Cobalt-Mediated Photochemical C-H Arylation of Pyrroles.

Precious metal complexes remain ubiquitous in photoredox catalysis (PRC) despite concerted efforts to find more earth-abundant catalysts and replacements based on 3d metals in particular. Most otherwise plausible 3d metal complexes are assumed to be unsuitable due to short-lived excited states, which has led researchers to prioritize the pursuit of longer excited-state lifetimes through careful molecular design. However, we report herein that the C-H arylation of pyrroles and related substrates (which are benchmark reactions for assessing the efficacy of photoredox catalysts) can be achieved using a simple and readily accessible octahedral bis(diiminopyridine) cobalt complex, [1-Co](PF6)2. Notably, [1-Co]2+ efficiently functionalizes both chloro- and bromoarene substrates despite the short excited-state lifetime of the key photoexcited intermediate *[1-Co]2+ (8 ps). We present herein the scope of this C-H arylation protocol and provide mechanistic insights derived from detailed spectroscopic and computational studies. These indicate that, despite its transient existence, reduction of *[1-Co]2+ is facilitated via pre-assembly with the NEt3 reductant, highlighting an alternative strategy for the future development of 3d metal-catalyzed PRC.

Illuminating Neuropeptide Y Y4 Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools.

The neuropeptide Y (NPY) Y4 receptor (Y4R), a member of the family of NPY receptors, is physiologically activated by the linear 36-amino acid peptide pancreatic polypeptide (PP). The Y4R is involved in the regulation of various biological processes, most importantly pancreatic secretion, gastrointestinal motility, and regulation of food intake. So far, Y4R binding affinities have been mostly studied in radiochemical binding assays. Except for a few fluorescently labeled PP derivatives, fluorescence-tagged Y4R ligands with high affinity have not been reported. Here, we introduce differently fluorescence-labeled (Sulfo-Cy5, Cy3B, Py-1, Py-5) Y4R ligands derived from recently reported cyclic hexapeptides showing picomolar Y4R binding affinity. With pKi values of 9.22-9.71 (radioligand competition binding assay), all fluorescent ligands (16-19) showed excellent Y4R affinity. Y4R saturation binding, binding kinetics, and competition binding with reference ligands were studied using different fluorescence-based methods: flow cytometry (Sulfo-Cy5, Cy3B, and Py-1 label), fluorescence anisotropy (Cy3B label), and NanoBRET (Cy3B label) binding assays. These experiments confirmed the high binding affinity to Y4R (equilibrium pKd: 9.02-9.9) and proved the applicability of the probes for fluorescence-based Y4R competition binding studies and imaging techniques such as single-receptor molecule tracking.

Structure and photochemistry of di-tert-butyldiphosphatetrahedrane.

Di-tert-butyldiphosphatetrahedrane (tBuCP)2 (1) is a mixed carbon- and phosphorus-based tetrahedral molecule, isolobal to white phosphorus (P4). However, despite the fundamental significance and well-explored reactivity of the latter molecule, the precise structure of the free (tBuCP)2 molecule (1) and a detailed analysis of its electronic properties have remained elusive. Here, single-crystal X-ray structure determination of 1 at low temperature confirms the tetrahedral structure. Furthermore, quantum chemical calculations confirm that 1 is isolobal to P4 and shows a strong largely isotropic diamagnetic response in the magnetic field and thus pronounced spherical aromaticity. A spectroscopic and computational study on the photochemical reactivity reveals that diphosphatetrahedrane 1 readily dimerises to the ladderane-type phosphaalkyne tetramer (tBuCP)4 (2) under irradiation with UV light. With sufficient thermal activation energy, the dimerisation proceeds also in the dark. In both cases, an isomerisation to a 1,2-diphosphacyclobutadiene 1' is the first step. This intermediate subsequently undergoes a [2 + 2] cycloaddition with a second 1,2-diphosphacyclobutadiene molecule to form 2. The 1,2-diphosphacyclobutadiene intermediate 1' can be trapped chemically by N-methylmaleimide as an alternative [2 + 2] cycloaddition partner.

Reactivity of Superbasic Carbanions Generated via Reductive Radical-Polar Crossover in the Context of Photoredox Catalysis.

