Aminophthalimide as a mimetic of purines and a fluorescent RNA base surrogate for RNA imaging.

Aminophthalimide and N,N-dimethylaminophthalimide are used as fluorescent mimetics of purines due to their similar size and their possibility for hydrogen bonding. Their C-nucleotides were synthetically incorporated into RNA by means of phosphoramidite chemistry, behave as nonspecific fluorescent base analogs with flexible hydrogen bonding capabilities, and show solvatochromic fluorescence that is suitable for RNA imaging in live cells.

Two-Factor Fluorogenicity of Tetrazine-Modified Cyanine-Styryl Dyes for Bioorthogonal Labelling of DNA.

Two green fluorescent tetrazine-modified cyanine-styryl dyes were synthesized for bioorthogonal labelling of DNA by means of the Diels-Alder reaction with inverse electron demand. With DNA as target biopolymer the fluorescence of these dyes is released by two factors: (i) sterically by their interaction with DNA, and (ii) structurally via the conjugated tetrazine as quencher moiety. As a result, the reaction with bicyclononyne-modified DNA is significantly accelerated up to ≥284,000 M-1 s-1 , and the fluorescence turn-on is enhanced up to 560 by the two-factor fluorogenicity. These dyes are cell permeable even in low concentrations and undergo fluorogenic reactions with BCN-modified DNA in living HeLa cells. The two-factor fluorescence release improves the signal-to-noise ratio such that washing procedures prior to cell imaging are not needed, which is a great advantage for live cell imaging of DNA and RNA in the future.

Fast and Efficient Postsynthetic DNA Labeling in Cells by Means of Strain-Promoted Sydnone-Alkyne Cycloadditions.

Sydnones are highly stable mesoionic 1,3-dipoles that react with cyclooctynes through strain-promoted sydnone-alkyne cycloaddition (SPSAC). Although sydnones have been shown to be valuable bioorthogonal chemical reporters for the labeling of proteins and complex glycans, nucleic acids have not yet been tagged by SPSAC. Evaluation of SPSAC kinetics with model substrates showed fast reactions with cyclooctyne probes (up to k=0.59 M-1 s-1 ), and two different sydnones were effectively incorporated into both 2'-deoxyuridines at position 5, and 7-deaza-2'-deoxyadenosines at position 7. These modified nucleosides were synthetically incorporated into single-stranded DNAs, which were successfully postsynthetically labeled with cyclooctyne probes both in vitro and in cells. These results show that sydnones are versatile bioorthogonal tags and have the premise to become essential tools for tracking DNA and potentially RNA in living cells.

Fluorescence Lifetime Imaging Microscopy (FLIM) of Intracellular Transport by Means of Doubly Labelled siRNA Architectures.

For monitoring the intracellular pathway of small interfering RNA (siRNA), both strands were labelled at internal positions by two ATTO dyes as an interstrand Förster resonance energy transfer pair. siRNA double strands show red emission and a short donor lifetime as readout, whereas siRNA antisense single strands show green emission and a long donor lifetime. This readout signals if GFP silencing can be expected (green) or not (red). We attached both dyes to three structurally different alkyne anchors by postsynthetic modifications. There is only a slight preference for the ribofuranoside anchors with the dyes at their 2'-positions. For the first time, the delivery and fate of siRNA in live HeLa cells was tracked by fluorescence lifetime imaging microscopy (FLIM), which revealed a clear relationship between intracellular transport using different transfection methods and knockdown of GFP expression, which demonstrates the potential of our siRNA architectures as a tool for future development of effective siRNA.

4-Aminophthalimide Amino Acids as Small and Environment-Sensitive Fluorescent Probes for Transmembrane Peptides.

Fluorescence probing of transmembrane (TM) peptides is needed to complement state-of-the art methods-mainly oriented circular dichroism and solid-state NMR spectroscopy-and to allow imaging in living cells. Three new amino acids incorporating the solvatofluorescent 4-aminophthalimide in their side chains were synthesized in order to examine the local polarity in the α-helical TM fragment of the human epidermal growth factor receptor. It was possible to distinguish their locations, either in the hydrophobic core of the lipid bilayer or at the membrane surface, by fluorescence readout, including blue shift and increased quantum yield. An important feature is the small size of the 4-aminophthalimide chromophore. It makes one of the new amino acids approximately isosteric to tryptophan, typically used as a very small fluorescent amino acid in peptides and proteins. In contrast to the only weakly fluorescent indole system in tryptophan, the 4-aminophthalimide moiety produces a significantly more informative fluorescence readout and is selectively excited outside the biopolymer absorption range.

