Synthesis of Fluorescent Pyrrolo[1,2-a]pyrimidines from Fischer Carbene Complexes as Building Blocks.

A novel synthetic methodology is reported for the synthesis of fluorescent pyrrolo[1,2-a]pyrimidines. Fischer carbene complexes served as the synthetic platform for (3+3) cyclization to form the heterocyclic moiety. The reaction process furnished two products, their ratio being modulated by the metal, base, and solvent. The selectivity exhibited was studied by analyzing the potential energy surface with density functional theory tools. The photophysical properties of absorption and emission were also evaluated. The dyes absorbed at wavelengths of 240-440 nm, depending on the substituents. The maximum emission wavelength was in the range of 470-513 nm, with quantum yields of 0.36-1.0 and a high Stokes shift range of 75-226 nm.

Extended BODIPYs as Red-NIR Laser Radiation Sources with Emission from 610 nm to 750 nm.

Herein, we report the synthetic access to a set of π-extended BODIPYs featuring a penta-arylated (phenyl and/or thiophene) dipyrrin framework. We take advantage of the full chemoselective control of 8-methylthio-2,3,5,6-tetrabromoBODIPY when we conduct the Liebeskind-Srogl cross-coupling (LSCC) to functionalize exclusively the meso-position, followed by the tetra-Suzuki reaction to arylate the halogenated sites. All these laser dyes display absorption and emission bands in the red edge of the visible spectrum reaching the near-infrared with thiophene functionalization. The emission efficiency, both fluorescence and laser, of the polyphenylBODIPYs can be enhanced upon decoration of the peripheral phenyls with electron donor/acceptor groups at para positions. Alternatively, the polythiopheneBODIPYs show an astonishing laser performance despite the charge transfer character of the emitting state. Therefore, these BODIPYs are suitable as a palette of stable and bright laser sources covering the spectral region from 610 nm to 750 nm.

Structure and Conformation of Novel BODIPY Ugi Adducts.

Two novel BODIPY-Ugi (boron dipyrromethene) adducts exhibit peculiar room temperature (T=20 °C) H-1 NMR spectra in that several protons located at the aromatic aniline-type ring are lost in the baseline. This observation revealed the existence of a dynamic conformational process where rotation around the C-N bond is hindered. Variable-temperature H-1 and C-13 NMR spectroscopic analysis confirmed this conclusion; that is, low-temperature spectra show distinct signals for all four aromatic protons below coalescence, whereas average signals are recorded above coalescence (T=+120 °C). Particularly interesting was the rather large difference in chemical shifts for the ortho protons below coalescence, Δδ=1.45 ppm, which was explained based on DFT computational analysis. Indeed, the calculated lowest-energy gas-phase conformation of the BODIPY Ugi adducts locates one half of the aniline-type ring in the shielding anisotropic cone of the bridge phenyl ring in the BODIPY segment. This is in contrast to the solid-state conformation established by X-ray diffraction analysis that shows a nearly parallel arrangement of the aromatic rings, probably induced by crystal packing forces.

Synthesis of Polysubstituted Symmetrical BODIPYs via Fischer Carbene Complexes: Theoretical, Photophysical and Electrochemical Evaluation.

A series of new symmetrical highly substituted BODIPYs 6 a-l was synthesized through a prefunctionalization approach in 35 %-89 % yields from the pyrrole core. This strategy allowed modulation of the substituents at the different positions based on the choice of Fischer's alkynyl carbenes, oxazolones and aldehydes used as precursors. The substituent variation at positions 2, 6, 3 and 5 had the greatest effect on the modulation of their photophysical properties such as absorption (λabs ) and emission (λem ) wavelengths, extinction coefficient (ϵ), quantum yields (ϕ), Stokes shifts (Δν), fluorescence decay, radiative (krad ) and non-radiative (knr ) constants and the CIE 1931 coordinates. Theoretical calculations allowed to corroborate the effect of the substituents of meso-position on the modification of the dihedral angles. Cyclic voltammetry studies revealed that the BODIPY series presents similar redox potential behavior, being electrochemically active even in successive cycles, which suggests that transport by diffusion is the dominant process.

Melanic pigmentation and light preference within and between two Drosophila species.

