Photoisomerization and Light-Controlled Antibacterial Activity of Fluoroquinolone-Azoisoxazole Hybrids.

Photopharmacology holds a huge untapped potential to locally treat diseases involving photoswitchable drugs via the elimination of drugs' off-target effects. The growth of this field has created a pressing demand to develop such light-active drugs. We explored the potential for creating photoswitchable antibiotic hybrids by attaching pharmacophores norfloxacin/ciprofloxacin and azoisoxazole (photoswitch). All compounds exhibited a moderate to a high degree of bidirectional photoisomerization, long thermal cis half-lives, and impressive photoresistance. Gram-negative pathogens were found to be insensitive to these hybrids, while against Gram-positive pathogens, all hybrids in their trans states exhibited antibacterial activity that is comparable to that of the parent drugs. Notably, the toxicity of the irradiated hybrid 6 was found to be 2-fold lower than the nonirradiated trans isomer, indicating that the pre-inactivated cis-enriched drug can be employed for the site-specific treatment of bacterial infection using light, which could potentially eliminate the unwanted exposure of toxic antibiotics to both beneficial and untargeted harmful microbes in our body. Molecular docking revealed different binding affinity of the cis and trans isomers with the topoisomerase IV enzyme, due to their different shapes.

Photoswitchable Antibiotic Hybrids: Spacer Length-Dependent Photochemical Control of Antibacterial Activity.

Photopharmacology holds huge potential for the permanent (long-term) eradication of antibiotic resistance by the application of photoswitchable antibiotics. To construct such antibiotics, various methods have been employed to modify known antibiotics with photoswitches, such that the irradiated state shows activity comparable to or higher than that of the parent antibiotic and that a large activity difference between irradiated and nonirradiated states is achieved. However, most of those methods are ineffective when dealing with more than one drug with dissimilar structures. Here, we have demonstrated a new approach, in which two pharmacophores, one being a photoswitch, are covalently linked via a spacer of variable lengths, leading to a set of azopyrazole-norfloxacin antibiotic hybrids. All compounds showed a high degree of bidirectional photoisomerization, long thermal cis half-lives, and excellent photoresistance. Notably, the hybrid with an optimal four-carbon spacer length enabled the irradiated state to become 12-fold more potent than its nonirradiated state without losing much antimicrobial activity of norfloxacin. Only Gram-positive bacteria were found to be sensitive to this hybrid, and the full antibacterial potency of its irradiated state was found to be retained for nearly 24 h.

Direct Growth Control of Antibiotic-Resistant Bacteria Using Visible-Light-Responsive Novel Photoswitchable Antibiotics.

In addition to the discovery of new (modified) potent antibiotics to combat antibiotic resistance, there is a critical need to develop novel strategies that would restrict their off-target effects and unnecessary exposure to bacteria in our body and environment. We report a set of new photoswitchable arylazopyrazole-modified norfloxacin antibiotics that present a high degree of bidirectional photoisomerization, impressive fatigue resistance and reasonably high cis half-lives. The irradiated isomers of most compounds were found to exhibit nearly equal or higher antibacterial activity than norfloxacin against Gram-positive bacteria. Notably, against norfloxacin-resistant S. aureus bacteria, the visible-light-responsive p-SMe-substituted derivative showed remarkably high antimicrobial potency (MIC of 0.25 µg/mL) in the irradiated state, while the potency was reduced by 24-fold in case of its non-irradiated state. The activity was estimated to be retained for more than 7 hours. This is the first report to demonstrate direct photochemical control of the growth of antibiotic-resistant bacteria and to show the highest activity difference between irradiated and non-irradiated states of a photoswitchable antibiotic. Additionally, both isomers were found to be non-harmful to human cells. Molecular modellings were performed to identify the underlying reason behind the high-affinity binding of the irradiated isomer to topoisomerase IV enzyme.

Noninnocent Azo-Aromatic Cobalt(II)-Catalyzed sp3 C-H Alkylation of Fluorenes with Alcohols.

