Steric Effects on the Thermal Processes of Hemithioindigo Based Molecular Motor Rotation.

Tuning the thermal behavior of light driven molecular motors is fundamentally important for their future rational design. In many molecular motors thermal ratcheting steps are comprised of helicity inversions, energetically stabilizing the initial photoproducts. In this work we investigated a series of five hemithioindigo (HTI) based molecular motors to reveal the influence of steric hindrance in close proximity to the rotation axle on this process. Applying a high yielding synthetic procedure, we synthesized constitutional isomeric derivatives to distinguish between substitution effects at the aromatic and aliphatic position on the rotor fragment. The kinetics of thermal helix inversions were elucidated using low temperature 1 H NMR spectroscopy and an in situ irradiation technique. In combination with a detailed theoretical description, a comparative analysis of substituent effects on the thermal helix inversions of the rotation cycle is now possible. Such deeper understanding of the rotational cycle of HTI molecular motors is essential for speed regulation and future applications of visible light triggered nanomachines.

Pyrrole Hemithioindigo Antimitotics with Near-Quantitative Bidirectional Photoswitching that Photocontrol Cellular Microtubule Dynamics with Single-Cell Precision*.

We report the first cellular application of the emerging near-quantitative photoswitch pyrrole hemithioindigo, by rationally designing photopharmaceutical PHTub inhibitors of the cytoskeletal protein tubulin. PHTubs allow simultaneous visible-light imaging and photoswitching in live cells, delivering cell-precise photomodulation of microtubule dynamics, and photocontrol over cell cycle progression and cell death. This is the first acute use of a hemithioindigo photopharmaceutical for high-spatiotemporal-resolution biological control in live cells. It additionally demonstrates the utility of near-quantitative photoswitches, by enabling a dark-active design to overcome residual background activity during cellular photopatterning. This work opens up new horizons for high-precision microtubule research using PHTubs and shows the cellular applicability of pyrrole hemithioindigo as a valuable scaffold for photocontrol of a range of other biological targets.

Hemithioindigos for Cellular Photopharmacology: Desymmetrised Molecular Switch Scaffolds Enabling Design Control over the Isomer-Dependency of Potent Antimitotic Bioactivity.

Druglike small molecules with photoswitchable bioactivity-photopharmaceuticals-allow biologists to perform studies with exquisitely precise and reversible, spatial and temporal control over critical biological systems inaccessible to genetic manipulation. The photoresponsive pharmacophores disclosed have been almost exclusively azobenzenes, which has limited the structural and substituent scope of photopharmacology. More detrimentally, for azobenzene reagents, it is not researchers' needs for adapted experimental tools, but rather protein binding site sterics, that typically force whether the trans (dark) or cis (lit) isomer is the more bioactive. We now present the rational design of HOTubs, the first hemithioindigo-based pharmacophores enabling photoswitchable control over endogenous biological activity in cellulo. HOTubs optically control microtubule depolymerisation and cell death in unmodified mammalian cells. Notably, we show how the asymmetry of hemithioindigos allows a priori design of either Z- or E- (dark- or lit)-toxic antimitotics, whereas the corresponding azobenzenes are exclusively lit-toxic. We thus demonstrate that hemithioindigos enable an important expansion of the substituent and design scope of photopharmacological interventions for biological systems.

Hemiindigo: Highly Bistable Photoswitching at the Biooptical Window.

Hemiindigo is a long known chromophore that absorbs in the blue part of the spectrum but has almost completely been ignored as potential photoswitch. Herein we show how the absorption of hemiindigo is shifted to the red part of the visible spectrum and how nearly perfect photoswitching can be achieved using blue or green and red light. Five derivatives were investigated giving very high isomeric yields in both switching directions, i.e. >90% E isomer after irradiation with 470 to 530 nm light and 99% Z isomer with 590 up to 680 nm light. At the same time the thermal bistability is extraordinarily high leading to half-lives of the pure isomeric states of up to 83 years at 25 °C. The herein developed photoswitches show photochromism in the visible enabling the two isomeric states to be distinguished by the naked eye. Substituted hemiindigos therefore constitute extremely promising new photoswitches with excellent properties for applications in biology, chemistry, or material sciences.

Synthesis of hemithioindigo-based chromopeptides by using the Tmb auxiliary in native chemical ligation studies.

