dye is proposed for nucleic acid detection in vitro and intracellular fluorescence imaging of live and fixed cells. In contrast, the benzoxazole dye T-5 is proposed as a good alternative to commercial dyes for mitochondria staining in the green-yellow region of the spectrum.

Detection of Lysozyme Amyloid Fibrils Using Trimethine Cyanine Dyes: Spectroscopic and Molecular Docking Studies

Due to their unique photophysical and photochemical properties and high sensitivity to the beta-pleated motifs, cyanine dyes have found numerical applications as molecular probes for the identification and characterization of amyloid fibrils in vitro and the visualization of amyloid inclusions in vivo. In the present study the spectroscopic and molecular docking techniques have been employed to evaluate the amyloid sensitivity and the mode of interaction between the trimethine cyanine dyes and native (LzN) and fibrillar (LzF) lysozyme. It was found that the trimethine association with non-fibrilar and fibrillar lysozyme is accompanied by the changes in dye aggregation extent. The molecular docking studies between trimethine dyes and lysozyme in the native and amyloid states indicate that: i) trimethines tend to form the most stable complexes with deep cleft of the native lysozyme; ii) the dye binding with non-fibrillar protein is governed by the hydrophobic interactions, π-stacking contacts between aromatic or cyclopentane ring of the cyanine and Trp in position 63 or 108 and hydrogen bonds between the OH-groups of the trimethines and acceptor atoms of Asp 101 (AK3-7) and Gln 57 (AK3-8) of LzN; iii) cyanine dyes form the energetically most favorable complexes with the groove Gly 2-Leu 4/Ser 8-Trp 10 of the lysozyme fibril core; iv) cyanines-LzF interaction is stabilised by hydrobhobic contacts, π-stacking interaction and hydrogen bonds. The dyes AK3-7, AK3-5 and AK3-11 were selected as the most prospective amyloid probes.

Photonics of Trimethine Cyanine Dyes as Probes for Biomolecules

Cyanine dyes are widely used as fluorescent probes in biophysics and medical biochemistry due to their unique photophysical and photochemical properties (their photonics). This review is focused on a subclass of the most widespread and studied cyanine dyes—trimethine cyanines, which can serve as potential probes for biomolecules. The works devoted to the study of the noncovalent interaction of trimethine cyanine dyes with biomolecules and changing the properties of these dyes upon the interaction are reviewed. In addition to the spectral-fluorescent properties, elementary photochemical properties of trimethine cyanines are considered, including: photoisomerization and back isomerization of the photoisomer, generation and decay of the triplet state, and its quenching by oxygen and other quenchers. The influence of DNA and other nucleic acids, proteins, and other biomolecules on these properties is covered. The interaction of a monomer dye molecule with a biomolecule usually leads to a fluorescence growth, damping of photoisomerization (if any), and an increase in intersystem crossing to the triplet state. Sometimes aggregation of dye molecules on biomolecules is observed. Quenching of the dye triplet state in a complex with biomolecules by molecular oxygen usually occurs with a rate constant much lower than the diffusion limit with allowance for the spin-statistical factor 1/9. The practical application of trimethine cyanines in biophysics and (medical) biochemistry is also considered. In conclusion, the prospects for further studies on the cyanine dye–biomolecule system and the development of new effective dye probes (including probes of a new type) for biomolecules are discussed.

An Insight into Symmetrical Cyanine Dyes as Promising Selective Antiproliferative Agents in Caco-2 Colorectal Cancer Cells.