Environmental adaptation and species divergence often involve suites of co-evolving traits. Pigmentation in insects presents a variable, adaptive, and well-characterized class of phenotypes for which correlations with multiple other traits have been demonstrated. In Drosophila, the pigmentation genes ebony and tan have pleiotropic effects on flies' response to light, creating the potential for correlated evolution of pigmentation and vision. Here, we investigate differences in light preference within and between two sister species, Drosophila americana and D. novamexicana, which differ in pigmentation in part because of evolution at ebony and tan and occupy environments that differ in many variables including solar radiation. We hypothesized that lighter pigmentation would be correlated with a greater preference for environmental light and tested this hypothesis using a habitat choice experiment. In a first set of experiments, using males of D. novamexicana line N14 and D. americana line A00, the light-bodied D. novamexicana was found slightly but significantly more often than D. americana in the light habitat. A second experiment, which included additional lines and females as well as males, failed to find any significant difference between D. novamexicana-N14 and D. americana-A00. Additionally, the other dark line of D. americana (A04) was found in the light habitat more often than the light-bodied D. novamexicana-N14, in contrast to our predictions. However, the lightest line of D. americana, A01, was found substantially and significantly more often in the light habitat than the two darker lines of D. americana, thus providing partial support for our hypothesis. Finally, across all four lines, females were found more often in the light habitat than their more darkly pigmented male counterparts. Additional replication is needed to corroborate these findings and evaluate conflicting results, with the consistent effect of sex within and between species providing an especially intriguing avenue for further research.

Effect of the substituents of new coumarin-imidazo[1,2-a]heterocyclic-3-acrylate derivatives on nonlinear optical properties: a combined experimental-theoretical approach.

A series of new coumarin-imidazo[1,2-a]heterocyclic-3-acrylate derivatives 7a-h were synthesized by the Heck reaction between the corresponding 3-(imidazo[1,2-a]pyrimidines)-(2-yl)-2H-chromen-2-ones 4a-e and methyl acrylate in 45-87% yields. The effect of the distinct substituents on third-order nonlinear optical properties was examined, experimentally measuring their nonlinear refractive indexes by the Z-scan technique. Density functional theory and time-dependent density functional theory were utilized with the B3LYP, CAM-B3LYP, PBE (PBEPBE), and M062X functionals on Gaussian09 software to calculate the vertical excitation, relaxation of the brightest excited states, conformation, HOMO-LUMO gaps, oscillator strength, polarizability, and hyperpolarizabilities of all derivatives. Although all acrylates showed a nonlinear response at a certain level of power, the compounds bearing a diethylamino electron-donating group exhibited higher nonlinear refractive index values (∼10-9 cm2 W-1), which is in agreement with the trend in the computational calculations of the first and second hyperpolarization. According to the structural analysis, the electron-withdrawing group (acrylate) is mainly responsible for the loss of coplanarity because of increasing the dihedral angle between the coumarin and imidazo[1,2-a]heterocyclic moieties (to 39.1°). On the other hand, the unsubstituted compound 4a presented the greatest nonlinearity due to its almost coplanar structure (n2 ∼ 10-8 cm2 W-1), highlighting the importance of this feature.

Organocatalytic Cascade Reactions for the Diversification of Thiopyrano-Piperidone Fused Rings Utilizing Trienamine Activation.

An aminocatalytic privileged diversity-oriented synthesis (ApDOS) strategy utilizing trienamine catalysis for the construction of diverse and complex thiopyrans-piperidone fused rings through a thia-Diels-Alder/nucleophilic ring-closing sequence by using dithioamides as activated heterodienophiles is reported. Following this strategy, a super cascade reaction to assemble nine fused rings can be achieved by employing a bis-dithioamide. Additionally, by linking an indole moiety on the dithioamide, a Pictet-Spengler type reaction can be promoted once the cascade sequence has been achieved, leading to more complex penta- hexa- and heptacyclic fused ring derivatives in a one-pot process. This investigation opens new perspectives for the synthesis of a new class of complex and diverse thiopyrans that contribute to populate new relevant regions in the chemical space.

A static and dynamic NMR study of 10-hydrazino-BODIPY.

10-Hydrazino-BODIPY, BoNHNH2 , presents slow rotation about the C10-NH bond that results in anisochronous 1 H and 13 C NMR signals. The assignment of the different signals has been made using traditional two-dimensional methods as well as spin-spin coupling constants and confirmed by DFT calculations (B3LYP) using the 6-311++G(d,p) basis set. The rotational barrier has been determined in three pairs of proton signals and compared with the calculated barrier.

