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Aromatically π-extended porphyrins possess exceptionally intense one-photon (1P) and sometimes two-photon (2P) absorption bands, presenting interest for construction of optical imaging probes and photodynamic agents. Here we investigated how breaking the molecular symmetry affects linear and 2PA properties of π-extended porphyrins. First, we developed the synthesis of porphyrins fused with two phthalimide fragments, termed syn-diarylphthalimidoporphyrins (DAPIP). Secondly, the photophysical properties of H(2), Zn, Pd and Pt DAPIP were measured and compared to those of fully-symmetric tetraarylphthalimidoporphyrins (TAPIP). The data were interpreted using DFT/TDDFT calculations and Sum-Over-States (SOS) formalism. Overall, the picture of 2PA in DAPIP was found to resemble that in centrosymmetric porphyrins, indicating that symmetry breaking, even as significant as by syn-phthalimido-fusion, induces a relatively small perturbation to the porphyrin electronic structure. Collectively, the compact size, versatile synthesis, high 1PA and 2PA cross-sections and bright luminescence makes DAPIP valuable chromophores for construction of imaging probes and other bio-applications.

Glycine betaine (GB) is a naturally occurring osmolyte that has been widely recognized as a protein protectant. Since GB consists of a methylated ammonium moiety, it can engage in strong cation-π interactions with aromatic amino acid sidechains. We hypothesize that such specific binding interactions would allow GB to decrease the stability of proteins that are predominantly stabilized by a cluster of aromatic amino acids. To test this hypothesis, we investigate the effect of GB on the stability of two β-hairpins (or mini-proteins) that contain such a cluster. We find that for both systems the stability of the folded state first decreases and then increases with increasing GB concentration. Such non-monotonic dependence not only confirms that GB can act as a protein denaturant, but also underscores the complex interplay between GB's stabilizing and destabilizing forces toward a given protein. While stabilizing osmolytes all have the tendency to be excluded from the protein surface which is the action underlying their stabilizing effect, our results suggest that in order to quantitatively assess the effect of GB on the stability of any given protein, specific cation-π binding interactions need to be explicitly considered. Moreover, our results show, consistent with other studies, that cation methylation can strengthen the respective cation-π interactions. Taken together, these findings provide new insight into the mechanism by which amino acid-based osmolytes interact with proteins.

Considerable efforts have been devoted to the development of spectroscopic probes that are sensitive to water and can be used to monitor, for example, biological and chemical processes involving dehydration or hydration. Continuing this line of research, herein we show that 7-cyanoindole can serve as a sensitive fluorescence probe of hydration as its fluorescence properties, including intensity, peak wavelength and lifetime, depend on the amount of water in nine water-organic solvent mixtures. Our results indicate that 7-cyanoindole is not only able to reveal the underlying microheterogeneity of these binary solvent systems, but also offers distinct advantages. These include: (1) its fluorescence intensity increases more than ten times upon going from a hydrated to a dehydrated environment; (2) its peak wavelength shifts as much as 35 nm upon dehydration; (3) its single-exponential fluorescence decay lifetime increases from 2.0 ns in water to 8-16 ns in water-organic binary mixtures, making it viable to distinguish between differently hydrated environments via fluorescence lifetime measurements; and (4) its absorption spectrum is significantly red-shifted from that of indole, making selective excitation of its fluorescence possible in the presence of naturally occurring amino-acid fluorophores. Moreover, we find that for seven binary mixtures the fluorescence lifetimes of 7-cyanoindole measured at solvent compositions where maximum microheterogeneity occurs correlate linearly with the peak wavenumbers of its fluorescence spectra obtained in the respective pure organic solvents. This suggests that the microheterogeneities of these binary mixtures bear certain similarity, a phenomenon that warrants further investigation.