Herein, employing well-defined redox noninnocent cobalt(II) complexes an efficient sp3 C-H alkylation of fluorenes using alcohols as alkylating agents to result in alkylated fluorenes is reported. The catalytic protocol was versatile with various fluorenes and benzyl alcohols. It also showed very good functional group tolerance with both alcohols and fluorenes. Moreover, an efficient single-step and simultaneous di C-C as well as both C-C and the C-N alkylation reaction of fluorenes was observed with this catalytic protocol. Such selective single-step dialkylation of fluorenes is indeed beneficial. Several control experiments, deuterium labeling, and 1H NMR kinetic studies have revealed a ligand radical-based borrowing hydrogen mechanism involving the azo-aromatic complexes of cobalt as catalysts for the alkylation of fluorenes.

A Phosphine-Oxide Cobalt(II) Complex and Its Catalytic Activity Studies toward Alcohol Dehydrogenation Triggered Direct Synthesis of Imines and Quinolines.

Herein, a new pincer-like amino phosphine donor ligand, H2L1, and its phosphine-oxide analog, H2L2, were synthesized. Subsequently, cobalt(II) complexes 1 and 2 were synthesized by the reaction of anhydrous Co(II)Cl2 with ligands H2L1 and H2L2, respectively. The ligands and complexes were fully characterized by various physicochemical and spectroscopic characterization techniques. Finally, the identity of the complexes 1 and 2 was confirmed by single crystal X-ray structure determination. The phosphine ligand containing complex 1 was converted to the phosphine oxide ligand containing complex 2 in air in acetonitrile solution. Both complexes 1 and 2 were investigated as precatalysts for alcohol dehydrogenation-triggered synthesis of imines in air. The phosphine-oxide complex 2 was more efficient than the phosphine complex 1. A wide array of alcohols and amines were successfully reacted in a mild condition to result in imines in good to excellent yields. Precatalyst 2 was also highly efficient for the synthesis of varieties of quinolines in air. As H2L2 in 2 has side arms that can be deprotonated, we investigated complex 2 for its base (KOtBu) promoted deprotonation events by various spectroscopic studies and DFT calculations. These studies have shown that mono deprotonation of the amine side arm attached to the pyridine is quite feasible, and deprotonation of complex 2 leads to a dearomatized pyridyl ring containing complex 2a. The mechanistic investigations of the catalytic reaction, by a combination of experimental and computational studies, have suggested that the dearomatized complex, 2a acted as an active catalyst. The reaction proceeded through the hydride transfer pathway. The activation barrier of this step was calculated to be 26.5 kcal/mol, which is quite consistent with the experimental reaction temperature under aerobic conditions. Although various pincer-like complexes are explored for such reactions, phosphine oxide ligand-containing complexes are still unexplored.

Alcohol Dehydrogenation-Triggered Selective C3-Alkylation of Indoles by Homogeneous Azo-aromatic Cobalt Catalysts.

Herein, we report azo-benzimidazole containing cobalt complexes (1-3) for alcohol dehydrogenation-triggered C3-alkylation of indoles. In complexes 1-3, ligands are redox noninnocent and showed facile irreversible L/L• reduction followed by Co(II)/Co(I) reduction in close-lying potentials. Taking advantage of facile redox events in 1-3, the first aerial dehydrogenation of alcohols to their corresponding carbonyl compounds is explored. Subsequently, C3-alkylation of indole was studied using alcohols as the alkylating agents. The developed catalytic protocol was found to be efficient and very selective. It has a broad substrate scope and good functional group tolerance. As far as we are aware, it is the first homogeneous cobalt catalyst for C3-alkylation of indole using alcohol as the alkylating agent. Detailed mechanistic studies, including a deuterium labeling experiment, have suggested a borrowing hydrogen method for the C3-alkylation of indole. The coordinated ligand, cooperatively with the Co(II)/Co(I) redox couple, oxidized the coordinated alkoxide in a radical pathway to result in the carbonyl compound (Scheme 1), which on subsequent condensation with indole generates the alkylideneindolenine intermediate "X". Reduction of "X" by an azo-anion radical Co(I) catalyst intermediate resulted in the C3-alkylated indole.