Sulfone derivatives enter the cytoplasm of Candida albicans sessile cells.

Since our study showed that sulfone derivatives' action mode creates a lesser risk of inducing widespread resistance among Candida spp., we continued verifying sulfones' antifungal activity using the following newly synthesized derivatives: bromodichloromethy-4-hydrazinyl-3-nitrophenyl sulfone (S1), difluoroiodomethyl-4-hydrazinyl-3-nitrophenyl sulfone (S2), and chlorodifluoromethyl-4-hydrazinyl-3-nitrophenyl sulfone (S3). As the mechanism by which sulfones gain access to the cytoplasm has not been elucidated yet, in order to track S1-3, we coupled their hydrazine group with BODIPY (final S1-3 BODIPY-labelled were named SB1-3). This approach allowed us to follow the vital internalization and endocytic routing of SB1-3, while BODIPY interacts primarily with fungal surfaces, thus confirming that S1-3 and their counterparts SB1-2 behaved as non-typical agents by damaging the cell membrane and wall after being endocytosed (SB1-3 fluorescence visible inside the unlysed sessile cells). Thus greatly decreasing the likelihood of the appearance of strains resistance. Core sulfones S1-3 are a promising alternative not only to treat planktonic C. albicans but also biofilm-embedded cells. In the flow cytometric analysis, the planktonic cell surface was digested by S1-3, which made the externalized PS accessible to AnnexinV binding and PI input (accidental cell death ACD). The occurrence of ACD as well as apoptosis (crescent-shaped nuclei) and anoikis of sessile cells (regulated cell death by 100%-reduction in attachment to epithelium) was assessed through monitoring the AO/PI/HO342 markers. CLSM revealed the invasion of S1-3 and SB1-3 in C. albicans without inducing cell lysis. This was a novel approach in which QCM-D was used for real-time in situ detection of viscoelastic changes in the C. albicans biofilm, and its interaction with S1 as a representative of the sulfones tested. S1 (not toxic in vivo) is a potent fungicidal agent against C. albicans and could be administered to treat invasive candidiasis as a monotherapy or in combination with antifungal agents of reference to treat C. albicans infections.

Ready Access to Molecular Rotors Based on Boron Dipyrromethene Dyes-Coumarin Dyads Featuring Broadband Absorption.

Herein we report on a straightforward access method for boron dipyrromethene dyes (BODIPYs)-coumarin hybrids linked through their respective 8- and 6- positions, with wide functionalization of the coumarin fragment, using salicylaldehyde as a versatile building block. The computationally-assisted photophysical study unveils broadband absorption upon proper functionalization of the coumarin, as well as the key role of the conformational freedom of the coumarin appended at the meso position of the BODIPY. Such free motion almost suppresses the fluorescence signal, but enables us to apply these dyads as molecular rotors to monitor the surrounding microviscosity.

Molecular Mechanism of Viscosity Sensitivity in BODIPY Rotors and Application to Motion-Based Fluorescent Sensors.

Viscosity in the intracellular microenvironment shows a significant difference in various organelles and is closely related to cellular processes. Such microviscosity in live cells is often mapped and quantified with fluorescent molecular rotors. To enable the rational design of viscosity-sensitive molecular rotors, it is critical to understand their working mechanisms. Herein, we systematically synthesized and investigated two sets of BODIPY-based molecular rotors to study the relationship between intramolecular motions and viscosity sensitivity. Through experimental and computational studies, two conformations (i.e., the planar and butterfly conformations) are found to commonly exist in BODIPY rotors. We demonstrate that the transformation energy barrier from the planar conformation to the butterfly conformation is strongly affected by the molecular structures of BODIPY rotors and plays a critical role in viscosity sensitivity. These findings enable rational structure modifications of BODIPY molecular rotors for highly effective protein detection and recognition.

BODIPY as electron withdrawing group for the activation of double bonds in asymmetric cycloaddition reactions.

In this work we have found that a BODIPY can be used as an electron withdrawing group for the activation of double bonds in asymmetric catalysis. The synthesis of cyclohexyl derivatives containing a BODIPY unit can easily be achieved via trienamine catalysis. This allows a new different asymmetric synthesis of BODIPY derivatives and opens the door to future transformation of this useful fluorophore. In addition, the Quantum Chemistry calculations and mechanistic studies provide insights into the role of BODIPY as an EWG.