Several cyanotryptophans have been shown to be useful biological fluorophores. However, how their fluorescence lifetimes vary with solvent has not been examined. In this regard, herein we measure the fluorescence decay kinetics as well as the absorption and emission spectra of six cyanoindoles in different solvents. In particular, we find, among other results, that only 4-cyanoindole affords a long fluorescence lifetime and hence high quantum yield in H2O. Therefore, our measurements provide not only a guide for choosing which cyanotryptophan to use in practice but also data for computational modeling of the substitution effect on the electronic transitions of indole.

Methods based on fluorescence resonance energy transfer (FRET) and photo-induced electron transfer (PET) are widely used in the biological sciences, employing mostly dye-based FRET and PET pairs. While very useful and important, dye-based reporters are not always applicable without concern, for example, in cases where the fluorophore size needs to be minimized. Therefore, development and characterization of smaller, ideally amino acid-based PET and FRET pairs will expand the biological spectroscopy toolbox to enable new applications. Herein, we show that, depending on the excitation wavelength, tryptophan and 4-cyanotrptophan can interact with each other via the mechanism of either energy or electron transfer, hence constituting a dual FRET and PET pair. The biological utility of this amino acid pair is further demonstrated by applying it to study the end-to-end collision rate of a short peptide, the mode of interaction between a ligand and BSA, and the activity of a protease.

We will discuss a family of recently developed tetraarylphalimidoporphyrins (TAPIP) and diarylphalimidoporphyrins (DAPIP), which possess unique photophysical properties and suggest new approaches to the design of superior biological sensors for pH and temperature in addition to their already established ability as sensors for oxygen. The synthesis of TAPIP has already been developed by our group previously. Here we present a synthetic route to asymmetric diarylphthalimidoporphyrins (DAPIP) based on [2+2] condensation of the corresponding dipyrromethanes, followed by oxidative aromatization. We report the properties of free-base (H2), Zn(II), Pt(II) and Pd(II) DAPIP's, including their two-photon absorption (2PA) spectra. Using a combination of phosphorescent Pt complexes of TAPIP and DAPIP we are designing a phosphorescent sensor for pH and oxygen, termed pHOx, which operates by measuring ratios of phosphorescence lifetimes. Unlike all existing optical pH sensors, measurements by pHOx are not affected by optical heterogeneities of the medium and provide unbiased pH readings in vivo simultaneously with pO2. At the same time, Pd complexes of TAPIP have been shown to emit both phosphorescence and thermally activated E-type delayed fluorescence, providing a functional element for optical sensing of temperature. However, to make the temperature readings unobstructed by optical heterogeneities of biological tissue, a PdTAPIP-based probe is supplemented by another probe system, based on H2DAPIP, which emits prompt fluorescence at the same wavelengths and can serve as a correction standard for unbiased temperature measurements.

Although N-acetylaspartate (NAA) has long been recognized as the most abundant amino acid in neurons by far, its primary role has remained a mystery. Based on its unique tertiary structure, we explored the potential of NAA to modulate aggregation of amyloid-beta (Aβ) peptide 1–42 via multiple corroborating aggregation assays along with electron microscopy. Thioflavin-T fluorescence assay demonstrated that at physiological concentrations, NAA substantially inhibited the initiation of Aβ fibril formation. In addition, NAA added after 25 minutes of Aβ aggregation was shown to break up preformed fibrils. Electron microscopy analysis confirmed the absence of mature fibrils following NAA treatment. Furthermore, fluorescence correlation spectroscopy and dynamic light scattering measurements confirmed significant reductions in Aβ fibril hydrodynamic radius following treatment with NAA. These results suggest that physiological levels of NAA could play an important role in controlling Aβ aggregation in vivo where they are both found in the same neuronal compartments.

