Two-Factor Fluorogenic Cyanine-Styryl Dyes with Yellow and Red Fluorescence for Bioorthogonal Labelling of DNA.

An orange- and a red-emitting tetrazine-modified cyanine-styryl dyes were synthesized for bioorthogonal labelling of DNA by means of the Diels-Alder reaction with inverse electron demand. Both dyes use the concept of the "two-factor" fluorogenicity for nucleic acids: (i) The dyes are nucleic-acid sensitive by their non-covalent binding to DNA, and (ii) their covalently attached tetrazine moiety quench the fluorescence. As a result, the reaction with bicyclononyne- and spirohexene-modified DNA is significantly accelerated up to k2 =280,000 M-1 s-1 , and the fluorescence turn-on is enhanced up to 305. Both dyes are cell permeable even in low concentrations and undergo fluorogenic reactions with spirohexene-modified DNA in living HeLa cells. The fluorescence is enhanced in living cells to such an extent that washing procedures before cell imaging are not required. Their large Stokes shifts (up to 0.77 eV) also makes them well suited for imaging because the wavelength ranges for excitation and emission can be best possible separated. Furthermore, the spirohexene-modified nucleosides and DNA extend and improve the toolbox of already existing "clickable" dyes for live cell imaging.

Photoredox Catalytic Access to N,O-Acetals from Enamides by Means of Electron-Poor Perylene Bisimides.

N,O-acetals are found as structural motifs in natural products and are important synthetic precursors for N-acylimines as building blocks in organic synthesis for C-C-bond formation and amines. For the synthesis of N,O-acetals, an acid-, base- and metal-free catalytic method is reported applying N,N-di-(2,6-diisopropyl)-1,7-dicyano-perylen-3,4,9,10-tetracarboxylic acid imide and N,N-di-(2,6-diisopropyl)-1,6,7,12-tetrabromo-2,5,8,11-tetracyano-perylen-3,4,9,10-tetracarboxylic acid imide as extremely electron-deficient photocatalysts. The first perylene bisimide highly selectively photocatalyzes the formation of the N,O-acetals as products in high yields, and the second and more electron-deficient perylene bisimide allows these reactions without thiophenol as an H-atom transfer reagent. Calculated electron density maps support this. The reaction scope comprises different substituents at the nitrogen of the enamides and different alcohols as starting material. Dehydroalanines are converted to non-natural amino acids which shows the usefulness of this method for organic and medicinal chemistry.

Nucleic Acid-Templated Synthesis of Cationic Styryl Dyes in Vitro and in Living Cells.

A novel method for synthesizing cationic styryl dyes through a nucleic acid-templated reaction has been developed. This approach overcomes issues associated with traditional synthesis methods, such as harsh conditions, low throughput, and wasteful chemicals. The presence of a nucleic acid template accelerated the styryl dye formation from quaternized heteroaromatic and cationic aldehyde substrates. These styryl dyes show remarkable optical properties change when bound to nucleic acids, hence the success of the synthesis could be readily monitored in situ by UV-Vis and fluorescence spectroscopy and the optical properties data were also observable at the same time. This method provides the desired products from a broad range of coupling partners. By employing different substrates and templates, it is possible to identify new dyes that can bind to a specific type of nucleic acid such as a G-quadruplex. The templated dye synthesis is also successfully demonstrated in live HeLa cells. This approach is a powerful tool for the rapid synthesis and screening of dyes specific for diverse types of nucleic acids or cellular organelles, facilitating new biological discoveries.

Cell-resistant wavelength-shifting molecular beacons made of L-DNA and a clickable L-configured uridine.

Wavelength-shifting molecular beacons were prepared from L-DNA. The clickable anchor for the two dyes, Cy3 and Cy5, was 2'-O-propargyl-L-uridine and was synthesized from L-ribose. Four clickable molecular beacons were prepared and double-modified with the azide dyes by a combination of click chemistry on a solid support for Cy3 during DNA synthesis and postsynthetic click chemistry for Cy5 in solution. Cy3 and Cy5 successfully formed a FRET pair in the beacons, and the closed form (red fluorescence) and the open form (green fluorescence) can be distinguished by the two-color fluorescence readout. Two molecular beacons were identified to show the greatest fluorescence contrast in temperature-dependent fluorescence measurements. The stability of the L-configured molecular beacons was demonstrated after several heating and cooling cycles as well as in the cell lysate. In comparison, D-configured molecular beacons showed a rapid decrease of fluorescence contrast in the cell lysate, which is caused by the opening of the beacons, probably due to degradation. This was confirmed in cell experiments using confocal microscopy. The L-configured molecular beacons are potential intracellular thermometers for future applications.