Photoisomerization of 2-Arylazoimidazoles under Visible Light: Identifying a Predictive Tool to Anticipate and Tune Likely Photoswitching Performance and Cis Half-Life.

Azopyrazoles are an emerging class of photoswitches, whereas analogous azoimidazole-based switches are unable to draw much attention because of their short cis half-lives, poor cis-trans photoreversion yields, and toxic ultraviolet (UV) light-assisted isomerization. A series of 24 various aryl-substituted N-methyl-2-arylazoimidazoles were synthesized, and their photoswitching performances and cis-trans isomerization kinetics were thoroughly investigated experimentally and theoretically. Para-π-donor-substituted azoimidazoles with highly twisted T-shaped cis conformations showed nearly complete bidirectional photoswitching, whereas di-o-substituted switches exhibited very long cis half-lives (days-years) with nearly ideal T-shaped conformations. This study demonstrates how the electron density in the aryl ring affects cis half-life and cis-trans photoreversion via twisting of the NNAr dihedral angle that can be used as a predictive metric for envisaging and tuning the likely switching performance and half-life of any given 2-arylazoimidazole. By applying this tool, two better-performing azoimidazole photoswitches were engineered. All switches permitted irradiation by violet (400-405 nm) and orange (>585 nm) light for forward and reverse isomerization, respectively, and displayed comparatively high quantum yields and impressive resistance to photobleaching.

Near-Complete Bidirectional Photoisomerization of para-Dialkylamino-Substituted Arylazopyrazoles under Violet and Green or Red Lights.

para-Dimethylamine- and para-pyrrolidine-substituted arylazopyrazoles display very high to near-quantitative or quantitative bidirectional isomerization under violet and green or red lights in both polar (DMSO and DMSO/aqueous buffer, pH 7.5) and nonpolar solvents. These switches confer a reasonable thermal stability to their cis-states (t1/2 ≈ 4-7 h in DMSO and DMSO/buffer) and also show a high level of resistance to photobleaching and an impressive stability to reduction by glutathione. Using DFT calculations, attempts have been made to decipher the photophysical properties and thermal stabilities of the cis isomers.

Hemilabile Amine-Functionalized Efficient Azo-Aromatic Cu-Catalysts Inspired by Galactose Oxidase: Impact of Amine Sidearm on Catalytic Aerobic Oxidation of Alcohols.

A series of azo-aromatic copper(II) complexes, [1a-g] and a Cu(I) complex, [1h], with varying amine-functionalized hemilabile pincer-like [HL1-3] and [L1,2], methyl-substituted azo [L3], and imine [L4] ligands, were synthesized and characterized. These complexes were investigated for aerobic oxidation of a variety of aromatic alcohols in the presence of 2.0 mol % precatalysts [1a-g], cobaltocene (2.0 mol %), N-methyl imidazole (NMI) (8.0 mol %), and TEMPOH (2.0 mol %) at room temperature. The Cu(I) complex (1h) acted as a catalyst in the absence of cobaltocene. To understand the mechanism, detailed experimental and theoretical studies have been performed with the representative complex [1a], which has suggested a new kind of mechanism involving a Cu(II)/Cu(I) redox couple. Cobaltocene acts as a reductant to [1a] to generate a Cu(I) complex, which activates dioxygen in the presence of NMI. TEMPOH transfers a hydrogen atom to the activated dioxygen with the generation of TEMPO•, which further participates in α-C-H bond activation in the Cu(II)-alkoxide intermediate in an intermolecular fashion in the catalytic cycle. The amine sidearm in the ligand backbone of the complexes has a significant role in catalytic activity. Complexes with amine sidearms are more effective than complexes without them. Moreover, the aliphatic secondary amine sidearm is more efficient among the amine sidearm than the aromatic secondary amine and tertiary amines. The amine sidearm that remained coordinated to the Cu(II) center is hemilabile, and it facilitates alcohol coordination in the catalytic process. Alcohol coordination was the rate-limiting step, and it was supported by the isotope effect study on benzyl alcohol, substitution effect on the amine moiety of the ligands, and DFT calculation. The hemilabile amine sidearm of the coordinated ligand also acted as a base in deprotonating the alcoholic O-H proton and acted as an acid in releasing H2O2 during the catalysis.