Tryptophan (Trp) fluorescence has been widely used to interrogate the structure, dynamics, and function of proteins. In particular, it provides a convenient and site-specific means to probe a protein's hydration status and dynamics. Herein, we show that a tryptophan analogue, 5-cyanotryptophan (TrpCN), can also be used for this purpose, but with the benefit of enhanced sensitivity to hydration. This conclusion is reached based on measurements of the static and time-resolved fluorescence properties of 5-cyanoindole, TrpCN, and TrpCN-containing peptides in different solvents, which indicate that upon dehydration the fluorescence quantum yield (QY) and lifetime (τF) of TrpCN undergo a much greater change in comparison to those of Trp. For example, in H2O the QY of TrpCN is less than 0.01, which increases to 0.11 in 1,4-dioxane. Consistently, the fluorescence decay kinetics of TrpCN in H2O are dominated by a 0.4 ns component, whereas in 1,4-dioxane the kinetics are dominated by a 6.0 ns component. The versatile utility of TrpCN as a sensitive fluorescence reporter is further demonstrated in three applications, where we used it (1) to probe the solvent property of a binary mixture consisting of dimethyl sulfoxide and H2O, (2) to monitor the binding interaction of an antimicrobial peptide with lipid membranes, and (3) to differentiate two differently hydrated environments in a folded protein.

The C≡N stretching frequency and fluorescence quantum yield of p-cyanophenylalanine are sensitive to environment. As such, this unnatural amino acid has found broad applications, ranging from studying how proteins fold to determining the local electric field of membranes. Herein, we demonstrate that the fluorescence of p-cyanophenylalanine can be quenched by selenomethionine through an electron transfer process occurring at short distances, thus further expanding its spectroscopic utility. Using this fluorophore-quencher pair, we are able to show that short polyproline peptides (1–4 prolines) are not rigid; instead, they sample a bimodal conformational distribution.

The M2 proton channel of the influenza A virus has been the subject of extensive studies because of its critical role in viral replication. As such, we now know a great deal about its mechanism of action, especially how it selects and conducts protons in an asymmetric fashion. The conductance of this channel is tuned to conduct protons at a relatively low biologically useful rate, which allows acidification of the viral interior of a virus entrapped within an endosome, but not so great as to cause toxicity to the infected host cell prior to packaging of the virus. The dynamic, structural and chemical features that give rise to this tuning are not fully understood. Herein, we use a tryptophan (Trp) analog, 5-cyanotryptophan, and various methods, including linear and nonlinear infrared spectroscopies, static and time-resolved fluorescence techniques, and molecular dynamics simulations, to site-specifically interrogate the structure and hydration dynamics of the Trp41 gate in the transmembrane domain of the M2 proton channel. Our results suggest that the Trp41 sidechain adopts the t90 rotamer, the χ2 dihedral angle of which undergoes an increase of approximately 35° upon changing the pH from 7.4 to 5.0. Furthermore, we find that Trp41 is situated in an environment lacking bulk-like water, and somewhat surprisingly, the water density and dynamics do not show a measurable difference between the high (7.4) and low (5.0) pH states. Since previous studies have shown that upon channel opening water flows into the cavity above the histidine tetrad (His37), the present finding thus provides evidence indicating that the lack of sufficient water molecules near Trp41 needed to establish a continuous hydrogen bonding network poses an additional energetic bottleneck for proton conduction.

Cancer Osaka thyroid (COT) kinase is an important regulator of pro-inflammatory cytokines in macrophages. Thus, pharmacologic inhibition of COT should be a valid approach to therapeutically intervene in the pathogenesis of macrophage-driven inflammatory diseases such as rheumatoid arthritis. We report the discovery and chemical optimization of a novel series of COT kinase inhibitors, with unprecedented nanomolar potency for the inhibition of TNFα. Pharmacological profiling in vivo revealed a high metabolism of these compounds in rats which was demonstrated to be predominantly attributed to aldehyde oxidase. Due to the very low activity of hepatic AO in the dog, the selected candidate 32 displayed significant blood exposure in dogs which resulted in a clear prevention of inflammation-driven lameness. Taken together, the described compounds both potently and selectively inhibit COT kinase in primary human cells and ameliorate inflammatory pathologies in vivo, supporting the notion that COT is an appropriate therapeutic target for inflammatory diseases.

