pH-Responsive Aminobenzocoumarins as Fluorescent Probes for Biological Acidity.

Regulation of pH plays an essential role in orchestrating the delicate cellular machinery responsible for life as we know it. Its abnormal values are indicative of aberrant cellular behavior and associated with pathologies including cancer progression or solid tumors. Here, we report a series of bent and linear aminobenzocoumarins decorated with different substituents. We investigate their photophysical properties and demonstrate that the probes display strong pH-responsive fluorescence "turn on" behavior in highly acidic environments, with enhancement up to 300-fold. In combination with their low cytotoxicity, this behavior enabled their application in bioimaging of acidic lysosomes in live human cells. We believe that these molecules serve as attractive lead structures for future rational design of novel biocompatible fluorescent pH probes.

Light in a Heartbeat: Bond Scission by a Single Photon above 800 nm.

Photocages enable scientists to take full control over the activity of molecules using light as a biocompatible stimulus. Their emerging applications in photoactivated therapies call for efficient uncaging in the near-infrared (NIR) window, which represents a fundamental challenge. Here, we report synthetically accessible cyanine photocages that liberate alcohol, phenol, amine, and thiol payloads upon irradiation with NIR light up to 820 nm in aqueous media. The photocages display a unique chameleon-like behavior and operate via two distinct uncaging mechanisms: photooxidation and heterolytic bond cleavage. The latter process constitutes the first example of a direct bond scission by a single photon ever observed in cyanine dyes or at wavelengths exceeding 800 nm. Modulation of the beating rates of human cardiomyocytes that we achieved by light-actuated release of adrenergic agonist etilefrine at submicromolar concentrations and low NIR light doses (∼12 J cm-2) highlights the potential of these photocages in biology and medicine.

Deuteration of heptamethine cyanine dyes enhances their emission efficacy.

The design of bright short-wave infrared fluorophores remains a grand challenge. Here we investigate the impact of deuteration on the properties in a series of heptamethine dyes, the absorption of which spans near-infrared and SWIR regions. We demonstrate that it is a generally applicable strategy that leads to enhanced quantum yields of fluorescence, longer-lived singlet excited states and suppressed rates of non-radiative deactivation processes.

Driving a Third Generation Molecular Motor with Electrons Across a Surface.

Excitation of single molecules with electrons tunneling between a sharp metallic tip of a scanning tunneling microscope and a metal surface is one way to study and control dynamics of molecules on surfaces. Electron tunneling induced dynamics may lead to hopping, rotation, molecular switching, or chemical reactions. Molecular motors that convert rotation of subgroups into lateral movement on a surface can in principle also be driven by tunneling electrons. For such surface-bound motor molecules the efficiency of motor action with respect to electron dose is still not known. Here, the response of a molecular motor containing two rotor units in the form of overcrowded alkene groups to inelastic electron tunneling has been examined on a Cu(111) surface in ultrahigh vacuum at 5 K. Upon vibrational excitation, switching between different molecular conformations is observed, including conversion of enantiomeric states of chiral conformations. Tunneling at energies in the range of electronic excitations causes activation of motor action and movement across the surface. The expected unidirectional rotation of the two rotor units causes forward movements but with a low degree of translational directionality.

Cyanine-flavonol hybrids as NIR-light activatable carbon monoxide donors in methanol and aqueous solutions.

Here we report on carbon monoxide-photoreleasable compounds (photoCORMs) that combine heptamethine cyanine and flavonol chromophores and are activated upon irradiation with near-infrared light. Excellent CO-release yields and uncaging cross sections in aqueous solutions, enhanced water solubilities thanks to polar substituents or a host-guest approach using cucurbit[7]uril are demonstrated. The hybrids display outstanding biocompatibility and diverse, structure-dependent cell penetrability and internalization.

Photouncaging of Carboxylic Acids from Cyanine Dyes with Near-Infrared Light.

Near-infrared light (NIR; 650-900 nm) offers unparalleled advantages as a biocompatible stimulus. The development of photocages that operate in this region represents a fundamental challenge due to the low energy of the excitation light. Herein, we repurpose cyanine dyes into photocages that are available on a multigram scale in three steps and efficiently release carboxylic acids in aqueous media upon irradiation with NIR light up to 820 nm. The photouncaging process is examined using several techniques, providing evidence that it proceeds via photooxidative pathway. We demonstrate the practical utility in live HeLa cells by delivery and release of the carboxylic acid cargo, that was otherwise not uptaken by cells in its free form. In combination with modularity of the cyanine scaffold, the realization of these accessible photocages will fully unleash the potential of the emerging field of NIR-photoactivation and facilitate its widespread adoption outside the photochemistry community.