Impact of sulfur substitution on biotin binding affinity to streptavidin.

In this study, we investigated how the replacement of the tetrahydrothiophene ring of biotin with either an oxolane or (methyl)pyrrolidine moiety may affect its molecular interactions, in an effort to identify alternative affinity ligands suitable for in vitro and in vivo applications in synthetic biology. Initial molecular dynamics (MD) simulations suggested the potential formation of a hydrogen bond between either the oxygen or nitrogen atom of the envisaged tetrahydroheteryl analogues and the Thr90 residue of streptavidin, mirroring the sulfur-centered hydrogen bond detected by the crystallographic analysis of the biotin-streptavidin interaction. Therefore, oxy-, aza-, and N-methylazabiotin were readily synthesized starting from chiral five- or six-carbon sugar precursors. Based on fluorescence-based titration experiments using the corresponding fluorescein conjugates, oxybiotin showed a binding behavior similar to biotin with streptavidin, while both amino analogues displayed lower binding capacities. Notably, azabiotin exhibited a pH-dependent interaction profile, demonstrating enhanced binding under acidic conditions but weaker binding under basic pH, which could be exploited for various purposes.

Cyclopropenes as Chemical Reporters for Dual Bioorthogonal and Orthogonal Metabolic Labeling of DNA.

Dual bioorthogonal labeling enables the investigation and understanding of interactions in the biological environment that are not accessible by a single label. However, applying two bioorthogonal reactions in the same environment remains challenging due to cross-reactivity. We developed a pair of differently modified 2'-deoxynucleosides that solved this issue for dual and orthogonal labeling of DNA. Inverse-electron demand Diels-Alder and photoclick reactions were combined to attach two different fluorogenic labels to genomic DNA in cells. Using a small synthetic library of 1- and 3-methylcyclopropenyl-modified 2'-deoxynucleosides, two 2'-deoxyuridines were identified to be the fastest-reacting ones for each of the two bioorthogonal reactions. Their orthogonal reactivity could be evidenced in vitro. Primer extension experiments were performed with both 2'-deoxyuridines investigating their replication properties as substitutes for thymidine and evaluating subsequent labeling reactions on the DNA level. Finally, dual, orthogonal and metabolic fluorescent labeling of genomic DNA was demonstrated in HeLa cells. An experimental procedure was developed combining intracellular transport and metabolic DNA incorporation of the two 2'-deoxyuridines with the subsequent dual bioorthogonal labeling using a fluorogenic cyanine-styryl tetrazine and a fluorogenic pyrene-tetrazole. These results are fundamental for advanced metabolic labeling strategies for nucleic acids in the future, especially for live cell experiments.

Photocatalytic Synthesis of Acetals and Ketals from Aldehydes and Silylenolethers without the Use of Acids.

Invited for the cover of this issue are Desirée Steuernagel and Hans-Achim Wagenknecht at the Karlsruhe Institute of Technology. The image depicts a traffic roundabout, which symbolizes the photocatalytic cycle for the conversion of aldehydes and silylenolethers as starting materials into their acetals. Read the full text of the article at 10.1002/chem.202203767.

Photocatalytic Synthesis of Acetals and Ketals from Aldehydes and Silylenolethers without the Use of Acids.

Acetals and ketals are among the most important protecting groups for carbonyl compounds. A new method for acetalization and ketalization by means of photoredox catalysis has been developed. A biscyanolated perylene bisimide is used as an electron-poor photocatalyst, together with green light (525 nm LED). Silylenolethers derived from aldehydes react efficiently to give acetals in good to excellent yields. A broad substrate range was shown with respect to both the aldehydes and the alcohols. The functional group tolerance is high; in particular, acid- and hydrogen-labile protecting groups are tolerated. Aldehydes can also be directly and selectively converted into the respective acetals. Only ketones must be converted to their silylenolethers before ketalization. This photocatalytic method works without any use of acids or photoacids, and does not need any additives or H-atom transfer reagents. Hence, it broadens the substrate scope and repertoire of photoredox catalysis with respect to carbonyl chemistry.