The 4,5,6-trimethoxy-2-mercaptobenzyl auxiliary was used in auxiliary-based native chemical ligation reactions with Boc-protected pHTI and mHTI ω-amino acid thioesters 2a,b for the construction of small hemithioindigo (HTI)-based chromopeptides 6a,b with a class 1 PDZ binding motif. While reversible tris(2-carboxyethyl)phosphine (TCEP)-HTI adduct formation required moderate use of access TCEP, the Na ascorbate concentration was broadly varied for optimization of the reaction conditions. In the studies presented, the mHTI ω-amino acid thioester 2b proved to be slightly less reactive than the pHTI ω-amino acid thioester 2a. Ligated products 5a-d were isolated in 35-81% yield, and also cleavage of the auxiliary proceeded smoothly, furnishing peptides 6a-d in 48-61% yield. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Light-Switchable Peptides with a Hemithioindigo Unit: Peptide Design, Photochromism, and Optical Spectroscopy.

This Minireview focuses on the hemithioindigo photoswitch and its use for the reversible control of three-dimensional peptide structure and related biological functions. Both the general design aspects and biophysical properties of various hemithioindigo-based chromopeptides are summarized. Hemithioindigo undergoes reversible Z→E photoisomerization after absorption of visible light. The unique ultrafast switching mechanism of hemithioindigo combines picosecond isomerization kinetics with strong double-bond torsion after light absorption, making it the ideal tool for instantaneous modulation of biological structure. Various inhibitors and model peptides based on hemithioindigo are described that can directly regulate biological signaling or allow the fastest events in peptide folding to be studied. Finally, a diverse range of chromopeptides with photoswitchable ß-hairpin structures based on azobenzenes, stilbenes, and hemithioindigo are compared to emphasize the unique properties of hemithioindigo.

A simple and robust preparation of N-acetylindoxyls: precursors for indigogenic substrates.

A generalised, simple and efficient synthesis of N-acetyl-5-bromo-4-chloroindoxyl and related analogues required for the synthesis of indigogenic substrates to probe for biological activities is reported. The method is both synthetically and operationally simple and represents a significant improvement on existing methods.

Making fast photoswitches faster--using Hammett analysis to understand the limit of donor-acceptor approaches for faster hemithioindigo photoswitches.

Hemithioindigo (HTI) photoswitches have a tremendous potential for biological and supramolecular applications due to their absorptions in the visible-light region in conjunction with ultrafast photoisomerization and high thermal bistability. Rational tailoring of the photophysical properties for a specific application is the key to exploit the full potential of HTIs as photoswitching tools. Herein we use time-resolved absorption spectroscopy and Hammett analysis to discover an unexpected principal limit to the photoisomerization rate for donor-substituted HTIs. By using stationary absorption and fluorescence measurements in combination with theoretical investigations, we offer a detailed mechanistic explanation for the observed rate limit. An alternative way of approaching and possibly even exceeding the maximum rate by multiple donor substitution is demonstrated, which give access to the fastest HTI photoswitch reported to date.

Photostability of 4,4'-dihydroxythioindigo, a mimetic of indigo.

The photochemical properties of indigo, a widely used industrial dye, has attracted both experimentalists and theoreticians from the beginning. Especially the high photostability of indigo has been the subject of intensive research. Recently, it was proposed that after photoexcitation an intramolecular proton transfer followed by a nonradiative relaxation to the ground state promote photostability. In indigo the hydrogen bond and the proton transfer occur between the opposing hemiindigo parts. Here, we provide experimental and theoretical evidence that a hydrogen transfer within one hemiindigo or hemithioindigo part is sufficient to attain photostability. This concept can serve as an interesting strategy towards new photostable dyes for the visible part of the spectrum.

What Happens to a Pyrrole Hemithioindigo Photoswitch Trapped in a Fluorescent Protein?

Artificial molecular photoswitches that can be reversibly controlled into different configurations by external light stimulation have broad application prospects in various fields, such as materials and chemical biology. Among them, the pyrrole hemithioindigo (PHT) photoswitch has a geometric structure similar to that of the fluorescent protein chromophore. What happens when the chromophore is replaced by PHT, and does it achieve similar functions to the original one? To answer these questions, we carried out ONIOM(QM/MM) and classical molecular dynamics studies on the photoisomerization mechanism and spectroscopic properties of PHT in the fluorescent protein. The results showed that in the protein environment, the fate of excited PHT is governed by the competition between fluorescence emission and hula-twist isomerization. Due to the strong steric hindrance effects caused by the interlacing residues in the protein that restrict the PHT conformation transformation, the cis-to-trans isomerization process of PHT needs to overcome a barrier of at least 4.9 kcal/mol; thus, fluorescence emission is more dominant in competition. It is also found that the intermolecular interaction between LYS67 and the carbonyl oxygen of PHT has a significant effect on the fluorescence emission. These results revealed the photochemical reaction mechanism of a light-driven molecular switch in the fluorescent protein and provided further theoretical support for the field of chemical biology.

