Andrea Fin

Oligothiophene Amphiphiles as Planarizable and Polarizable Fluorescent Membrane Probes

Lessons from lobster coloration and the chemistry of vision suggested an approach to responsive fluorescent probes that can sense membrane potential, fluidity, and tension. Fluorophore deplanarization by lateral crowding along the scaffold (red circles) and fluorophore polarization by terminal donors (D), acceptors (A), and charges (+) are coupled to provide such membrane probes, as studies with unilamellar vesicles of phospholipids (DOPC, DPPC) show.

Planarizable push–pull oligothiophenes: in search of the perfect twist

The concept to couple fluorophore planarization and fluorophore polarization for the construction of innovative fluorescent membrane probes is elaborated comprehensively in the context of oligothiophenes. Increasing length with different degree of twist from ter- to quinquethiophenes results in increasing extinction coefficients, decreasing quantum yields and relatively minor red shifts. Quaterthiophenes show maximal Stokes shifts and are thus preserved to further elaborate on deplanarization. Increasing quaterthiophene deplanarization results in increasing blue shifts and decreasing quantum yields in solution, whereas planarization in solid-ordered lipid bilayer membranes gives the respective red shifts with fluorescence recovery. An extensive screening reveals that intermediate global deplanarization with strong individual twists near the membrane interface are best. Weaker and stronger global twisting and strong individual twists deeper in the membrane are less convincing because planarization becomes either too easy or too difficult. The best probe reports decreasing membrane fluidity with a red shift of 44 nm and a fluorescence increase of almost 500%. These insights are important because they cover significant chemical space to help improving our understanding of chromophore twisting and promise bright perspectives with regard to biological applications and refined probe design.

A Fluorescent Adenosine Analogue as a Substrate for an A-to-I RNA Editing Enzyme†

Adenosine to inosine RNA editing catalyzed by ADAR enzymes is common in humans, and altered editing is associated with disease. Experiments using substrate RNAs with adenosine analogues at editing sites are useful for defining features of the ADAR reaction mechanism. The reactivity of ADAR2 was evaluated with RNA containing the emissive adenosine analogue thieno[3,4-d]-6-aminopyrimidine ((th)A). This nucleoside was incorporated into a mimic of the glutamate receptor B (GluR B) mRNA R/G editing site. We found that (th)A is recognized by AMV reverse transcriptase as A, and is deaminated rapidly by human ADAR2 to give (th)I. Importantly, ADAR reaction progress can be monitored by following the deamination-induced change in fluorescence of the (th)A-modified RNA. The observed high (th)A reactivity adds to our understanding of the structural features that are necessary for an efficient hADAR2 reaction. Furthermore, the new fluorescent assay is expected to accelerate mechanistic studies of ADARs.

Pyrene‐Based Quantitative Detection of the 5‐Formylcytosine Loci Symmetry in the CpG Duplex Content during TET‐Dependent Demethylation

Methylcytosine (5mC) is mostly symmetrically distributed in CpG sites. Ten-eleven-translocation (TET) proteins are the key enzymes involved in active DNA demethylation through stepwise oxidation of 5mC. However, oxidation pathways of TET enzymes in the symmetrically methylated CpG context are still elusive. Employing the unique fluorescence properties of pyrene group, we designed and synthesized a sensitive fluorescence-based probe not only to target 5-formylcytosine (5fC) sites, but also to distinguish symmetric from asymmetric 5fC sites in the double stranded DNA context during TET-dependent 5mC oxidation process. Using this novel probe, we revealed dominant levels of symmetric 5fC among total 5fC sites during in vitro TET-dependent 5mC oxidation and novel mechanistic insights into the TET-dependent 5mC oxidation in the mCpG context.

Recent Progress with Functional Biosupramolecular Systems

The objective of this account is to summarize our recent progress with functional biosupramolecular systems concisely. The functions covered are artificial photosynthesis, anion transport, and sensing in lipid bilayer membranes. With artificial photosynthesis, the current emphasis is on the construction of ordered and oriented architectures on solid surfaces. Recent examples include the zipper assembly of photosystems with supramolecular n/p-heterojunctions and oriented antiparallel redox gradients. Current transport systems in lipid bilayers reveal new interactions at work. Examples include anion-macrodipole or anion-π interactions. Current attention with membrane-based sensing systems shifts from biosensor approaches with enzymatic signal generation to aptamers (i.e., the DNA version of immunosensing) and differential sensing with dynamic polyion-counterion transporters. The functional diversity accessible with biosupramolecular systems is highlighted, as is the critical importance of cross-fertilization at intertopical convergence zones.

Conceptually new entries into cells.