Cancer remains one of the diseases with the highest worldwide incidence. Several cytotoxic approaches have been used over the years to overcome this public health threat, such as chemotherapy, radiotherapy, and photodynamic therapy (PDT). Cyanine dyes are a class of compounds that have been extensively studied as PDT sensitisers; nevertheless, their antiproliferative potential in the absence of a light source has been scarcely explored. Herein, the synthesis of eighteen symmetric mono-, tri-, and heptamethine cyanine dyes and their evaluation as potential anticancer agents is described. The influences of the heterocyclic nature, counterion, and methine chain length on the antiproliferative effects and selectivities were analysed, and relevant structure–activity relationship data were gathered. The impact of light on the cytotoxic activity of the most promising dye was also assessed and discussed. Most of the monomethine and trimethine cyanine dyes under study demonstrated a high antiproliferative effect on human tumour cell lines of colorectal (Caco-2), breast (MCF-7), and prostate (PC-3) cancer at the initial screening (10 µM). However, concentration–viability curves showed higher potency and selectivity for the Caco-2 cell line. A monomethine cyanine dye derived from benzoxazole was the most promising compound (IC50 for Caco-2 = 0.67 µM and a selectivity index of 20.9 for Caco-2 versus normal human dermal fibroblasts (NHDF)) and led to Caco-2 cell cycle arrest at the G0/G1 phase. Complementary in silico studies predicted good intestinal absorption and oral bioavailability for this cyanine dye.

Intrinsic Mitochondrial Reactive Oxygen Species (ROS) Activate the In Situ Synthesis of Trimethine Cyanines in Cancer Cells

AbstractEnvironment‐responsive in situ synthesis of molecular fluorescent dyes is challenging. Herein, we develop a photoextension strategy to make trimethine cyanines with decent conversion efficiency (up to 81 %) using 1‐butyl 2,3,3‐trimethyl 3H‐indole derivatives as the sole precursors, and demonstrate a free radical mechanism. In the inducer‐extension stage, free radicals and reactive oxygen species (ROS) were able to mediate similar reactions with no assistance of light. We explored a Mito‐extension strategy to in situ synthesize trimethine cyanines in the living cells. The cellular ROS‐dependence provided a foundation for preferential cyanine expression in cancer cells. Finally, we applied an iodized precursor as an intrinsic ROS‐activated theranostic agent that integrated mitochondria‐targeted cyanine synthesis, cell imaging and phototherapy.

Intrinsic Mitochondrial Reactive Oxygen Species (ROS) Activate the In Situ Synthesis of Trimethine Cyanines in Cancer Cells.

Environment-responsive in situ synthesis of molecular fluorescent dyes is challenging. Herein, we develop a photoextension strategy to make trimethine cyanines with decent conversion efficiency (up to 81 %) using 1-butyl 2,3,3-trimethyl 3H-indole derivatives as the sole precursors, and demonstrate a free radical mechanism. In the inducer-extension stage, free radicals and reactive oxygen species (ROS) were able to mediate similar reactions with no assistance of light. We explored a Mito-extension strategy to in situ synthesize trimethine cyanines in the living cells. The cellular ROS-dependence provided a foundation for preferential cyanine expression in cancer cells. Finally, we applied an iodized precursor as an intrinsic ROS-activated theranostic agent that integrated mitochondria-targeted cyanine synthesis, cell imaging and phototherapy.

Effect of Trimethine Cyanine Dye- and Folate-Conjugation on the In Vitro Biological Activity of Proapoptotic Peptides.

Despite continuous advances, anticancer therapy still faces several technical hurdles, such as selectivity on cellular and subcellular targets of therapeutics. Toward addressing these limitations, we have combined the use of proapoptotic peptides, trimethine cyanine dye, and folate to target the mitochondria of tumor cells. A series of proapoptotic peptides and their conjugates with a cyanine dye and/or folate were synthesized in the solid phase, and their toxicity in different human cell lines was assessed. Cyanine-bearing conjugates were found to be up to 100-fold more cytotoxic than the parent peptides and to localize in mitochondria. However, the addition of a folate motif did not enhance the potency or selectivity of the resulting conjugates toward tumor cells that overexpress folate receptor α. Furthermore, while dual-labeled constructs were also found to localize within the target organelle, they were not generally selective towards folate receptor α-positive cell lines in vitro.