Photocatalytic reactions involving a reductive radical-polar crossover (RRPCO) generate intermediates with carbanionic reactivity. Many of these proposed intermediates resemble highly reactive organometallic compounds. However, conditions of their formation are generally not tolerated by their isolated organometallic versions and often a different reactivity is observed. Our investigations on their nature and reactivity under commonly used photocatalytic conditions demonstrate that these intermediates are indeed best described as free, superbasic carbanions capable of deprotonating common polar solvents usually assumed to be inert such as acetonitrile, dimethylformamide, and dimethylsulfoxide. Their basicity not only towards solvents but also towards electrophiles, such as aldehydes, ketones, and esters, is comparable to the reactivity of isolated carbanions in the gas-phase. Previously unsuccessful transformations thought to result from a lack of reactivity are explained by their high reactivity towards the solvent and weakly acidic protons of reaction partners. An intuitive explanation for the mode of action of photocatalytically generated carbanions is provided, which enables methods to verify reaction mechanisms proposed to involve an RRPCO step and to identify the reasons for the limitations of current methods.

Characterization of Fluorescent Dyes Frequently Used for Bioimaging: Photophysics and Photocatalytical Reactions with Proteins.

Derivatives of the rhodamine-based dye 5-TAMRA (5-carboxy-tetramethylrhodamine) and the indocarbocyanine-type Cy3B (cyclized derivative of the cyanine dye Cy3), both representing important fluorophores frequently used for the labeling of biomolecules (proteins, nucleic acids) and bioactive compounds, such as receptor ligands, were photophysically investigated in aqueous solution, i.e., in neat phosphate-buffered saline (PBS) and in PBS supplemented with 1 wt % bovine serum albumin (BSA). The dyes exhibit comparable absorption (λabs,max: 550-569 nm) and emission wavelengths (λem,max: 580-582 nm), and similar S1 lifetimes (2.27-2.75 ns), and their excited state deactivation proceeds mainly via the lowest excited singlet state (triplet quantum yield ca. 1%). However, the probes show marked differences with respect to their fluorescence quantum yield and photostability. While 5-TAMRA shows a lower quantum yield (37-39%) than the Cy3B derivative (ca. 57%), its photostability is considerably higher compared to Cy3B. Generally, the impact of the protein on the photophysics is low. However, on prolonged illumination, both fluorescent dyes undergo a photocatalytic reaction with tryptophan residues of BSA mediated by sensitized singlet oxygen resulting in a tryptophan photoproduct with an absorption maximum around 330 nm. The overall results of this work will assist in choosing the right dye for the labeling of bioactive compounds, and the study demonstrates that experiments performed with 5-TAMRA or Cy3B-labeled compounds in a biological environment may be influenced by photochemical modification of experimentally relevant proteins at aromatic amino acid residues.

Creating a Defined Chirality in Amino Acids and Cyclic Dipeptides by Photochemical Deracemization.

2,5-Diketopiperazines are cyclic dipeptides displaying a wide range of applications. Their enantioselective preparation has now been found possible from the respective racemates by a photochemical deracemization (53 examples, 74 % to quantitative yield, 71-99 % ee). A chiral benzophenone catalyst in concert with irradiation at λ=366 nm enables to establish the configuration at the stereogenic carbon atom C6 at will. If other stereogenic centers are present in the diketopiperazines they remain unaffected and a stereochemical editing is possible at a single position. Consecutive reactions, including the conversion into N-aryl or N-alkyl amino acids or the reduction to piperazines, occur without compromising the newly created stereogenic center. Transient absorption spectroscopy revealed that the benzophenone catalyst processes one enantiomer of the 2,5-diketopiperazines preferentially and enables a reversible hydrogen atom transfer that is responsible for the deracemization process. The remarkably long lifetime of the protonated ketyl radical implies a yet unprecedented mode of action.

Multifaceted View on the Mechanism of a Photochemical Deracemization Reaction.