This article summarizes the background and a few preliminary results concerning project 7 of the NCCR Chemical Biology. The general objective is to explore new concepts for cellular uptake, membrane tunneling, sensing and labeling. Emphasis is on the use of dynamic covalent chemistry for counterion activation, slow release of polyions and fluorescent probes, and the generation of activator libraries and polyions that grow and shrink.

Emissive Synthetic Cofactors: An Isomorphic, Isofunctional, and Responsive NAD+ Analogue

The synthesis, photophysics, and biochemical utility of a fluorescent NAD+ analogue based on an isothiazolo[4,3-d]pyrimidine core (NtzAD+) are described. Enzymatic reactions, photophysically monitored in real time, show NtzAD+ and NtzADH to be substrates for yeast alcohol dehydrogenase and lactate dehydrogenase, respectively, with reaction rates comparable to that of the native cofactors. A drop in fluorescence is seen as NtzAD+ is converted to NtzADH, reflecting a complementary photophysical behavior to that of the native NAD+/NADH. NtzAD+ and NtzADH serve as substrates for NADase, which selectively cleaves the nicotinamides glycosidic bond yielding tzADP-ribose. NtzAD+ also serves as a substrate for ribosyl transferases, including human adenosine ribosyl transferase 5 (ART5) and Cholera toxin subunit A (CTA), which hydrolyze the nicotinamide and transfer tzADP-ribose to an arginine analogue, respectively. These reactions can be monitored by fluorescence spectroscopy, in stark contrast to the corresponding processes with the nonemissive NAD+.

Off-line and real-time monitoring of acetaminophen photodegradation by an electrochemical sensor

The photochemistry of N-acetyl-para-aminophenol (acetaminophen, APAP) is here investigated by using differential pulse voltammetry (DPV) analysis to monitor APAP photodegradation upon steady-state irradiation. The purpose of this work is to assess the applicability of DPV to monitor the photochemical behaviour of xenobiotics, along with the development of an electrochemical set-up for the real-time monitoring of APAP photodegradation. We here investigated the APAP photoreactivity towards the main photogenerated reactive transients species occurring in sunlit surface waters (hydroxyl radical HO, carbonate radical CO3-, excited triplet state of anthraquinone-2-sulfonate used as proxy of the chromophoric DOM, and singlet oxygen 1O2), and determined relevant kinetic parameters. A standard procedure based on UV detection coupled with liquid chromatography (HPLC-UV) was used under identical experimental conditions to compare and verify the DPV-based results. The latter were in agreement with HPLC data, with the exception of the triplet-sensitized processes. In the other cases, DPV could be used as an alternative to the well-tested but more costly and time-consuming HPLC-UV technique. We have also assessed the reaction rate constant between APAP and HO by real-time DPV, which allowed for the monitoring of APAP photodegradation inside the irradiation chamber. Unfortunately, real-time DPV measurements are likely to be affected by temperature variations of the irradiated samples. Overall, DPV appeared as a fast, cheap and reasonably reliable technique when used for the off-line monitoring of APAP photodegradation. When a suitable real-time procedure is developed, it could become a very straightforward method to study the photochemical behaviour of electroactive xenobiotics.

Emissive 5-Substituted Uridine Analogues

Chemical biology and medicinal chemistry applications require new nucleoside analogues with well-defined photophysical properties in order to visualize, monitor, and advance the understanding of nucleic acids. To impart favorable photophysical properties upon the native nucleosides and decipher structure–property relationships, robust and flexible synthetic procedures are required. Modification at the 5-position of uridine likely comprises the largest number of chemical variations investigated to enhance and tune the photophysical properties of this practically non-emissive nucleoside. The chapter discusses the design, synthesis, and characterization of diverse emissive uridine analogues.

Solubilising groups: a conceptual equivalent of protecting groups in organic synthesis

In this brief essay, one of the most common and least appreciated challenge in the field is addressed. Few chemists, particularly supramolecular chemists, exist who are not all too familiar with solubility problems during the synthesis of new molecules. Solubility problems are inherent to the synthesis of molecules that are made to build supramolecular architectures because the same intermolecular interactions that cause the problem are later on essential for the final self-assembly of the system of interest. Naturally, many solutions exist for a problem that occurs so frequently. They are used as daily routine in many laboratories, the temporary attachment of hydrophobic bulk of various size and nature being the most common. However, contrary to the comparable situation with protecting groups, these solubilising groups are generally underappreciated, often communicated orally as one of those precious ‘lab secrets’ that nobody really takes serious but everybody really needs to get things running and reach the relevant part of the research project. Here, we briefly try to summarise latent concepts concerning solubilising groups, focusing particularly on questions concerning quantitative aspects and the removal of solubilising groups for self-assembly with pre-, post- or in situ desolubilisation, and provide a simple practical example with tert-butyldiphenylsilyl as an illustrative solubilising group.