Structure-Photoreactivity Relationship of 3-Hydroxyflavone-Based CO-Releasing Molecules.

Carbon monoxide (CO) is an endogenous signaling molecule that regulates diverse physiological processes. The therapeutic potential of CO is hampered by its intrinsic toxicity, and its administration poses a significant challenge. Photoactivatable CO-releasing molecules (photoCORMs) are an excellent tool to overcome the side effects of untargeted CO administration and provide precise spatial and temporal control over its release. Here, we studied the CO release mechanism of a small library of derivatives based on 3-hydroxy-2-phenyl-4H-benzo[g]chromen-4-one (flavonol), previously developed as an efficient photoCORM, by steady-state and femto/nanosecond transient absorption spectroscopies. The main objectives of the work were to explore in detail how to enhance the efficiency of CO photorelease from flavonols, bathochromically shift their absorption bands, control their acid-base properties and solubilities in aqueous solutions, and minimize primary or secondary photochemical side-reactions, such as self-photooxygenation. The best photoCORM performance was achieved by combining substituents, which simultaneously bathochromically shift the chromophore absorption spectrum, enhance the formation of the productive triplet state, and suppress the singlet oxygen production by shortening flavonol triplet-state lifetimes. In addition, the cell toxicity of selected flavonol compounds was analyzed using in vitro hepatic HepG2 cells.

Cyanine Renaissance: Tailoring the Properties to Applications.

In this account, we provide an overview of the applications that arose from the recently developed synthetic methodology that delivers heptamethine cyanines (Cy7) substituted at the central chain. The ability to easily introduce and manipulate various substituents in different substitution patterns along the cyanine chain enabled rational tailoring of the photophysical and photochemical properties. Exercising this control over the structure-property relationship proved to have a substantial impact in the field of cyanine dyes and was swiftly harnessed in a number of emerging applications in distinct areas, including fluorescent probes, biosensors, dye-sensitized upconversion nanoparticles, phototruncation of cyanines and photocages. While this method unlocked a number of new avenues, many synthetic challenges remain to be conquered in order to fully capitalize on the potential of cyanines, and we provide a short perspective that summarizes them at the end of this manuscript.

Structure-Activity Studies of Nitroreductase-Responsive Near-Infrared Heptamethine Cyanine Fluorescent Probes.

Two new classes of near-infrared molecular probes were prepared and shown to exhibit "turn on" fluorescence when cleaved by the nitroreductase enzyme, a well-known biomarker of cell hypoxia. The fluorescent probes are heptamethine cyanine dyes with a central 4'-carboxylic ester group on the heptamethine chain that is converted by a self-immolative fragmentation mechanism to a 4'-caboxylate group that greatly enhances the fluorescence brightness. Each compound was prepared by ring opening of a Zincke salt. The chemical structures have either terminal benzoindolinenes or propargyloxy auxochromes, which provide favorable red-shifted absorption/emission wavelengths and a hyperchromic effect that enhances the photon output when excited by 808 nm light. A fluorescent probe with terminal propargyloxy-indolenines exhibited less self-aggregation and was rapidly activated by nitroreductase with large "turn on" fluorescence; thus, it is the preferred choice for translation towards in vivo applications.

Photoremovable Protecting Groups: Across the Light Spectrum to Near- Infrared Absorbing Photocages.

We discuss the past decade of progress in the field of photoremovable protecting groups that allowed the development of photocages activatable by near-IR light and highlight the individual conceptual advancements that lead to general guidelines to design new such photoremovable protecting groups. We emphasize the importance of understanding the individual photochemical reaction mechanisms that was necessary to achieve this progress and provide an outlook of the subsequent steps to facilitate a swift translation of this research into clinical praxis. Since this issue of CHIMIA is dedicated to the late Prof. Thomas Bally, we decided to provide a personal perspective on the field to which he contributed himself. We tried to write this review with the general readership of CHIMIA in mind in a hope to pay a tribute to the extraordinary dedication and clarity with which Thomas Bally used to explain abstract chemical concepts to his students or colleagues. We are uncertain whether we matched such challenge but we believe that he would have liked such approach very much.

