Superior Photoprotection of Cyanine Dyes with Thio-imidazole Amino Acids.

Preventing fluorophore photobleaching and unwanted blinking is crucial for single-molecule fluorescence (SMF) studies. Reductants achieve photoprotection via quenching excited triplet states, yet either require counteragents or, for popular alkyl-thiols, are limited to cyanine dye Cy3 protection. Here, we provide mechanistic and imaging results showing that the naturally occurring amino acid ergothioneine and its analogue dramatically enhance photostability for Cy3, Cy5, and their conformationally restrained congeners, providing a biocompatible universal solution for demanding fluorescence imaging.

Binding and Sliding Dynamics of the Hepatitis C Virus Polymerase: Hunting the 3' Terminus.

The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) polymerase catalyzes the replication of the (+) single-stranded RNA genome of HCV. In vitro studies have shown that replication can be performed in the absence of a primer. However, the dynamics and mechanism by which NS5B locates the 3'-terminus of the RNA template to initiate de novo synthesis remain elusive. Here, we performed single-molecule fluorescence studies based on protein-induced fluorescence enhancement reporting on NS5B dynamics on a short model RNA substrate. Our results suggest that NS5B exists in a fully open conformation in solution wherefrom it accesses its binding site along RNA and then closes. Our results revealed two NS5B binding modes: an unstable one resulting in rapid dissociation, and a stable one characterized by a larger residence time on the substrate. We associate these bindings to an unproductive and productive orientation, respectively. Addition of extra mono (Na+)- and divalent (Mg2+) ions increases the mobility of NS5B along its RNA substrate. However, only Mg2+ ions induce a decrease in NS5B residence time. Dwell times of residence increase with the length of the single-stranded template, suggesting that NS5B unbinds its substrate by unthreading the template rather than by spontaneous opening.

Chemically Tuned, Reversible Fluorogenic Electrophile for Live Cell Nanoscopy.

We report a chemically tuned fluorogenic electrophile designed to conduct live-cell super-resolution imaging by exploiting its stochastic reversible alkylation reaction with cellular nucleophiles. Consisting of a lipophilic BODIPY fluorophore tethered to an electrophilic cyanoacrylate warhead, the new probe cyanoAcroB remains nonemissive due to internal conversion along the cyanoacrylate moiety. Intermittent fluorescence occurs following thiolate Michael addition to the probe, followed by retro-Michael reaction, tuned by the cyano moiety in the acrylate warhead and BODIPY decoration. This design enables long-term super-resolved imaging of live cells by preventing fluorescent product accumulation and background increase, while preserving the pool of the probe. We demonstrate the imaging capabilities of cyanoAcroB via two methods: (i) single-molecule localization microscopy imaging with nanometer accuracy by stochastic chemical activation and (ii) super-resolution radial fluctuation. The latter tolerates higher probe concentrations and low imaging powers, as it exploits the stochastic adduct dissociation. Super-resolved imaging with cyanoAcroB reveals that electrophile alkylation is prevalent in mitochondria and endoplasmic reticulum. The 2D dynamics of these organelles within a single cell are unraveled with tens of nanometers spatial and sub-second temporal resolution through continuous imaging of cyanoAcroB extending for tens of minutes. Our work underscores the opportunities that reversible fluorogenic probes with bioinspired warheads bring toward illuminating chemical reactions with super-resolved features in live cells.

Subcellular Targeted Nanohoop for One- and Two-Photon Live Cell Imaging.

Fluorophores are powerful tools for interrogating biological systems. Carbon nanotubes (CNTs) have long been attractive materials for biological imaging due to their near-infrared excitation and bright, tunable optical properties. The difficulty in synthesizing and functionalizing these materials with precision, however, has hampered progress in this area. Carbon nanohoops, which are macrocyclic CNT substructures, are carbon nanostructures that possess ideal photophysical characteristics of nanomaterials, while maintaining the precise synthesis of small molecules. However, much work remains to advance the nanohoop class of fluorophores as biological imaging agents. Herein, we report an intracellular targeted nanohoop. This fluorescent nanostructure is noncytotoxic at concentrations up to 50 µM, and cellular uptake investigations indicate internalization through endocytic pathways. Additionally, we employ this nanohoop for two-photon fluorescence imaging, demonstrating a high two-photon absorption cross-section (65 GM) and photostability comparable to a commercial probe. This work further motivates continued investigations into carbon nanohoop photophysics and their biological imaging applications.