Copper-free dual labeling of DNA by triazines and cyclopropenes as minimal orthogonal and bioorthogonal functions.

Two different and small functions for inverse electron demand Diels-Alder reactions were applied for dual labeling of DNA: the 1,2,4-triazine was attached to the 5-position of 2'-deoxyuridine triphosphate, and the 1-methylcyclopropene to the 7-position of 7-deaza-2'-deoxyadenosine triphosphate. These two modified nucleotides were sequence-selectively incorporated into oligonucleotides by DNA polymerases. These products were labeled by two different fluorescent dyes using postsynthetic reactions that are not only bioorthogonal in general, but also mutually orthogonal.

A new structure-activity relationship for cyanine dyes to improve photostability and fluorescence properties for live cell imaging.

A new set of cyanine-indole dyes was synthesized, characterized by optical and cytotoxic properties and subsequently applied for live cell imaging. Furthermore, these dyes were postsynthetically linked covalently to the 2'-position of uridine anchors in presynthesized oligonucleotides using the copper(i)-catalyzed azide-alkyne cycloaddition in order to evaluate their photostability and imaging properties in living cells. The nucleophilicity at position C-2 of the indole part of the dyes was elucidated as key for a new structure-activity relationship that served as a rational guide to improve the photostability and optical properties of these green-emitting dyes for live cell imaging of nucleic acids. While the photostability rises exponentially with decreasing nucleophilicity, thermal bleaching experiments confirmed an opposite trend supposing that the superoxide radical anion is mainly responsible for the photobleaching of the dyes. Furthermore, the cytotoxicities of the dyes were tested in HeLa cells and moderate to low LD50 values were obtained. This interdisciplinary strategy allowed us to identify one dye with excellent optical properties and even better photostability and decreased cytotoxicity compared to a cyanine-indole dye that bears an additional cyclooctatetraene group as a triplet state quencher.

DNA Primer Extension with Cyclopropenylated 7-Deaza-2'-deoxyadenosine and Efficient Bioorthogonal Labeling in Vitro and in Living Cells.

A deoxyadenosine triphosphate (dATP) analogue for DNA labeling was synthesized with the 1-methylcyclopropene (1MCP) group at the 7-position of 7-deaza-2'-deoxyadenosine and applied for primer extension experiments. The real-time kinetic data reveals that this 1MCP-modified dATP analogue is incorporated into DNA much faster than that of the similarly 1MCP-modified deoxyuridine triphosphate (dUTP) analogue. The postsynthetic fluorescent labeling of these oligonucleotides works efficiently according to PAGE analysis, and can be applied for immobilization of a functional antibody on a surface. Site-specific labeling at two different positions in DNA was achieved and the bioorthogonality of the postsynthetic fluorescent labeling was demonstrated in living HeLa cells.

"siRNA traffic lights": arabino-configured 2'-anchors for fluorescent dyes are key for dual color readout in cell imaging.

Two fluorescent dyes covalently attached in diagonal interstrand orientation to siRNA undergo energy transfer and thereby enable a dual color fluorescence readout (red/green) for hybridization. Three different structural variations were carried out and compared by their optical properties, including (i) the base surrogate approach with an acyclic linker as a substitute of the 2-deoxyriboside between the phosphodiester bridges, (ii) the 2'-modification of conventional ribofuranosides and (iii) the arabino-configured 2'-modification. The double stranded siRNA with the latter type of modification delivered the best energy transfer efficiency, which was explained by molecular dynamics simulations that showed that the two dyes are more flexible at the arabino-configured sugars compared to the completely stacked situation at the ribo-configured ones. Single molecule fluorescence lifetime measurements indicate their application in fluorescence cell imaging, which reveals a red/green fluorescence contrast in particular for the arabino-configured 2'-modification by the two dyes, which is key for tracking of siRNA transport into HeLa cells.

