Thiazole orange was synthetically incorporated into oligonucleotides by using the corresponding phosphoramidite as the building block for automated DNA synthesis. Due to the covalent fixation of the TO dye as a DNA base surrogate, the TO-modified oligonucleotides do not exhibit a significant increase of fluorescence upon hybridization with the counterstrand. However, if 5-nitroindole (NI) is present as a second artificial DNA base (two base pairs away from the TO dye) a fluorescence increase upon DNA hybridization can be observed. That suggests that a short-range photoinduced electron transfer causes the fluorescence quenching in the single strand. The latter result represents a concept that can be transferred to the commercially available Cy3 label. It enables the Cy3 fluorophore to display the DNA hybridization by a fluorescence increase that is normally not observed with this dye.
Fluorescent bioanalytics and fluorescent cell imaging of nucleic acids demands the design and synthesis of new dyes that exhibit potentially interesting optical properties particularly in combination with other dyes.[1] Dual covalent labeling of nucleic acids has turned out to be very useful for creating tailor-made fluorescent probes for such applications. [2] For instance, hybridization-sensitive probes can be created by two covalently attached thiazole orange (TO) fluorophores that interact excitonically only in the single strand. [3] On the other hand, observable fluorescence color changes can be achieved by applying an energy transfer that occurs between two adjacent fluorescent labels that are forced into close proximity by the surrounding DNA architecture. [4] With respect to the preparation of such dual labeled oligonucleotides recent developments of the "click"-type Huisgen-Sharpless-Meldal cycloaddition for nucleic acids offers great advantages since (unexpectedly) complicated and time-consuming syntheses of DNA building blocks for potentially interesting dyes can be avoided.[5]The cyanine-styryl-type dyes represent very promising candidates for nucleic acids based primarily on their recently reported property as bright noncovalent binders to RNA.[6] Herein, we present the synthesis of a "clickable" CyIQ (cyanine indole quinoline) fluorophore that can be easily attached covalently to any desired 2'-position in nucleic acids. Moreover, the optical properties of the CyIQ dye can be tuned by excitonic interaction with a second adjacent label, or by energy transfer processes with thiazole red (TR) [7] as a second fluorescence base surrogate. The CyIQ dye consists of a quinoline part that is conjugated with an indole by a styryl-type bridge. The dye can be prepared in a four-step synthesis including the introduction of the "clickable" azide functionality (Scheme 1). It starts with commercially available 2-methyl quinoline (1) that is alkylated with 3-iodopropanole (2) as the short linker between the dye and the azide group. Condensation of the methyl group of 3 in the presence of piperidine yields dye 5. In the next steps, an Appel reaction leads to iodide 6, and the azide is introduced by nucleophilic substitution to yield 7, ready for postsynthesis DNA modification.Due to its positive charge the CyIQ dye 5 is sufficiently water soluble for titration experiments with a random-sequence double-stranded DNA (Figure 1). The absorption spectra of this titration reveal significant changes; especially the absorption shift from 465 nm (without DNA) to 472 nm (with DNA) indicates excitonic dye-dye interactions of 5 (in the absence of DNA), which are interrupted by the interaction of the dye with an increasing amount of DNA. These dye-dye interactions cause a significant fluorescence quenching of 5 in aqueous solution without DNA. With increasing amounts of DNA the fluorescence intensity of the dye is recovered and finally enhanced to the 17-fold when the saturation plateau is reached. This fluorescence enhancemen...
Upconverting nanoparticles (UCNPs) are a highly attractive tool owing to their unique property of showing visible luminescence when excited in the near‐infrared (NIR) region. Plain UCNPs have no biorecognition capabilities, but functionalization of their surface with azido groups renders them conjugatable to ethynyl‐modified oligonucleotides in a bioorthogonal fashion. Single‐stranded DNA was covalently attached to the surface of UCNPs by click chemistry and purified by size exclusion chromatography (SEC) at elevated temperature. Covalent attachment was evidenced by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy. DNA conjugation makes the particle soluble in water and enables it to recognize its counter strand. Such UCNPs are capable of nonspecifically crossing cell membranes. Confocal microscopy reveals the high potential of the bright UCNPs for live cell imaging in the NIR, where the UCNP–DNA conjugates can be considered to act as a kind of nano‐sized lamp. Furthermore, cross‐linking of those DNA nanolamps yields highly emissive aggregates.
The insertion of cyanine dye azides as energy donor dyes via postsynthetic "click"-type cycloaddition chemistry with e.g. a new thiazole orange azide combined with thiazole red yields dual emitting DNA probes with good fluorescence readout properties.
A holistic, nontargeted mass spectrometric analysis of any herbal material and preparation is intimately connected to fast chemical profiling and visualization of secondary plant metabolite classes or single compounds. High-resolution mass spectral data enable a broad variety of analytical possibilities. Often a fast and comprehensive overview on compound classes (phytochemical profiling) is needed before single-substance considerations. We present a fast approach for the initial characterization and substance class profiling using relative mass defect plots for the visualization of herbal compositions. From a dataset of 1160 common plant metabolites that represent a varied mixture of molecular classes in polarity, glycosylation, and alkylation, manually annotated for substance classes, the relative mass defects were calculated using theoretical molecular masses. For the calculation of the relative mass defect, a new approach incorporating two correction functions to obtain correct relative mass defect results also for large hydrocarbons, and a multitude of polyhalogenated molecules was developed. Using the Khachyan algorithm, elliptical areas clustering substance classes within the relative mass defect plots were calculated. The resulting novel relative mass defect plots provide a quick way of two-dimensional substance class mapping directly from high-resolution mass spectral data and may be considered as a unique fingerprint for herbals, part of them or herbal preparations. We show that adding the retention time as a third dimension improves the resolution power of the two-dimensional relative mass defect plot and offers the possibility for a more detailed substance class mapping.
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