The non-natural amino acid p-cyanophenylalanine (PheCN) has recently emerged as a useful fluorescent probe of proteins; however, its photophysical properties have not been systematically examined. Herein, we measure the fluorescence quantum yield and the fluorescence lifetime of PheCN in a series of solvents. It is found that the fluorescence lifetime of PheCN shows a linear dependence on the Kamlet–Taft parameter α of the protic solvents used, indicating that the solute-solvent hydrogen bonding interactions mediate the non-radiative decay rate. Thus, results of this study provide a basis for quantitative application of PheCN fluorescence in protein conformational studies.

High throughput screening led to the identification of nicotinamide derivative 2 as a structurally novel mGluR5 antagonist. Optimization of the modular scaffold led to the discovery of 16m, a compound with high affinity for mGluR5 and excellent selectivity over other glutamate receptors. Compound 16m exhibits a favorable PK profile in rats, robust anxiolytic-like effects in three different animal models of fear and anxiety, as well as a good PK/PD correlation.

Shape-memory materials (including polymers, metals, and ceramics) are those that are processed into a temporary shape and respond to some external stimuli (e.g., temperature) to undergo a transition back to a permanent shape.[1, 2] Shape memory polymers are finding use in a range of applications from aerospace to fabrics, to biomedical devices and microsystem components.[3–5] For many applications, it would be beneficial to initiate heating with an external trigger (e.g., transdermal light exposure). In this work, we formulated composites of gold nanorods (

Most antianxiety drugs (anxiolytics) work by modulating neurotransmitters in the brain. Benzodiazepines are fast and effective anxiolytic drugs; however, their long-term use is limited by the development of tolerance and withdrawal symptoms. Ligands of the translocator protein [18 kilodaltons (kD)] may promote the synthesis of endogenous neurosteroids, which also exert anxiolytic effects in animal models. Here, we found that the translocator protein (18 kD) ligand XBD173 enhanced gamma-aminobutyric acid-mediated neurotransmission and counteracted induced panic attacks in rodents in the absence of sedation and tolerance development. XBD173 also exerted antipanic activity in humans and, in contrast to benzodiazepines, did not cause sedation or withdrawal symptoms. Thus, translocator protein (18 kD) ligands are promising candidates for fast-acting anxiolytic drugs with less severe side effects than benzodiazepines.

High-resolution images of oxygen distributions in microheterogeneous samples are obtained by two-photon laser scanning microscopy (2P LSM), using a newly developed dendritic nanoprobe with internally enhanced two-photon absorption (2PA) cross-section. In this probe, energy is harvested by a 2PA antenna, which passes excitation onto a phosphorescent metalloporphyrin via intramolecular energy transfer. The 2P LSM allows sectioning of oxygen gradients with near diffraction-limited resolution, and lifetime-based acquisition eliminates dependence on the local probe concentration. The technique is validated on objects with a priori known oxygen distributions and applied to imaging of pO(2) in cells.

The SAR of over 50 derivatives of octahydrobenzo[g]quinoline (obeline)-type somatostatin sst1 receptor antagonist 1 is presented, focusing on the modification of its arylpiperazine moiety. Sst1 affinities in this series cover a range of five orders of magnitude with the best derivatives displaying subnanomolar sst1 affinities and >10,000-fold selectivities over the sst2 receptor subtype as well as promising pharmacokinetic properties.

A novel class of non-peptide somatostatin receptor ligands bearing the octahydrobenzo[g]quinoline (obeline) structural element has been identified. SAR studies have been performed that led to the discovery of derivatives with high affinity (pK(d) r sst(1)or = 9) and selectivity (or = 150-fold for h sst(1) over h sst(2)-h sst(5)) for somatostatin receptor subtype sst(1). In a functional assay, the compounds act as antagonists at human recombinant sst(1) receptors.