Azide-Alkyne "Click" Reaction in Water Using Parts-Per-Million Amine-Functionalized Azoaromatic Cu(I) Complex as Catalyst: Effect of the Amine Side Arm.

A series of Cu(II) complexes, 1-4 and 6, were synthesized through a reaction of amine-functionalized pincer-like ligands, HL1,2, La,b, and a bidentate ligand L1 with CuCl2·2H2O. The chemical reduction of complex 1 using 1 equiv of sodium l-ascorbate resulted in a dimeric Cu(I) complex 5 in excellent yield. All of the complexes, 1-6, were thoroughly characterized using various physicochemical characterization techniques, single-crystal X-ray structure determination, and density functional theory calculations. Ligands HL1,2 and La,b behaved as tridentated donors by the coordination of the amine side arm in their respective Cu(II) complexes, and the amine side arm remained as a pendant in Cu(I) complexes. All of these complexes (1-6) were explored for copper(I)-catalyzed 1,3-dipolar azide-alkyne cycloaddition (CuAAC) reaction at room temperature in water under air. Complex 5 directly served as an active catalyst; however, complexes 1-4 and 6 required 1 equiv of sodium l-ascorbate to generate their corresponding active Cu(I) catalyst. It has been observed that azo-based ligand-containing Cu(I)-complexes are air-stable and were highly efficient for the CuAAC reaction. The amine side arm in the ligand backbone has a dramatic role in accelerating the reaction rate. Mechanistic investigations showed that the alkyne C-H deprotonation was the rate-limiting step and the pendant amine side arm intramolecularly served as a base for Cu-coordinated alkyne deprotonation, leading to the azide-alkyne 2 + 3 cycloaddition reaction. Thus, variation of the amine side arm in complexes 1-4 and use of the most basic diisopropyl amine moiety in complex 4 has resulted in an unique amine-functionalized azoaromatic Cu(I) system for CuAAC reaction upon sodium l-ascorbate reduction. The complex 4 has shown excellent catalysis at its low parts-per-million level loading in water. The catalytic protocol was versatile and exhibited very good functional group tolerance. It was also employed efficiently to synthesize a number of useful functional triazoles having medicinal, catalytic, and targeting properties.

Reversible and Tunable Photoswitching of Protein Function through Genetic Encoding of Azobenzene Amino Acids in Mammalian Cells.

The genetic encoding of three different azobenzene phenylalanines with different photochemical properties was achieved in human cells by using an engineered pyrrolysyl tRNA/tRNA synthetase pair. In order to demonstrate reversible light control of protein function, azobenzenes were site-specifically introduced into firefly luciferase. Computational strategies were applied to guide the selection of potential photoswitchable sites that lead to a reversibly controlled luciferase enzyme. In addition, the new azobenzene analogues provide enhanced thermal stability, high photoconversion, and responsiveness to visible light. These small-molecule photoswitches can reversibly photocontrol protein function with excellent spatiotemporal resolution, and preferred sites for incorporation can be computationally determined, thus providing a new tool for investigating biological processes.

Effects of Ancillary Ligands on Redox and Chemical Properties of Ruthenium Coordinated Azoaromatic Pincer.