Analysis of some immunogenic properties of the recombinant Sporothrix schenckii Gp70 expressed in Escherichia coli.

AIM: Sporothrix schenckii is the causative agent of sporotrichosis. A 70-kDa glycoprotein, Gp70, is a candidate for the development of prophylactic alternatives to control the disease, and its gene (GP70) is predicted to encode for a protein of 43 kDa, contrasting with the molecular weight of the native protein. MATERIALS & METHODS: The GP70 was expressed in bacteria, the recombinant protein purified, used in immunoassays and injected to Galleria mellonella. RESULTS & CONCLUSION: The recombinant protein was detected by anti-Gp70 antibodies, confirming that the Gp70 backbone is a 43-kDa peptide. This protein showed enzyme activity of cyclase and was recognized by sera of patients with sporotrichosis. Although it was not useful for serodiagnosis of sporotrichosis, it conferred protection to animals against experimental sporotrichosis.

Synthetic Approach to Readily Accessible Benzofuran-Fused Borondipyrromethenes as Red-Emitting Laser Dyes.

We took advantage of the chemoselective meso-functionalization of 2,3,5,6-tetrabromo-8-methylthioBODIPY 6 to prepare a series of 2,3,5,6-tetrabromo-8-arylBODIPY derivatives suitable for SNAr substitution reactions with phenols exclusively at positions 3 and 5. Pd(0)-catalyzed intramolecular arylation reaction ensued on the remaining brominated positions 2 and 6 to give a new family of benzofuran-fused BODIPY dyes. This method utilizes readily available starting materials and allows for the preparation of the title compounds with excellent functional group tolerance. Moreover, it was demonstrated that the methodology described herein is amenable for the incorporation of biomolecules. The photophysical and lasing properties of the benzofuran-fused BODIPY dyes were thoroughly analyzed with the aid of electrochemical measurements and quantum mechanical simulations. These dyes show bright and intriguing emission (both fluorescence and laser) toward the red edge of the visible spectrum with remarkable tolerance under strong and continuous irradiation.

Fully Functionalizable ß,ß'-BODIPY Dimer: Synthesis, Structure, and Photophysical Signatures.

The versatility in the synthesis of BODIPY derivatives in terms of functionalization is further demonstrated. In particular, in this work ß-ß'-BODIPY dimers with varied functional groups in the meso positions were synthesized in very efficient yields and short reaction times from a single platform. A photophysical study was carried out in all of the compounds. The resultant dimers show absorption bands at around 600 nm as a consequence of electronically coupled monomers disposed with a dihedral angle of around 30°, which is supported by theoretical simulations. The emission properties of these molecules are distinguished by the appearance of an ICT state as the polarity of the solvent increases.

Synthesis, Photophysical Study, and Biological Application Analysis of Complex Borondipyrromethene Dyes.

A series of complex boronic acids were prepared through multicomponent reactions (MCRs). Both Passerini and Ugi MCRs were carried out in which one component was an arylboronic acid. The resulting highly functionalized boronic acids participated efficiently in the Liebeskind-Srogl cross-coupling reaction with meso-methylthioBODIPY derivatives to yield complex borondipyrromethene (BODIPY) dyes in good yields. The joined spectroscopic and computational study points out the deep impact of the arylated chromophoric position on the photophysical signatures. Thus, unconstrained aryls grafted at the meso position did not sway the spectral band positions but switched on new nonradiative relaxation channels, whereas additional arylation at the opposite α-pyrrolic position softened such fluorescence quenching and shifted the emission to the red-edge of the visible spectrum. The conducted biological analysis revealed that peripheral blood mononuclear cells incubated with these new compounds showed reduced cytotoxicity and retained their normal activities. Additionally, the dyes remained stable inside the cells after 24 h of incubation. These results demonstrated that these novel fluorescent probes based on BODIPY can be applied for cell imaging and analysis, expanding their applications.

Development of a Fluorescent Bodipy Probe for Visualization of the Serotonin 5-HT1A Receptor in Native Cells of the Immune System.