The surface properties of carbon black microparticles in aqueous solution are characterized through adsorption of a hydrophobic ion (malachite green) monitored by optical second-harmonic generation (SHG). Carbon black particle surfaces with 14 different O/C ratios (from 0.028 to 0.270) have been examined. The adsorption isotherm of MG on the surface of carbon black particles weighing as little as 0.010 g can be measured using SHG measured during continuous titration of the MG molecule into the solution. The SHG data allows determination of the adsorption free energy and reveals coherent interference between the SH fields from the surface-adsorbed MG molecules and the carbon black particle. The change in the coherent interference implies a change of adsorption orientation of MG on surfaces with different O/C ratios. There is strong correlation found between the observed adsorption characteristics and the O/C ratio of the surfaces. Furthermore, MG adsorption isotherms of surfaces with higher O/C ratios appear similar to the ones of surfaces preadsorbed with surfactants that can increase surface hydrophilicity and dispersion in aqueous solution. All observations indicate that particle surfaces with higher O/C ratios appear more hydrophilic. This study demonstrates once again the many advantages of SHG as an effective surface analytical technique of colloidal particles.

Fluorescence imaging of cells and tissue can be used to evaluate beta-NADH redox and location. At low temperature, beta-NADH fluorescence intensity increases and therefore sensitivity of imaging increases. In this paper, the temperature dependence of fluorescence was evaluated for beta-NADH in glycerol/water solution and in trehalose/sucrose glass. The average fluorescence lifetime for NADH in glycerol/water is 0.66 ns, compared with 5.3 ns in trehalose/ sucrose at 20 degrees C. Emission spectra were recorded from 290 to 12 K. The fluorescence of beta-NADH in glycerol/water increases approximately 16 fold and the emission shifts about 35 nm to the blue as temperature decreases. Much smaller change is seen for fluorescence of beta-NADH in sugar glass. Below 77 K, the beta-NADH spectral features did not change significantly with temperature change, and so no increase in sensitivity is obtained by going to very low temperatures. It is suggested that the sensitivity of beta-NADH fluorescence is related to water relaxation around the excited state molecule. Differences in water in various tissues may contribute to beta-NADH fluorescence changes when cells are altered.

Imaging oxygen in 3D with submicron spatial resolution can be made possible by combining phosphorescence quenching technique with multiphoton laser scanning microscopy. Because Pt and Pd porphyrin-based phosphorescent dyes, traditionally used as phosphors in biological oxygen measurements, exhibit extremely low two-photon absorption (2PA) cross-sections, we designed a nanosensor for oxygen, in which a 2P absorbing antenna is coupled to a metalloporphyrin core via intramolecular energy transfer (ET) with the purpose of amplifying the 2PA induced phosphorescence of the metalloporphyrin. The central component of the device is a polyfunctionalized Pt porphyrin, whose triplet state emission at ambient temperatures is strong, occurs in the near infrared and is sensitive to O2. The 2PA chromophores are chosen in such a way that their absorption is maximal in the near infrared (NIR) window of tissue (e.g., 700−900 nm), while their fluorescence is overlapped with the absorption band(s) of the core metalloporphyrin, ensuring an efficient antenna-core resonance ET. The metalloporphyrin-antenna construct is embedded inside the protecting dendritic jacket, which isolates the core from interactions with biological macromolecules, controls diffusion of oxygen and makes the entire sensor water-soluble. Several Pt porphyrin-coumarin based sensors were synthesized and their photophyics studied to evaluate the proposed design.

The conversion of prothrombin to thrombin is catalyzed by prothrombinase, an enzyme complex composed of the serine proteinase factor Xa and a cofactor protein, factor Va, assembled on membranes. Kinetic studies indicate that interactions with extended macromolecular recognition sites (exosites) rather than the active site of prothrombinase are the principal determinants of binding affinity for substrate or product. We now provide a model-independent evaluation of such ideas by physical studies of the interaction of substrate derivatives and product with prothrombinase. The enzyme complex was assembled using Xa modified with a fluorescent peptidyl chloromethyl ketone to irreversibly occlude the active site. Binding was inferred by prethrombin 2-dependent perturbations in the fluorescence of Oregon Green488 at the active site of prothrombinase. Active site-independent binding was also unequivocally established by fluorescence resonance energy transfer between 2,6-dansyl tethered to the active site of Xa and eosin tethered to the active sites of either thrombin or meizothrombin des fragment 1. Comparable interprobe distances obtained from these measurements suggest that substrate and product interact equivalently with the enzyme. Competition established the ability of a range of substrate or product derivatives to bind in a mutually exclusive fashion to prothrombinase. Equilibrium dissociation constants obtained for the active site-independent binding of prothrombin, prethrombin 2, meizothrombin des fragment 1 and thrombin to prothrombinase were comparable with their affinities inferred from kinetic studies using active enzyme. Our findings directly establish that binding affinity is principally determined by the exosite-mediated interaction of either the substrate, both possible intermediates, or product with prothrombinase. A single type of exosite binding interaction evidently drives affinity and binding specificity through the stepwise reactions necessary for the two cleavage reactions of prothrombin activation and product release.