Upon irradiation in the presence of a chiral benzophenone catalyst (5 mol %), a racemic mixture of a given chiral imidazolidine-2,4-dione (hydantoin) can be converted almost quantitatively into the same compound with high enantiomeric excess (80-99% ee). The mechanism of this photochemical deracemization reaction was elucidated by a suite of mechanistic experiments. It was corroborated by nuclear magnetic resonance titration that the catalyst binds the two enantiomers by two-point hydrogen bonding. In one of the diastereomeric complexes, the hydrogen atom at the stereogenic carbon atom is ideally positioned for hydrogen atom transfer (HAT) to the photoexcited benzophenone. Detection of the protonated ketyl radical by transient absorption revealed hydrogen abstraction to occur from only one but not from the other hydantoin enantiomer. Quantum chemical calculations allowed us to visualize the HAT within this complex and, more importantly, showed that the back HAT does not occur to the carbon atom of the hydantoin radical but to its oxygen atom. The achiral enol formed in this process could be directly monitored by its characteristic transient absorption signal at λ ≅ 330 nm. Subsequent tautomerization leads to both hydantoin enantiomers, but only one of them returns to the catalytic cycle, thus leading to an enrichment of the other enantiomer. The data are fully consistent with deuterium labeling experiments and deliver a detailed picture of a synthetically useful photochemical deracemization reaction.

Synthesis, Characterization, and Application of Muscarinergic M3 Receptor Ligands Linked to Fluorescent Dyes.

Through the linkage of two muscarinergic M3 receptor ligands to fluorescent tetramethylrhodamine- and cyanine-5-type dyes, two novel tool compounds, OFH5503 and OFH611, have been developed. Based on the suitable binding properties and kinetics related to the M3 subtype, both ligand-dye conjugates were found to be useful tools to determine binding affinities via flow cytometric measurements. In addition, confocal microscopy underlined the comparably low unspecific binding and the applicability for studying M3 receptor expression in cells. Along with the proven usefulness regarding studies on the M3 subtype, the conjugates OFH5503 and OFH611 could, due to their high affinity to the M1 receptor, evolve as even more versatile tools in the field of research on muscarinergic receptors.

Monitoring the photochemistry of a formazan over 15 orders of magnitude in time.

2,3,5-triphenyltetrazolium chloride (TTC) may convert into phenyl-benzo[c]tetrazolocinnolium chloride (PTC) and 1,3,5-triphenylformazan (TPF) under irradiation with light. The latter reaction, albeit enzymatically rather than photochemically, is used in so-called TTC assays indicating cellular respiration and cell growth. In this paper, we address the photochemistry of TPF with time-resolved spectroscopy on various time scales. TPF is stabilized by an intramolecular hydrogen bond and switches photochemically via an E-Z isomerization around an N=N double bond into another TPF-stereoisomer, from which further isomerizations around the C=N double bond of the phenylhydrazone group are possible. We investigate the underlying processes by time-resolved spectroscopy in dependence on excitation wavelength and solvent environment, resolving several intermediates over a temporal range spanning 15 orders of magnitude (hundreds of femtoseconds to hundreds of seconds) along the reaction path. In a quantum-chemical analysis, we identify 16 stable ground-state isomers and discuss which ones are identified in the experimental data. We derive a detailed scheme how these species are thermally and photochemically interconnected and conclude that proton transfer processes are involved.

Ultrafast transient absorption and solvation of a super-photoacid in acetoneous environments.

The phenomenon of photoacidity, i.e., an increase in acidity by several orders of magnitude upon electronic excitation, is frequently encountered in aromatic alcohols capable of transferring a proton to a suitable acceptor. A promising new class of neutral super-photoacids based on pyranine derivatives has been shown to exhibit pronounced solvatochromic effects. To disclose the underlying mechanisms contributing to excited-state proton transfer (ESPT) and the temporal characteristics of solvation and ESPT, we scrutinize the associated ultrafast dynamics of the strongest photoacid of this class, namely tris(1,1,1,3,3,3-hexafluoropropan-2-yl)8-hydroxypyrene-1,3,6-trisulfonate, in acetoneous environment, thereby finding experimental evidence for ESPT even under these adverse conditions for proton transfer. Juxtaposing results from time-correlated single-photon counting and femtosecond transient absorption measurements combined with a complete decomposition of all signal components, i.e., absorption of ground and excited states as well as stimulated emission, we disclose dynamics of solvation, rotational diffusion, and radiative relaxation processes in acetone and identify the relevant steps of ESPT along with the associated time scales.