In this work, the effect of the electronically different ancillary ligands on the overall properties of the RuIIL moiety (L = 2,6-bis(phenylazo)pyridine) in heteroleptic complexes of general formula [RuLQCl]0/+ was investigated. Four different ancillary ligands (Q) with different electronic effects were used to prepare the heteroleptic compounds from the precursor complex, [RuL(CH3CN)Cl2] (1); Q = pcp: 2-(4-chloro-phenylazo)pyridine (strong π-acceptor), [2]+; bpy: 2,2'-bipyridyl (moderate π-acceptor), [3]+; acac-: acetylacetonate (strong σ-donor), 4; and DTBCat2-: 3,5-di- tert-butyl catecholate (strong π-donor), 5. The complexes [2]+, [3]+, 4, and 5 were fully characterized and structurally identified. The electronic structures of these complexes along with their redox partners were elucidated by using a host of physical measurements: nuclear magnetic resonance, cyclic voltammetry, electronic paramagnetic resonance, UV-vis-NIR spectroscopy, and density functional theory. The studies revealed significant effects of the coligands on azo bond lengths of the RuL moiety and their redox behavior. Aerobic alcohol oxidation reactions using these Ru complexes as catalysts were scrutinized. It was found that the catalytic efficiency is primarily controlled by the electronic effect of the coligand. Accordingly, the complex [2]+ (containing a strong π-acceptor coligand, pcp) brings about oxidation efficiently, producing 86% of benzaldehyde. In comparison, however, the complexes 4 and 5 (containing electron donating coligand) furnished only 15-20% of benzaldehyde under identical reaction conditions. Investigations of the reaction mechanism suggest that an unstable Ru-H species is formed, which is transformed to a Ru-hydrazo intermediate by H-walking as reported by Hall et al. ( J. Am. Chem. Soc., 2015, 137, 12330). Aerial O2 regenerates the catalyst via oxidation of the hydrazo intermediate.

Genetic Code Expansion in Zebrafish Embryos and Its Application to Optical Control of Cell Signaling.

Site-specific incorporation of unnatural amino acids into proteins provides a powerful tool to study protein function. Here we report genetic code expansion in zebrafish embryos and its application to the optogenetic control of cell signaling. We genetically encoded four unnatural amino acids with a diverse set of functional groups, which included a photocaged lysine that was applied to the light-activation of luciferase and kinase activity. This approach enables versatile manipulation of protein function in live zebrafish embryos, a transparent and commonly used model organism to study embryonic development.

Optical Control of DNA Helicase Function through Genetic Code Expansion.

Nucleotide excision repair (NER) is a general DNA repair mechanism that is capable of removing a wide variety of DNA lesions induced by physical or chemical insults. UvrD, a member of the helicase SF1 superfamily, plays an essential role in bacterial NER by unwinding the duplex DNA in the 3' to 5' direction to displace the lesion-containing strand. In order to achieve conditional control over NER, we generated a light-activated DNA helicase. This was achieved through a site-specific incorporation of a genetically encoded hydroxycoumarin lysine at a crucial position in the ATP-binding pocket of UvrD. The resulting caged enzyme was completely inactive in several functional assays. Moreover, enzymatic activity of the optically triggered UvrD was comparable to that of the wild-type protein, thus demonstrating excellent OFF to ON switching of the helicase. The developed approach provides optical control of NER, thereby laying a foundation for the regulation of ATP-dependent helicase functions in higher organisms. In addition, this methodology is applicable to the light-activation of a wide range of ATPases.

Role of Mediator and Effects of Temperature on ortho-C-N Bond Fusion Reactions of Aniline Using Ruthenium Templates: Isolation and Characterization of New Ruthenium Complexes of the in-Situ-Generated Ligands.