Cylcodextrin sugars are cyclic sugars that have a hydrophilic exterior and a hydrophobic center. This enables cyclodextrins to solubilize hydrophobic molecules in aqueous media. Cyclodextrins may inhibit aggregation by intercalating surface aromatic residues and competing with interprotein aromatic clusters (pi-pi interactions). In order to investigate this concept, the interaction of hydroxypropyl-beta-cyclodextrin (HPBCD) with melittin is studied with steady-state and time-resolved fluorescence, fluorescence polarization, circular dichroism, and IR spectroscopy. HPBCD inhibits the aggregation of melittin. This inhibition and the spectroscopic results are consistent with the lone aromatic tryptophan of the peptide being intercalated within HPBCD.

The decahydroisoquinoline derivative NVP-ACQ090 is a potent and selective antagonist at the somatostatin sst3 receptor. The original research synthesis of NVP-ACQ090 comprises a main chain of nine linear steps and two side chains of three and steps, respectively. This synthesis is highly convergent, but very complex and expensive, and involves several reagents that are not acceptable for a large scale synthesis. In chemical development, all the unacceptables could be replaced, and the overall efficiency of the synthesis was much improved.

Somatostatin (SRIF, somatotropin release inhibiting factor), discovered for its inhibitory action on growth hormone (GH) secretion from pituitary, is an abundant neuropeptide. Two forms, SRIF14 and SRIF28 exist. Recently, a second family of peptides with very similar sequences and features was described; the cortistatins (CST), CST17 and CST29 which are brain selective. The five cloned SRIF receptors (sst1-5) belong to the G-protein coupled/ heptathelical receptor family. Structural and operational features distinguish two classes of receptors; SRIF1 - sst2/sst3/sst5 (high affinity for octreotide or seglitide) and SRIF2 = sst1/sst4(very low affinitty for the aforementioned ligands). The affinity of SRIF receptors for somatostatins and cortistatins is equally high, and it is not clear whether selective receptors do exist for one or the other of the peptides. Several radiologlands label all SRIF receptors, e.g., [125]LTT-SRIF28' [l25I]CGP23996, [125]Tyr10cortistatin or [125I]Tyr11SRIF14. In contrast, [125I]Tyr3octreotide, [125I]BIM23027, [125I]MK678 or [125I]D-Trp8SRIF14 label predominantly SRIF1 sites, especially sst2 and possibly sst5 receptors. In brain, [125I]Tyr3octreotide binding equates with sst2 receptor mRNA distribution. Native SRIF2receptors can be labeled with [125I]SRIF14 in the presence of high NaCl in brain (sst1) or lung (sst4) tissue. Short cyclic or linear peptide analogs show selectivity for sst2/sst5 (octreotide, lanreotide, BIM 23027), sst1 (CH-275), sst3 (sst3-ODN-8), or sst5 receptors (BIM 23268); although claims for selectivity have not always been confirmed. Beta peptides ith affinity for SRIF receptors are also reported. The general lack of SRIF receptor antagonists is unique for peptide receptors, although CYN 154806 is a selective and potent sst2 antagonist. Nonpeptide ligands are still rare, although a number of molecules have been reported with selectivity and potency for sst1 (L 757,519), sst2 (L 779,976), sst3 (L 796,778), sst4 (NNC 26-9100, L 803,087) or sst1/sst5 receptors (L 817,018). Such molecules are essential to establish the role of SRIF receptors, e.g., sst1 in hypothalamic glutamate currents: sst2 in inhibiting release of GH, glucagon, TSH, gastric acid secretion, pain, seizures and tumor growth, and sst5 in vascular remodeling and inhibition of insulin and GH release.