Insights into the different mechanistic stages of light-induced hydrogen evolution of a 5,5'-bisphenanthroline linked RuPt complex.

Herein, the synthesis in conjunction with the structural, electrochemical, and photophysical characterization of a 5,5'-bisphenanthroline (phenphen) linked heterodinuclear RuPt complex (Ru(phenphen)Pt) and its light-driven hydrogen formation activity are reported. A single crystal X-ray diffraction (SC-XRD) analysis identified a perpendicular orientation of the two directly linked 1,10-phenanthroline moieties. The disruption of π-conjugation blocks intramolecular electron transfer as evidenced by a comparative time-resolved optical spectroscopy study of Ru(phenphen)Pt and the reference complexes Ru(phenphen) and Ru(phenphen)Ru. However, reductive quenching is observed in the presence of an external electron donor such as triethylamine. Irradiating Ru(phenphen)Pt with visible light (470 nm) leads to H2 formation. We discuss a potential mechanism that mainly proceeds via Pt colloids and provide indications that initial hydrogen generation may also proceed via a molecular pathway. As previous reports on related heterodinuclear RuPt-based photocatalysts revealed purely molecular hydrogen evolution, the present work thus highlights the role of the bridging ligand in stabilizing the catalytic center and consequently determining the mechanism of light-induced hydrogen evolution in these systems.

Tuning Deazaflavins Towards Highly Potent Reducing Photocatalysts Guided by Mechanistic Understanding - Enhancement of the Key Step by the Internal Heavy Atom Effect.

Deazaflavins are well suited for reductive chemistry acting via a consecutive photo-induced electron transfer, in which their triplet state and semiquinone - the latter is formed from the former after electron transfer from a sacrificial electron donor - are key intermediates. Guided by mechanistic investigations aiming to increase intersystem crossing by the internal heavy atom effect and optimising the concentration conditions to avoid unproductive excited singlet reactions, we synthesised 5-aryldeazaflavins with Br or Cl substituents on different structural positions via a three-component reaction. Bromination of the deazaisoalloxazine core leads to almost 100 % triplet yield but causes photo-instability and enhances unproductive side reactions. Bromine on the 5-phenyl group in ortho position does not affect the photostability, increases the triplet yield, and allows its efficient usage in the photocatalytic dehalogenation of bromo- and chloroarenes with electron-donating methoxy and alkyl groups even under aerobic conditions. Reductive powers comparable to lithium are achieved.

Photocatalytic Oxidative [2+2] Cycloelimination Reactions with Flavinium Salts: Mechanistic Study and Influence of the Catalyst Structure.

Flavinium salts are frequently used in organocatalysis but their application in photoredox catalysis has not been systematically investigated to date. We synthesized a series of 5-ethyl-1,3-dimethylalloxazinium salts with different substituents in the positions 7 and 8 and investigated their application in light-dependent oxidative cycloelimination of cyclobutanes. Detailed mechanistic investigations with a coumarin dimer as a model substrate reveal that the reaction preferentially occurs via the triplet-born radical pair after electron transfer from the substrate to the triplet state of an alloxazinium salt. The very photostable 7,8-dimethoxy derivative is a superior catalyst with a sufficiently high oxidation power (E*=2.26 V) allowing the conversion of various cyclobutanes (with Eox up to 2.05 V) in high yields. Even compounds such as all-trans dimethyl 3,4-bis(4-methoxyphenyl)cyclobutane-1,2-dicarboxylate can be converted, whose opening requires a high activation energy due to a missing pre-activation caused by bulky adjacent substituents in cis-position.

Complex Evolution of Light-Dependent Protochlorophyllide Oxidoreductases in Aerobic Anoxygenic Phototrophs: Origin, Phylogeny, and Function.