In this work, ortho-C-N bond fusion reactions of aniline are followed by the use of two different ruthenium mediators. Reaction of aniline with [RuIII(terpy)Cl3] (terpy = 2,2':6',2″-terpyridine) resulted in a trans bis-aniline ruthenium(II) complex [1]+ which upon oxidation with H2O2 produced compound [2]+ of a bidentate ligand, N-phenyl-1,2-benzoquinonediimine, due to an oxidative ortho-C-N bond fusion reaction. Complex [1]+ and aniline (neat) at 185 °C produced a bis-chelated ruthenium complex (3). A previously reported complex [RuII(N-phenyl-1,2-benzoquinonediimine)(aniline)2(Cl)2] (5) undergoes similar oxidation by air at 185 °C to produce complex [3]. A separate chemical reaction between aniline and strongly oxidizing tetra-n-propylammonium perruthenate [(n-pr)4N]+[RuO4]- in air produced a ruthenium complex [4] of a N4-tetraamidophenylmacrocycle ligand via multiple ortho-C-N bond fusion reaction. Notably, the yield of this product is low (5%) at 100 °C but increases to 25% in refluxing aniline. All these complexes are characterized fully by their physicochemical characterizations and X-ray structure determination. From their structural parameters and other spectroscopic studies, complex [2]+ is assigned as [RuII(terpy)(N-phenyl-1,2-benzoquinonediimine)(Cl)]+ whereas complex [4] is described as a ruthenium(VI) complex comprised of a reduced deprotonated N-phenyl-1,2-diamidobenzene and N4-tetraamidophenylmacrocyclic ligand. Complex [2]+ exhibits one reversible oxidation at 1.32 V and one reversible reduction at -0.75 V vs Ag/AgCl reference electrode. EPR of the electrogenerated complexes has revealed that the oxidized complex is a ruthenium(III) complex with an axial EPR spectrum at gav= 2.06. The reduced complex [2], on the other hand, shows a single-line EPR signal at gav= 1.998. In contrast, complex [4] shows two successive one-electron oxidation waves at 0.5 and 0.8 V and an irreversible reduction wave at -0.9 V. EPR studies of the oxidized complexes [4]+ and [4]2+ reveal that oxidations are ligand centered. DFT calculations were employed to elucidate the electronic structures as well as the redox processes associated with the above complexes. Aerial ortho-C-N bond fusion reactions of aniline using two different mediators, viz. [RuIII(terpy)Cl3] and [(n-pr)4N]+[RuO4]-, have been followed. It is found that in the case of oxidizable Ru(III) mediator complex, C-N bond fusion is limited only to dimerization reaction whereas the high-valent Ru(VII) salt mediates multiple C-N bond fusion reactions leading to the formation of a novel tetradentate N4-tetraamidophenylmacrocyclic ligand. Valence ambiguity in the complexes of the resultant redox-active ligands is scrutinized.

Red-Shifting Azobenzene Photoswitches for in Vivo Use.

Recently, there has been a great deal of interest in using the photoisomerization of azobenzene compounds to control specific biological targets in vivo. These azo compounds can be used as research tools or, in principle, could act as optically controlled drugs. Such "photopharmaceuticals" offer the prospect of targeted drug action and an unprecedented degree of temporal control. A key feature of azo compounds designed to photoswitch in vivo is the wavelength of light required to cause the photoisomerization. To pass through tissue such as the human hand, wavelengths in the red, far-red, or ideally near infrared region are required. This Account describes our attempts to produce such azo compounds. Introducing electron-donating or push/pull substituents at the para positions delocalizes the azobenzene chromophore and leads to long wavelength absorption but usually also lowers the thermal barrier to interconversion of the isomers. Fast thermal relaxation means it is difficult to produce a large steady state fraction of the cis isomer. Thus, specifically activating or inhibiting a biological process with the cis isomer would require an impractically bright light source. We have found that introducing substituents at all four ortho positions leads to azo compounds with a number of unusual properties that are useful for in vivo photoswitching. When the para substituents are amide groups, these tetra-ortho substituted azo compounds show unusually slow thermal relaxation rates and enhanced separation of n-π* transitions of cis and trans isomers compared to analogues without ortho substituents. When para positions are substituted with amino groups, ortho methoxy groups greatly stabilize the azonium form of the compounds, in which the azo group is protonated. Azonium ions absorb strongly in the red region of the spectrum and can reach into the near-IR. These azonium ions can exhibit robust cis-trans isomerization in aqueous solutions at neutral pH. By varying the nature of ortho substituents, together with the number and nature of meta and para substituents, long wavelength switching, stability to photobleaching, stability to hydrolysis, and stability to reduction by thiols can all be crafted into a photoswitch. Some of these newly developed photoswitches can be used in whole blood and show promise for effective use in vivo. It is hoped they can be combined with appropriate bioactive targets to realize the potential of photopharmacology.

Conditional control of alternative splicing through light-triggered splice-switching oligonucleotides.