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.

Reductive Activation of Aryl Chlorides by Tuning the Radical Cation Properties of N-Phenylphenothiazines as Organophotoredox Catalysts.

Aryl chlorides as substrates for arylations present a particular challenge for photoredox catalytic activation due to their strong C(sp2 )-Cl bond and their strong reduction potential. Electron-rich N-phenylphenothiazines, as organophotoredox catalysts, are capable of cleaving aryl chlorides simply by photoinduced electron transfer without the need for an additional electrochemical activation setup or any other advanced photocatalysis technique. Due to the extremely strong reduction potential in the excited state of the N-phenylphenothiazines the substrate scope is high and includes aryl chlorides both with electron-withdrawing and electron-donating substituents. We evidence this reactivity for photocatalytic borylations and phosphonylations. Advanced time-resolved transient absorption spectroscopy in combination with electrochemistry was the key to elucidating and comparing the unusual photophysical properties not only of the N-phenylphenothiazines, but also of their cation radicals as the central intermediates in the photocatalytic cycle. The revealed photophysics allowed the excited-state and radical-cation properties to be fine-tuned by the molecular design of the N-phenylphenothiazines; this improved the photocatalytic activity.

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.

Remote Photodamaging of DNA by Photoinduced Energy Transport.

Local DNA photodamaging by light is well-studied and leads to a number of structurally identified direct damage, in particular cyclobutane pyrimidine dimers, and indirect oxidatively generated damage, such as 8-oxo-7,8-hydroxyguanine. Similar damages have now been found at remote sites, at least more than 105 Š(30 base pairs) away from the site of photoexcitation. In contrast to the established mechanisms of local DNA photodamaging, the processes of remote photodamage are only partially understood. Known pathways include those to remote oxidatively generated DNA photodamages, which were elucidated by studying electron hole transport through the DNA about 20 years ago. Recent studies with DNA photosensitizers and mechanistic proposals on photoinduced DNA-mediated energy transport are summarized in this minireview. These new mechanisms to a new type of remote DNA photodamaging provide an important extension to our general understanding to light-induced DNA damage and their mutations.

Hybridization-Sensitive Fluorescent Probes for DNA and RNA by a Modular "Click" Approach.

Fluorescent DNA probes were prepared in a modular approach using the "click" post-synthetic modification strategy. The new glycol-based module and DNA building block place just two carbons between the phosphodiester bridges and anchor the dye by an additional alkyne group. This creates a stereocenter in the middle of this artificial nucleoside substitute. Both enantiomers and a variety of photostable cyanine-styryl dyes as well as thiazole orange derivatives were screened as "clicked" conjugates in different surrounding DNA sequences. The combination of the (S)-configured DNA anchor and the cyanylated cyanine-styryl dye shows the highest fluorescence light-up effect of 9.2 and a brightness of approximately 11,000 M-1 cm-1. This hybridization sensitivity and fluorescence readout were further developed utilizing electron transfer and energy transfer processes. The combination of the hybridization-sensitive DNA building block with the nucleotide of 5-nitroindole as an electron acceptor and a quencher increases the light-up effect to 20 with the DNA target and to 15 with the RNA target. The fluorescence readout could significantly be enhanced to values between 50 and 360 by the use of energy transfer to a second DNA probe with commercially available dyes, like Cy3.5, Cy5, and Atto590, as energy acceptors at the 5'-end. The latter binary probes shift the fluorescent readout from the range of 500-550 nm to the range of 610-670 nm. The optical properties make these fluorescent DNA probes potentially useful for RNA imaging. Due to the strong light-up effect, they will not require washing procedures and will thus be suitable for live-cell imaging.

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.

Photoredox Catalytic Pentafluorosulfanylative Domino Cyclization of α-Substituted Alkenes to Oxaheterocycles by Using SF6.