Synthesis, Characterization, and Computational Investigation of Bright Orange-Emitting Benzothiadiazole [10]Cycloparaphenylene.

Conjugated aromatic macrocycles are attractive due to their unique photophysical and optoelectronic properties. In particular, the cyclic radially oriented π-system of cycloparaphenylenes (CPPs) gives rise to photophysical properties unlike any other small molecule or carbon nanomaterial. CPPs have tunable emission, possess large extinction coefficients, wide effective Stokes shifts, and high quantum yields. However, accessing bright CPPs with emissions beyond 500 nm remains difficult. Herein, we present a novel and bright orange-emitting CPP-based fluorophore showing a dramatic 105 nm red-shift in emission and striking 237 nm effective Stokes shift while retaining a large quantum yield of 0.59. We postulate, and experimentally and theoretically support, that the quantum yield remains large due to the lack of intramolecular charge transfer.

Exploration of the Solid-State Sorption Properties of Shape-Persistent Macrocyclic Nanocarbons as Bulk Materials and Small Aggregates.

Porous molecular materials combine benefits such as convenient processability and the possibility for atom-precise structural fine-tuning which makes them remarkable candidates for specialty applications in the areas of gas separation, catalysis, and sensing. In order to realize the full potential of these materials and guide future molecular design, knowledge of the transition from molecular properties into materials behavior is essential. In this work, the class of compounds termed cycloparaphenylenes (CPPs)-shape-persistent macrocycles with built-in cavities and radially oriented π-systems-was selected as a conceptually simple class of intrinsically porous nanocarbons to serve as a platform for studying the transition from analyte sorption properties of small aggregates to those of bulk materials. In our detailed investigation, two series of CPPs were probed: previously reported hoop-shaped [n]CPPs and a novel family of all-phenylene figure-8 shaped (lemniscal) bismacrocycles, termed spiro[n,n]CPPs. A series of nanocarbons with different macrocycle sizes and heteroatom content have been prepared by atom-precise organic synthetic methods, and their structural, photophysical, and electronic attributes were disclosed. Detailed experimental studies (X-ray crystallography, gas sorption, and quartz-crystal microbalance measurements) and quantum chemical calculations provided ample evidence for the importance of the solid-state arrangement on the porosity and analyte uptake ability of intrinsically porous molecular nanocarbons. We demonstrate that this molecular design principle, i.e., incorporation of sterically demanding spiro junctions into the backbone of nanohoops, enables the manipulation of solid-state morphology without significantly changing the nature and size of the macrocyclic cavities. As a result, the novel spiro[n,n]CPPs showed a remarkable performance as high affinity material for vapor analyte sensing.

Effect of curvature and placement of donor and acceptor units in cycloparaphenylenes: a computational study.

Cycloparaphenylenes have promise as novel fluorescent materials. However, shifting their fluorescence beyond 510 nm is difficult. Herein, we computationally explore the effect of incorporating electron accepting and electron donating units on CPP photophysical properties at the CAM-B3LYP/6-311G** level. We demonstrate that incorporation of donor and acceptor units may shift the CPP fluorescence as far as 1193 nm. This computational work directs the synthesis of bright red-emitting CPPs. Furthermore, the nanohoop architecture allows for interrogation of strain effects on common conjugated polymer donor and acceptor units. Strain results in a bathochromic shift versus linear variants, demonstrating the value of using strain to push the limits of low band gap materials.

Symmetry breaking and the turn-on fluorescence of small, highly strained carbon nanohoops.

[n]Cycloparaphenylenes, or "carbon nanohoops," are unique conjugated macrocycles with radially oriented π-systems similar to those in carbon nanotubes. The centrosymmetric nature and conformational rigidity of these molecules lead to unusual size-dependent photophysical characteristics. To investigate these effects further and expand the family of possible structures, a new class of related carbon nanohoops with broken symmetry is disclosed. In these structures, referred to as meta[n]cycloparaphenylenes, a single carbon-carbon bond is shifted by one position in order to break the centrosymmetric nature of the parent [n]cycloparaphenylenes. Advantageously, the symmetry breaking leads to bright emission in the smaller nanohoops, which are typically non-fluorescent due to optical selection rules. Moreover, this simple structural manipulation retains one of the most unique features of the nanohoop structures-size dependent emissive properties with relatively large extinction coefficients and quantum yields. Inspired by earlier theoretical work by Tretiak and co-workers, this joint synthetic, photophysical, and theoretical study provides further design principles to manipulate the optical properties of this growing class of molecules with radially oriented π-systems.