Serotonin (5-HT) modulates key aspects of the immune system. However, its precise function and the receptors involved in the observed effects have remained elusive. Among the different serotonin receptors, 5-HT1A plays an important role in the immune system given its presence in cells involved in both the innate and adaptive immune responses, but its actual levels of expression under different conditions have not been comprehensively studied due to the lack of suitable tools. To further clarify the role of 5-HT1A receptor in the immune system, we have developed a fluorescent small molecule probe that enables the direct study of the receptor levels in native cells. This probe allows direct profiling of the receptor expression in immune cells using flow cytometry. Our results show that important subsets of immune cells including human monocytes and dendritic cells express functional 5-HT1A and that its activation is associated with anti-inflammatory signaling. Furthermore, application of the probe to the experimental autoimmune encephalomyelitis model of multiple sclerosis demonstrates its potential to detect the specific overexpression of the 5-HT1A receptor in CD4+ T cells. Accordingly, the probe reported herein represents a useful tool whose use can be extended to study the levels of 5-HT1A receptor in ex vivo samples of different immune system conditions.

A palette of background-free tame fluorescent probes for intracellular multi-color labelling in live cells.

A multi-color labelling technique for visualizing multiple intracellular apparatuses in their native environment using small fluorescent probes remains challenging. This approach requires both orthogonal and biocompatible coupling reactions in heterogeneous biological systems with minimum fluorescence background noise. Here, we present a palette of BODIPY probes containing azide and cyclooctyne moieties for copper-free click chemistry in living cells. The probes, referred to as 'tame probes', are highly permeable and specific in nature, leaving no background noise in cells. Such probes, which are rationally designed through optimized lipophilicity, water solubility and charged van der Waals surface area, allow us to demonstrate rapid and efficient concurrent multi-labelling of intracellular target components. We show that these probes are capable of not only labelling organelles and engineered proteins, but also showing the intracellular glycoconjugates' dynamics, through the use of metabolic oligosaccharide engineering technology in various cell types. The results demonstrated in this study thus provide flexibility for multi-spectral labelling strategies in native systems in a high spatiotemporal manner.

Exploring viscosity, polarity and temperature sensitivity of BODIPY-based molecular rotors.

Microviscosity is a key parameter controlling the rate of diffusion and reactions on the microscale. One of the most convenient tools for measuring microviscosity is by fluorescent viscosity sensors termed 'molecular rotors'. BODIPY-based molecular rotors in particular proved extremely useful in combination with fluorescence lifetime imaging microscopy, for providing quantitative viscosity maps of living cells as well as measuring dynamic changes in viscosity over time. In this work, we investigate several new BODIPY-based molecular rotors with the aim of improving on the current viscosity sensing capabilities and understanding how the structure of the fluorophore is related to its function. We demonstrate that due to subtle structural changes, BODIPY-based molecular rotors may become sensitive to temperature and polarity of their environment, as well as to viscosity, and provide a photophysical model explaining the nature of this sensitivity. Our data suggests that a thorough understanding of the photophysics of any new molecular rotor, in environments of different viscosity, temperature and polarity, is a must before moving on to applications in viscosity sensing.

Adapting BODIPYs to singlet oxygen production on silica nanoparticles.

A modified Stöber method is used to synthesize spherical core-shell silica nanoparticles (NPs) with an external surface functionalized by amino groups and with an average size around 50 nm. Fluorescent dyes and photosensitizers of singlet oxygen were fixed, either separately or conjointly, respectively in the core or in the shell. Rhodamines were encapsulated in the core with relatively high fluorescence quantum yields (Φfl ≥ 0.3), allowing fluorescence tracking of the particles. Various photosensitizers of singlet oxygen (PS) were covalenty coupled to the shell, allowing singlet oxygen production. The stability of NP suspensions strongly deteriorated upon grafting the PS, affecting their apparent singlet oxygen quantum yields. Agglomeration of NPs depends both on the type and on the amount of grafted photosensitizer. New, lab-made, halogenated 4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes (BODIPY) grafted to the NPs achieved higher singlet oxygen quantum yields (ΦΔ âˆ¼ 0.35-0.40) than Rose Bengal (RB) grafted NPs (ΦΔ âˆ¼ 0.10-0.27). Finally, we combined both fluorescence and PS functions in the same NP, namely a rhodamine in the silica core and a BODIPY or RB grafted in the shell, achieving the performance Φfl ∼ 0.10-0.20, ΦΔ âˆ¼ 0.16-0.25 with a single excitation wavelength. Thus, proper choice of the dyes, of their concentrations inside and on the NPs and the grafting method enables fine-tuning of singlet oxygen production and fluorescence emission.