Virtually inert sulfur hexafluoride becomes a precious pentafluorosulfanylation agent, if properly activated by photoredox catalysis, to access α-fluoro and α-alkoxy SF5 -compounds. This advanced protocol converts SF6 in the presence of alkynols as bifunctional C-C- and C-O-bond forming reagents directly into pentafluorosulfanylated oxygen-containing heterocycles in a single step from α-substituted alkenes. The proposed mechanism is supported by theoretical calculations and gives insights not only in the pentafluorosulfanylation step but also into formation of the carbon-carbon bond and is in full agreement with Baldwin's cyclization rules. The key step is a radical type 5-, 6- respectively 7-exo-dig-cyclization. The synthesized oxaheterocycles cannot be simply prepared by other synthetic methods, show a high level of structural complexity and significantly expand the scope of pentafluorosulfanylated building blocks valuable for medicinal and material chemistry.

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.

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.

Postsynthetic Modifications of DNA and RNA by Means of Copper-Free Cycloadditions as Bioorthogonal Reactions.

Bioorthogonal chemistry has mainly been developed for proteins and carbohydrates. The chemistry of nucleic acids is different, and bioorthogonal labeling strategies that were successfully applied for proteins and carbohydrates cannot be simply transferred to DNA and RNA. Cycloadditions play a central role for bioorthogonal chemistry with nucleic acids. In vivo postsynthetic labeling of DNA and RNA requires copper-free variants of cycloaddition chemistry to achieve "bio"orthogonality that can be applied even in living cells. Currently, there are three major types of copper-free cycloadditions available for nucleic acids: (i) the ring-strain-promoted azide-alkyne cycloadditions, (ii) the "photoclick" 1,3-dipolar cycloadditions, and (iii) the Diels-Alder reactions with inverse electron demand. In principle, bioorthogonally reactive building blocks for postsynthetic modifications of nucleic acids by cycloaddition can be prepared by three different ways: (i) The organic synthesis of DNA and RNA applies phosphoramidites as building blocks for solid-phase automated chemistry. (ii) The biochemical preparation of DNA and RNA by primer extension (PEX) and PCR applies triphosphates as building blocks together with DNA/RNA polymerases, and works in aqueous buffer. (iii) DNA and RNA is labeled by the intrinsic metabolism in cells using bioorthogonally reactive nucleosides. In contrast to proteins and carbohydrates, for which metabolic labeling strategies are well developed, there are only a few examples in the literature for metabolic labeling of nucleic acids. In this review, we summarize the currently available DNA and RNA building blocks, both phosphoramidites and nucleotide triphosphates, for copper-free and bioorthogonal postsynthetic modification strategies.

Photoredox Catalytic α-Alkoxypentafluorosulfanylation of α-Methyl- and α-Phenylstyrene Using SF6.

SF6 was applied as pentafluorosulfanylation reagent to prepare ethers with a vicinal SF5 substituent through a one-step method involving photoredox catalysis. This method shows a broad substrate scope with respect to applicable alcohols for the conversion of α-methyl and α-phenyl styrenes. The products bear a new structural motif with two functional groups installed in one step. The alkoxy group allows elimination and azidation as further transformations into valuable pentafluorosulfanylated compounds. These results confirm that non-toxic SF6 is a useful SF5 transfer reagent if properly activated by photoredox catalysis, and toxic reagents are completely avoided. In combination with light as an energy source, a high level of sustainability is achieved. Through this method, the proposed potential of the SF5 substituent in medicinal chemistry, agrochemistry, and materials chemistry may be exploited in the future.

How Far Does Energy Migrate in DNA and Cause Damage? Evidence for Long-Range Photodamage to DNA.

A new DNA architecture addresses the question, how far energy migrates in DNA and forms cyclobutane pyrimidine dimers (CPDs) as photodamages causing skin cancer. The 3-methoxyxanthone nucleoside allows site-selective photoenergy injection into DNA. The designated CPD site lacks the phosphodiester bond and can be placed in defined distances. The CPD formation links two oligonucleotides together and allows probing by gel electrophoresis. We obtained a sigmoidal distance dependence with R0 of 25±3 Å. Below R0 , short-range energy migration occurs with high CPD yields and shallow distance dependence, characteristic for a coherent process. 5-methyl-C as epigenetic modification on the 3'-side facilitates CPD formation. Above R0 , long-range incoherent energy migration occurs over 30 A-T pairs (105.4 Å). The evidence of long-range CPD formation is fundamental for our understanding of DNA photodamaging. Open access funding enabled and organized by Projekt DEAL.