Light-dependent protochlorophyllide oxidoreductase (LPOR) and dark-operative protochlorophyllide oxidoreductase are evolutionary and structurally distinct enzymes that are essential for the synthesis of (bacterio)chlorophyll, the primary pigment needed for both anoxygenic and oxygenic photosynthesis. In contrast to the long-held hypothesis that LPORs are only present in oxygenic phototrophs, we recently identified a functional LPOR in the aerobic anoxygenic phototrophic bacterium (AAPB) Dinoroseobacter shibae and attributed its presence to a single horizontal gene transfer event from cyanobacteria. Here, we provide evidence for the more widespread presence of genuine LPOR enzymes in AAPBs. An exhaustive bioinformatics search identified 36 putative LPORs outside of oxygenic phototrophic bacteria (cyanobacteria) with the majority being AAPBs. Using in vitro and in vivo assays, we show that the large majority of the tested AAPB enzymes are genuine LPORs. Solution structural analyses, performed for two of the AAPB LPORs, revealed a globally conserved structure when compared with a well-characterized cyanobacterial LPOR. Phylogenetic analyses suggest that LPORs were transferred not only from cyanobacteria but also subsequently between proteobacteria and from proteobacteria to Gemmatimonadetes. Our study thus provides another interesting example for the complex evolutionary processes that govern the evolution of bacteria, involving multiple horizontal gene transfer events that likely occurred at different time points and involved different donors.

The sacrificial inactivation of the blue-light photosensor cryptochrome from Drosophila melanogaster.

Drosophila melanogaster cryptochrome functions as the primary blue-light receptor that mediates circadian photo entrainment. Absorption of a photon leads to reduction of the protein-bound FAD via consecutive electron transfer along a conserved tryptophan tetrad resembling the signalling state required for conformational changes and induction of subsequent signalling cascades. However, how the initial photochemistry and subsequent dark processes leading to downstream signalling are linked to each other at the molecular level is still poorly understood. Here, we investigated in detail the initial photochemical events in DmCRY by time-resolved and stationary absorption spectroscopy combined with quantum chemical and molecular dynamics calculations. We resolved the early events along the conserved tryptophan tetrad and the final deprotonation of the terminal tryptophanyl radical cation. These initial events lead to conformational changes, such as the known C-terminal tail release, Trp decomposition, and finally FAD release providing evidence that DmCRY does not undergo a photocycle. We propose that light is a negative regulator of DmCRY stability even under in vitro conditions where the proteasomal machinery is missing, that is in line with its biological function, i.e. entrainment of the circadian clock.

DNA-Based Oligochromophores as Light-Harvesting Systems.

The chromophores ethynyl pyrene as blue, ethynyl perylene as green and ethynyl Nile red as red emitter were conjugated to the 5-position of 2'-deoxyuridine via an acetylene bridge. Using phosphoramidite chemistry on solid phase labelled DNA duplexes were prepared that bear single chromophore modifications, and binary and ternary combinations of these chromophore modifications. The steady-state and time-resolved fluorescence spectra of all three chromophores were studied in these modified DNA duplexes. An energy-transfer cascade occurs from ethynyl pyrene over ethynyl perylene to ethynyl Nile red and subsequently an electron-transfer cascade in the opposite direction (from ethynyl Nile red to ethynyl perylene or ethynyl pyrene, but not from ethynyl perylene to ethynyl pyrene). The electron-transfer processes finally provide charge separation. The efficiencies by these energy and electron-transfer processes can be tuned by the distances between the chromophores and the sequences. Most importantly, excitation at any wavelength between 350 and 700 nm finally leads to charge separated states which make these DNA samples promising candidates for light-harvesting systems.

A search for radical intermediates in the photocycle of LOV domains.