The spliceosome machinery is composed of several proteins and multiple small RNA molecules that are involved in gene regulation through the removal of introns from pre-mRNAs in order to assemble exon-based mRNA containing protein-coding sequences. Splice-switching oligonucleotides (SSOs) are genetic control elements that can be used to specifically control the expression of genes through correction of aberrant splicing pathways. A current limitation with SSO methodologies is the inability to achieve conditional control of their function paired with high spatial and temporal resolution. We addressed this limitation through site-specific installation of light-removable nucleobase-caging groups as well as photocleavable backbone linkers into synthetic SSOs. This enables optochemical OFF → ON and ON → OFF switching of their activity and thus precise control of alternative splicing. The use of light as a regulatory element allows for tight spatial and temporal control of splice switching in mammalian cells and animals.

Regioselective ortho Amination of Coordinated 2-(Arylazo)pyridine. Isolation of Monoradical Palladium Complexes of a New Series of Azo-Aromatic Pincer Ligands.

In an unusual reaction of [Pd(L(1))Cl2] (L(1) = 2-(arylazo)pyridine) with amines, a new series of palladium complexes [Pd(L(2•-))Cl] (L(2) = 2-((2-amino)arylazo)pyridine) (1a-1h) were isolated. The complexes were formed via N-H and N-C bond cleavage reactions of 1°/2° and 3° amines, respectively, followed by regioselective aromatic ortho-C-N bond formation reaction and are associated with ortho-C-H/ortho-C-Cl bond activation. A large variety of amines including both aromatic and aliphatic were found to be effective in producing air-stable complexes. Identity of the resultant complexes was confirmed by their X-ray structure determination. Efforts were also made to understand the mechanism of the reaction. A series of experiments were performed, which point toward initial ligand reduction followed by intraligand electron transfer. Examination of the structural parameters of these complexes (1) indicates that the in situ generated ligand coordinated to the Pd(II) center serves as the backbone of these air-stable monoradical complexes. Molecular and electronic structures of the isolated complexes were further scrutinized by various spectroscopic techniques including cyclic voltammetry, variable temperature magnetic susceptibility measurements, electron paramagnetic resonance, and UV-vis spectroscopy. Finally the electronic structure was confirmed by density functional theory calculations. The isolated monoradical complexes adopt an unusual π-stacked array, which leads to a relatively strong antiferromagnetic interaction (J = -40 cm(-1) for the representative complex 1c).

A two-in-one pincer ligand and its diiron(II) complex showing spin state switching in solution through reversible ligand exchange.

A novel pyrazolate-bridged ligand providing two {PNN} pincer-type compartments has been synthesized. Its diiron(II) complex LFe2(OTf)3(CH3CN) (1; Tf = triflate) features, in solid state, two bridging triflate ligands, with a terminal triflate and a MeCN ligand completing the octahedral coordination spheres of the two high-spin metal ions. In MeCN solution, 1 is shown to undergo a sequential, reversible, and complete spin transition to the low-spin state upon cooling. Detailed UV/Vis and (19)F NMR spectroscopic studies as well as magnetic measurements have unraveled that spin state switching correlates with a rapid multistep triflate/MeCN ligand exchange equilibrium. The spin transition temperature can be continuously tuned by varying the triflate concentration in solution.

Genetic encoding of caged cysteine and caged homocysteine in bacterial and mammalian cells.

We report the genetic incorporation of caged cysteine and caged homocysteine into proteins in bacterial and mammalian cells. The genetic code of these cells was expanded with an engineered pyrrolysine tRNA/tRNA synthetase pair that accepts both light-activatable amino acids as substrates. Incorporation was validated by reporter assays, western blots, and mass spectrometry, and differences in incorporation efficiency were explained by molecular modeling of synthetase-amino acid interactions. As a proof-of-principle application, the genetic replacement of an active-site cysteine residue with a caged cysteine residue in Renilla luciferase led to a complete loss of enzyme activity; however, upon brief exposure to UV light, a >150-fold increase in enzymatic activity was observed, thus showcasing the applicability of the caged cysteine in live human cells. A simultaneously conducted genetic replacement with homocysteine yielded an enzyme with greatly reduced activity, thereby demonstrating the precise probing of a protein active site. These discoveries provide a new tool for the optochemical control of protein function in mammalian cells and expand the set of genetically encoded unnatural amino acids.