Molecular driving forces for Z/E isomerization mediated by heteroatoms: the example hemithioindigo.

A combined experimental and theoretical investigation of photoinduced Z/E isomerizations is presented. Unsubstituted Hemithioindigo is selected as a representative minimal model to unravel the reaction mechanism in the presence of heteroatoms on an atomic level. Time-resolved spectroscopy reveals multiexponential reaction dynamics on the few picoseconds time scale, which are interpreted by quantum chemical calculations at the CASSCF/CASPT2 level of theory. Detailed insight into the processes governing the ultrafast decay from the first excited state, mediated by a number of conical intersections, is provided. Charge separation and charge balance recovery on the reaction pathway play the leading role and are controlled by the electron-donating or -withdrawing character of the heteroatoms. The electronic and geometric structures of the individual minimum energy conical intersections governing the reaction are rationalized, and an extended energetically low lying conical intersection seam is extracted. Comparison to the experimental results permits linking the observed time constants to molecular intermediates and pathways. An explanation is provided for the pronounced differences of Z → E and the E → Z photoreactions upon excitation to the first excited singlet state.

Organic/inorganic complex pigments: ancient colors Maya Blue.

Maya Blue is an ancient blue pigment composed of palygorskite clay and indigo. It was used by the ancient Maya and provides a dramatic background for some of the most impressive murals throughout Mesoamerica. Despite exposure to acids, alkalis, and chemical solvents, the color of the Maya Blue pigment remains unaltered. The chemical interaction between palygorskite and indigo form an organic/inorganic complex with the carbonyl oxygen of the indigo bound to a surface Al(3+) in the Si-O lattice. In addition indigo will undergo an oxidation to dehydroindigo during preparation. The dehydro-indigo molecule forms a similar but stronger complex with the Al(3+). Thus, Maya Blue varies in color due to the mixed indigo/dehydroindigo complex. The above conclusions are the result of application of multiple techniques (X-ray diffraction, differential thermal analysis/thermal gravimetric analysis, high resolution transmission electron microscopy, scanning electron microscopy, infrared and Raman spectroscopy) to the characterization of the organic/inorganic complex. A picture of the bonding of the organic molecule to the palygorskite surface forming a surface complex is developed and supported by the results of density functional theory calculations. We also report that other organic molecules such as thioindigo form similar organic/inorganic complexes thus, opening an entirely new class of complex materials for future applications.

Design and biological evaluation of photo-switchable inhibitors.

Photo-switchable compounds are becoming increasingly popular for a series of biological applications based on the reversible photo-control of structure and function of biomolecules. Three applications for the usage of BODTCM and hemithioindigo as photo-reactive compounds are described here. The structure of the villin headpiece was modified by replacing a part of the backbone with hemithioindigo, aiming at induction of the folding process by irradiation with a defined wavelength. The E-isomer of BODTCM was applied as potential inhibitor of the 12/15-lipoxygenase (12/15-LOX), which is implicated in the pathogenesis of inflammatory diseases. A required death domain for the binding of proapoptotic proteins (e.g. Bak) to the hydrophobic groove of antiapoptotic proteins is the BH3 helix. Inserting hemithioindigo into this short peptide, stabilization towards proteolytic degradation is achieved. Such photo-reactive compounds might be developed as potential drugs for a great variety of diseases.

Production of superoxide radical in reductive metabolism of a synthetic food-coloring agent, indigocarmine, and related compounds.

Indigocarmine, which is widely used as a synthetic colouring agent for foods and cosmetics in many countries, was reduced to its leuco form and decolorized by rat liver microsomes with NADPH under anaerobic conditions. The reductase activity was enhanced in liver microsomes of phenobarbital-treated rats, and inhibited by diphenyliodonium chloride, a NADPH-cytochrome P450 reductase (P450 reductase) inhibitor, but was not inhibited by SKF 525-A or carbon monoxide. Indigocarmine reductase activity was exhibited by purified rat P450 reductase. In contrast, when indigocarmine was incubated with rat liver microsomes and NADPH under aerobic conditions, superoxide radical was produced and its production was inhibited by superoxide dismutase and diphenyliodonium chloride. When indigocarmine was incubated with purified rat P450 reductase in the presence of NADPH, superoxide radical production was enhanced 17.7-fold (similar to the enhancement of indigocarmine-reducing ability) as compared with that of rat liver microsomes. A decrease of one molecule of NADPH was accompanied with formation of about two molecules of superoxide radical. P450 reductase exhibited little reductase activity towards indigo and tetrabromoindigo, which also afforded little superoxide radical under aerobic conditions. These results indicate that indigocarmine is reduced by P450 reductase to its leuco form, and superoxide radical is produced by autoxidation of the leuco form, through a mechanism known as futile redox cycling.