LOV domains are the light sensitive parts of phototropins and many other light-activated enzymes that regulate the response to blue light in plants and algae as well as some fungi and bacteria. Unlike all other biological photoreceptors known so far, the photocycle of LOV domains involves the excited triplet state of the chromophore. This chromophore is flavin mononucleotide (FMN) which forms a covalent adduct with a cysteine residue in the signaling state. Since the formation of this adduct from the triplet state involves breaking and forming of two bonds as well as a change from the triplet to the singlet spin state, various intermediates have been proposed, e.g. a protonated triplet state (3)FMNH(+), the radical anion (2)FMN˙(-), or the neutral semiquinone radical (2)FMNH˙. We performed an extensive search for these intermediates by two-dimensional transient absorption (2D-TA) with a streak camera. However, no transient with a rate constant between the decay of fluorescence and the decay of the triplet state could be detected. Analysis of the decay associated difference spectra results in quantum yields for the formation of the adduct from the triplet of ΦA(LOV1) ≈ 0.75 and ΦA(LOV2) ≈ 0.80. This is lower than the values ΦA(LOV1) ≈ 0.95 and ΦA(LOV2) ≈ 0.99 calculated from the rate constants, giving indirect evidence of an intermediate that reacts either to form the adduct or to decay back to the ground state. Since there is no measurable delay between the decay of the triplet and the formation of the adduct, we conclude that this intermediate reacts much faster than it is formed. The LOV1-C57S mutant shows a weak and slowly decaying (τ > 100 µs) transient whose decay associated spectrum has bands at 375 and 500 nm, with a shoulder at 400 nm. This transient is insensitive to the pH change in the range 6.5-10.0 but increases on addition of ß-mercaptoethanol as the reducing agent. We assign this intermediate to the radical anion which is protected from protonation by the protein. We propose that the adduct is formed via the same intermediate by combination of the radical ion pair.

An inhibitor of NADPH oxidase-4 attenuates established pulmonary fibrosis in a rodent disease model.

Idiopathic pulmonary fibrosis is a chronic progressive disease of increasing prevalence for which there is no effective therapy. Increased oxidative stress associated with an oxidant-antioxidant imbalance is thought to contribute to disease progression. NADPH oxidases (Nox) are a primary source of reactive oxygen species within the lung and cardiovascular system. We demonstrate that the Nox4 isoform is up-regulated in the lungs of patients with IPF and in a rodent model of bleomycin-induced pulmonary fibrosis and vascular remodeling. Nox4 is constitutively active, and therefore increased expression levels are likely to contribute to disease pathology. Using a small molecule Nox4/Nox1 inhibitor, we demonstrate that targeting Nox4 results in attenuation of an established fibrotic response, with reductions in gene transcripts for the extracellular matrix components collagen 1α1, collagen 3α1, and fibronectin and in principle pathway components associated with pulmonary fibrosis and hypoxia-mediated vascular remodeling: transforming growth factor (TGF)-ß1, plasminogen activator inhibitor-1, hypoxia-inducible factor, and Nox4. TGF-ß1 is a principle fibrotic mediator responsible for inducing up-regulation of profibrotic pathways associated with disease pathology. Using normal human lung-derived primary fibroblasts, we demonstrate that inhibition of Nox4 activity using a small molecule antagonist attenuates TGF-ß1-mediated up-regulation in expression of profibrotic genes and inhibits the differentiation of fibroblast to myofibroblasts, that is associated with up-regulation in smooth muscle actin and acquisition of a contractile phenotype. These studies support the view that targeting Nox4 may provide a therapeutic approach for attenuating pulmonary fibrosis.

Attenuation of leukocyte recruitment via CXCR1/2 inhibition stops the progression of PAH in mice with genetic ablation of endothelial BMPR-II.

Previous studies from our group have demonstrated that bone morphogenetic protein receptor-II (BMPR-II), expressed on pulmonary artery endothelial cells, imparts profound anti-inflammatory effects by regulating the release of proinflammatory cytokines and promoting barrier function by suppressing the transmigration of leukocytes into the pulmonary vessel wall. Here we demonstrate that, in mice with endothelial-specific loss of BMPR-II expression (L1Cre(+);Bmpr2(f/f)), reduction in barrier function and the resultant pulmonary hypertension observed in vivo are the result of increased leukocyte recruitment through increased CXCR1/2 signaling. Loss of endothelial expressed BMPR-II leads to elevated plasma levels of a wide range of soluble mediators important in regulating leukocyte migration and extravasation, including the CXCR1/2 ligand, KC. Treatment of L1Cre(+);Bmpr2(f/f) mice with the CXCR1/2 antagonist SCH527123 inhibits leukocyte transmigration into lung and subsequently reverses the pulmonary hypertension. Our data have uncovered a previously unrecognized regulatory function of BMPR-II, which acts to regulate the expression of CXCR2 on endothelial cells, suggesting that increased CXCR2 signaling may also be a feature of the human pathology and that CXCR1/2 pathway antagonists may represent a novel therapeutic approach for treating pulmonary hypertension because of defects in BMPR-II expression.