Cyanine-Flavonol Hybrids for Near-Infrared Light-Activated Delivery of Carbon Monoxide.

Carbon monoxide (CO) is an endogenous signaling molecule that controls a number of physiological processes. To circumvent the inherent toxicity of CO, light-activated CO-releasing molecules (photoCORMs) have emerged as an alternative for its administration. However, their wider application requires photoactivation using biologically benign visible and near-infrared (NIR) light. In this work, a strategy to access such photoCORMs by fusing two CO-releasing flavonol moieties with a NIR-absorbing cyanine dye is presented. These hybrids liberate two molecules of CO in high chemical yields upon activation with NIR light up to 820 nm and exhibit excellent uncaging cross-sections, which surpass the state-of-the-art by two orders of magnitude. Furthermore, the biocompatibility and applicability of the system in vitro and in vivo are demonstrated, and a mechanism of CO release is proposed. It is hoped that this strategy will stimulate the discovery of new classes of photoCORMs and accelerate the translation of CO-based phototherapy into practice.

Deciphering the Structure-Property Relations in Substituted Heptamethine Cyanines.

Heptamethine cyanines (Cy7) are fluorophores essential for modern bioimaging techniques and chemistry. Here, we systematically evaluated the photochemical and photophysical properties of a library of Cy7 derivatives containing diverse substituents in different positions of the heptamethine chain. A single substitution allows modulation of their absorption maxima in the range of 693-805 nm and photophysical properties, such as quantum yields of singlet-oxygen formation, decomposition, and fluorescence or affinity to singlet oxygen, within 2-3 orders of magnitude. The same substituent in different positions of the chain often exhibits distinctly contradictory effects, demonstrating that both the type and position of the substituent are pivotal for the design of Cy7-based applications. The combination of experimental results with quantum-chemical calculations provides insights into the structure-property relationship, the elucidation of which will accelerate the development of cyanines with properties tailored for specific applications, such as fluorescent probes and sensors, photouncaging, photodynamic therapy, or singlet-oxygen detection.

Mechanisms of Orthogonal Photodecarbonylation Reactions of 3-Hydroxyflavone-Based Acid-Base Forms.

Carbon monoxide is a naturally occurring gasotransmitter combining inherent toxicity with a remarkable therapeutic potential and arduous administration. Photoactivatable carbon monoxide-releasing molecules (photoCORMs) are chemical agents that allow for precise spatial and temporal control over the CO release. In this work, we present a comprehensive mechanistic study of the photochemical CO release from 3-hydroxy-2-phenyl-4H-chromen-4-one, a π-extended 3-hydroxyflavone photoCORM, in methanol using steady-state and transient absorption spectroscopies and quantum chemical calculations. The multiplicity of the productive excited states and the role of oxygen (O2) in the CO production are emphasized, revealing a photoreaction dichotomy of the 3-hydroxyflavone acid and base forms. The utilization of three major orthogonal mechanistic pathways, all of which lead to the CO release, can fuel future endeavors to improve the CO release efficacy of 3-hydroxyflavone-based derivatives and refine their potential medical applications as photoCORMs.

Approach to a Substituted Heptamethine Cyanine Chain by the Ring Opening of Zincke Salts.

Cyanine dyes play an indispensable and central role in modern fluorescence-based biological techniques. Despite their importance and widespread use, the current synthesis methods of heptamethine chain modification are restricted to coupling reactions and nucleophilic substitution at the meso position in the chain. Herein, we report the direct transformation of Zincke salts to cyanine dyes under mild conditions, accompanied by the incorporation of a substituted pyridine residue into the heptamethine scaffold. This work represents the first general approach that allows the introduction of diverse substituents and different substitution patterns at the C3'-C5' positions of the chain. High yields, functional tolerance, versatility toward the condensation partners, and scalability make this method a powerful tool for accessing a new generation of cyanine derivatives.

Central-to-Helical-to-Axial-to-Central Transfer of Chirality with a Photoresponsive Catalyst.