Synthesis of Wavelength-Shifting Fluorescent DNA and RNA with Two Photostable Cyanine-Styryl Dyes as the Base Surrogate Pair.

Two nucleic acid building blocks were synthesized, consisting of two photostable green- and red-emitting cyanine-styryl dyes and (S)-3-amino-1,2-propanediol as a substitute for the ribofuranoside, and incorporated as base-pair surrogates by using automated phosphoramidte chemistry in the solid phase. The optical properties and, in particular, the energy-transfer properties were screened in a range of DNA duplexes, in which the "counter bases" of the two dyes were varied and the distance between the two dyes was enlarged to up to three intervening adenosine-thymidine pairs. The DNA duplex with the best optical properties and the best red/green emission ratio as the readout bore adenosine and thymidine opposite to the dyes, and the two dyes directly adjacent to each other as the base surrogate pair. This structural arrangement can be transferred to RNA to obtain similarly fluorescent RNA probes. Representatively, the positively evaluated DNA duplex was applied to verify the fluorescence readout in living HeLa cells by using fluorescence confocal microscopy.

A Modular Class of Fluorescent Difluoroboranes: Synthesis, Structure, Optical Properties, Theoretical Calculations and Applications for Biological Imaging.

Ten borylated bipyridines (BOBIPYs) have been synthesized and selected structural modifications have been made that allow useful structure-optical property relationships to be gathered. These systems have been further investigated using DFT calculations and spectroscopic measurements, showing blue to green fluorescence with quantum yields up to 41 %. They allow full mapping of the structure to determine where selected functionalities can be implemented, to tune the optical properties or to incorporate linking groups. The best derivative was thus functionalised with an alkyne linker, which would enable further applications through click chemistry and in this optic, the stability of the fluorophores has been evaluated.

Highly Luminescent, Water-Soluble Lanthanide Fluorobenzoates: Syntheses, Structures and Photophysics, Part I: Lanthanide Pentafluorobenzoates.

Highly luminescent, photostable, and soluble lanthanide pentafluorobenzoates have been synthesized and thoroughly characterized, with a focus on Eu(III) and Tb(III) complexes as visible emitters and Nd(III) , Er(III) , and Yb(III) complexes as infrared emitters. Investigation of the crystal structures of the complexes in powder form and as single crystals by using X-ray diffraction revealed five different structural types, including monomeric, dimeric, and polymeric. The local structure in different solutions was studied by using X-ray absorption spectroscopy. The photoluminescence quantum yields (PLQYs) of terbium and europium complexes were 39 and 15 %, respectively; the latter value was increased almost twice by using the heterometallic complex [Tb0.5 Eu0.5 (pfb)3 (H2 O)] (Hpfb=pentafluorobenzoic acid). Due to the effectively utilized sensitization strategy (pfb)(-) →Tb→Eu, a pure europium luminescence with a PLQY of 29 % was achieved.

Functionalized triazolopeptoids--a novel class for mitochondrial targeted delivery.

Here we introduce linear 1,4-triazolopeptoids as a novel class of cell penetrating peptidomimetics suitable as organ targeting molecular transporters of bioactive cargo. Repetitive triazole moieties with up to three residues were assembled on solid supports using copper-catalyzed alkyne-azide cycloadditions (CuAAC) in a submonomer approach. Depending on the lipophilicity of their side chain appendages the 1,4-triazolopeptoids showed either endosomal localization or a strong colocalization with the mitochondria of HeLa cells with moderate toxicity. While the basic triazolopeptoids mainly target the neuromast cells in zebrafish embryos, the lipophilic ones colocalize with either cartilage in the jaws and the blood vessel system.

Cell-penetrating peptoids: introduction of novel cationic side chains.

During the last decade peptoid-based molecular transporters have been broadly applied. They are highly valued for their easy synthesis and their superior stability against enzymatic degradation. The special structure of peptoids generally allows introducing a variety of different side chains. Yet, the cationic side chains of cell-penetrating peptoids displayed solely lysine- or arginine-like structures. Thus this report is intended to extend the spectrum of cationic peptoid side chains. Herein, we present novel functional groups, like polyamines, aza-crown ethers, or triphenylphosphonium ions that are introduced into peptoids for the first time. In addition, the obtained peptoids were tested for their cell-penetrating properties.