On the spectroscopic analyses of thioindigo dye.

A combined experimental and theoretical study on molecular structure and vibrational frequencies of thioindigo was reported. The FT-IR spectrum of thioindigo is recorded in the solid phase. The equilibrium geometries, harmonic vibrational frequencies, thermo-chemical parameters, total dipole moment and HOMO-LUMO energies are calculated by density functional theory DFT/B3LYP utilizing 6-311G(d,p) basis set. Results showed that cis-isomer is highly recommended to be a promising structure for many applications in optoelectronic devices due to its high calculated dipole moment value (3.44 Debye) which indicates its high reactivity to interact with the surrounding molecules as compared to the trans-isomer which has no dipole moment. Both isomers have a similar HOMO-LUMO energy gap, of 3.02 eV (cis-isomer) and 2.78 eV (trans-isomer).

Light-switchable hemithioindigo-hemistilbene-containing peptides: ultrafast spectroscopy of the Z → E isomerization of the chromophore and the structural dynamics of the peptide moiety.

Two hemithioindigo-hemistilbene (HTI) derivatives, designed to operate as structural switches in peptides, as well as two HTI peptides are characterized by ultrafast spectroscopy in the visible and the infrared. The two HTI switches follow the reaction scheme published for other HTI compounds with a picosecond excited state reaction (τ(1) ≈ 6 ps) and isomerization from Z to E with τ(2) = 13 and 51 ps. As compared to the isolated chromophores, the isomerization reaction is slowed down in the chromopeptides to τ(2) = 24 and 69 ps. For the smaller peptide containing 6 amino acids, the structural changes of the peptide moiety observed via the IR spectrum in the amide I band follow the isomerization of the molecular switch closely. In the larger cyclic chromopeptide, containing 20 amino acids and mimicking a ß-hairpin structure in the Z-form of the chromophore, the peptide moiety also changes its structure during isomerization of the chromophore. However, the IR spectrum at the end of the observation period of 3 ns deviates significantly from the stationary difference spectrum. These signatures indicate that strong additional structural changes, e.g., breaking of interchain hydrogen bonds, also occur on longer time scales.

The Hammett relationship and reactions in the excited electronic state: hemithioindigo Z/E-photoisomerization.

The photochemical reaction dynamics of a set of photochromic compounds based on thioindigo and stilbene molecular parts (hemithioindigos, HTI) are presented. Photochemical Z/E isomerization around the central double bond occurs with time constants of 216 ps (Z --> E) and 10 ps (E --> Z) for a 5-methyl-hemithioindigo. Chemical substitution on the stilbene moiety causes unusually strong changes in the reaction rate. Electron-donating substituents in the position para to the central double bond (e.g., para-methoxy) strongly accelerate the reaction, while the reaction is drastically slowed by electron-withdrawing groups in this position (e.g., para-nitrile). We correlate the experimental data of seven HTI-compounds in a quantitative manner using the Hammett equation and present a qualitative explanation for the application of ground-state Hammett constants to describe the photoisomerization reaction.

Purification, stability, and mineralization of 3-hydroxy-2- formylbenzothiophene, a metabolite of dibenzothiophene.

3-Hydroxy-2-formylbenzothiophene (HFBT) is a metabolite found in many bacterial cultures that degrade dibenzothiophene (DBT) via the Kodama pathway. The fate of HFBT in cultures and in the environment is unknown. In this study, HFBT was produced by a DBT-degrading bacterium and purified by sublimation. When stored in organic solvent or as a crystal, the HFBT slowly decomposed, yielding colored products. Two of these were identified as thioindigo and cis-thioindigo. The supernatant of the DBT-degrading culture contained thioindigo, which has not been reported previously as a product of DBT biodegradation. In mineral salts medium, HFBT was sufficiently stable to allow biodegradation studies with a mixed microbial culture over a 3- to 4-week period. High-performance liquid chromatography analyses showed that HFBT was removed from the medium. 2-Mercaptophenylglyoxalate, detected as benzothiophene-2,3-dione, was found in an HFBT-degrading mixed culture, and the former appears to be a metabolite of HFBT. This mixed culture also mineralized HFBT to CO2.