Recent advances in molecular design have displayed striking examples of dynamic chirality transfer between various elements of chirality, e.g., from central to either helical or axial chirality and vice versa. While considerable progress in atroposelective synthesis has been made, it is intriguing to design chiral molecular switches able to provide selective and dynamic control of axial chirality with an external stimulus to modulate stereochemical functions. Here, we report the synthesis and characterization of a photoresponsive bis(2-phenol)-substituted molecular switch 1. The unique design exhibits a dynamic hybrid central-helical-axial transfer of chirality. The change of preferential axial chirality in the biaryl motif is coupled to the reversible switching of helicity of the overcrowded alkene core, dictated by the fixed stereogenic center. The potential for dynamic control of axial chirality was demonstrated by using ( R)-1 as switchable catalyst to direct the stereochemical outcome of the catalytic enantioselective addition of diethylzinc to aromatic aldehydes, with successful reversal of enantioselectivity for several substrates.

Visible to NIR Light Photoactivation of Hydrogen Sulfide for Biological Targeting.

The synthesis and photochemical properties of H2S-releasing BODIPY thiocarbamate photocage scaffolds activatable by visible-to-NIR (up to 700 nm) light to release carbonyl sulfide (COS), which is transformed to H2S using either isolated or natural carbonic anhydrase, is reported. The excellent uncaging cross section and high H2S release yields in in vitro experiments, including live-cell imaging, suggest that these photocages can serve as a platform for the bio-orthogonal phototriggered release within the tissue-transparent window.

Photosensitized Cross-Linking of Tryptophan and Tyrosine Derivatives by Rose Bengal in Aqueous Solutions.

Photosensitized cross-linking of N-acetyl derivatives of tryptophan and tyrosine by rose bengal in phosphate buffer saline was studied by steady-state absorption and emission spectroscopy, nanosecond transient absorption spectroscopy, and chemical analyses. These amino acids undergo cross-linking to form dimeric and higher oligomeric products under anoxic conditions with rose bengal as a sacrificial oxidant, whereas they react predominantly with singlet oxygen produced by rose bengal in aerated solutions. This investigation provides a comprehensive view into the first steps of rose bengal photosensitized cross-linking of these two amino acids. The results can be helpful for future applications of rose bengal and related dyes, such as photochemical tissue bonding or visible light photocatalysis.

Braking of a Light-Driven Molecular Rotary Motor by Chemical Stimuli.

Artificial molecular motors hold great promise for application in responsive functional materials as well as to control the properties of biohybrid systems. Herein a strategy is reported to modulate the rotation of light-driven molecular motors. That is, the rotary speed of a molecular motor, functionalized with a biphenol moiety, could be decreased in situ by non-covalent substrate binding, as was established by 1 H NMR and UV/Vis spectroscopy. These findings constitute an important step in the development of multi-responsive molecular machinery.

Locked synchronous rotor motion in a molecular motor.

Biological molecular motors translate their local directional motion into ordered movement of other parts of the system to empower controlled mechanical functions. The design of analogous geared systems that couple motion in a directional manner, which is pivotal for molecular machinery operating at the nanoscale, remains highly challenging. Here, we report a molecular rotary motor that translates light-driven unidirectional rotary motion to controlled movement of a connected biaryl rotor. Achieving coupled motion of the distinct parts of this multicomponent mechanical system required precise control of multiple kinetic barriers for isomerization and synchronous motion, resulting in sliding and rotation during a full rotary cycle, with the motor always facing the same face of the rotor.

Third-Generation Light-Driven Symmetric Molecular Motors.

Symmetric molecular motors based on two overcrowded alkenes with a notable absence of a stereogenic center show potential to function as novel mechanical systems in the development of more advanced nanomachines offering controlled motion over surfaces. Elucidation of the key parameters and limitations of these third-generation motors is essential for the design of optimized molecular machines based on light-driven rotary motion. Herein we demonstrate the thermal and photochemical rotational behavior of a series of third-generation light-driven molecular motors. The steric hindrance of the core unit exerted upon the rotors proved pivotal in controlling the speed of rotation, where a smaller size results in lower barriers. The presence of a pseudo-asymmetric carbon center provides the motor with unidirectionality. Tuning of the steric effects of the substituents at the bridgehead allows for the precise control of the direction of disrotary motion, illustrated by the design of two motors which show opposite rotation with respect to a methyl substituent. A third-generation molecular motor with the potential to be the fastest based on overcrowded alkenes to date was used to visualize the equal rate of rotation of both its rotor units. The autonomous rotational behavior perfectly followed the predicted model, setting the stage for more advanced motors for functional dynamic systems.