Ergolines were recently identified as a novel class of H3 receptor (H3R) inverse agonists. Although their optimization led to drug candidates with encouraging properties for the treatment of narcolepsy, brain penetration remained low. To overcome this issue, ergoline 1 ((6aR,9R,10aR)-4-(2-(dimethylamino)ethyl)-N-phenyl-9-(pyrrolidine-1-carbonyl)-6,6a,8,9,10,10a-hexahydroindolo[4,3-fg]quinoline-7(4H)-carboxamide)) was transformed into a series of indole derivatives with high H3R affinity. These new molecules were profiled by simultaneous determination of their brain receptor occupancy (RO) levels and pharmacodynamic (PD) effects in mice. These efforts culminated in the discovery of 15 m ((R)-1-isopropyl-5-(1-(2-(2-methylpyrrolidin-1-yl)ethyl)-1H-indol-4-yl)pyridin-2(1H)-one), which has an ideal profile showing a strong correlation of PD effects with RO, and no measurable safety liabilities. Its desirably short duration of action was confirmed by electroencephalography (EEG) measurements in rats.

Ergoline derivative (6aR,9R)-4-(2-(dimethylamino)ethyl)-N-phenyl-9-(pyrrolidine-1-carbonyl)-6,6a,8,9-tetrahydroindolo[4,3-fg]quinoline-7(4H)-carboxamide (1), a CXCR3 antagonist, also inhibits human histamine H3 receptors (H3R) and represents a structurally novel H3R inverse agonist chemotype. It displays favorable pharmacokinetic and in vitro safety profiles, and served as a lead compound in a program to explore ergoline derivatives as potential drug candidates for the treatment of narcolepsy. A key objective of this work was to enhance the safety and efficacy profiles of 1, while minimizing its duration of action to mitigate the episodes of insomnia documented with previously reported clinical candidates during the night following administration. Modifications to the ergoline core at positions 1, 6 and 8 were systematically investigated, and derivative 23 (1-((4aR,8R,9aR)-8-(hydroxymethyl)-1-(2-((R)-2-methylpyrrolidin-1-yl)ethyl)-4,4a,7,8,9,9a-hexahydroindolo[1,14-fg]quinolin-6(1H)-yl)ethanone) was identified as a promising lead compound. Derivative 23 has a desirable pharmacokinetic profile and demonstrated efficacy by enhancing brain concentrations of tele-methylhistamine, a major histamine metabolite. This validates the potential of the ergoline scaffold to serve as a template for the development of H3R inverse agonists.

The spectroscopy and excited-state lifetimes of 3- and 4-aminophthalimides (3AP, 4AP), N-methyl-4AP (NM4AP) and some complexes with polar molecules have been explored under supersonic jet conditions. Spectroscopic data, MM2-level structure calculations and rotational coherence spectroscopy show that a single water molecule attaches to 3AP by accepting a hydrogen bond from the imide > NH group. Whereas 3AP/H2O is present as a single species, the case of 4AP is complicated by the presence of two isomers, having significantly different fluorescence decay times and radiative lifetimes. This observation, which is repeated for methanol complexation, is evidence for the sensitivity of the electronic structure of 4AP to a site-specific interaction.

Fluorescence excitation spectra of SO 2 Ar generated in a supersonic expansion near the SO 2 C → X , 0 0 0 , 2 0 1 , and 3 0 2 bands have been obtained. Three features red-shifted by 82, 60 and 41 cm −1 from monomer bands are assigned to the SO 2 Ar complex. The rotational constants of SO 2 (C)Ar are determined to be A = 0.2769 cm −1 , B = 0.0520 cm −1 and C = 0.0488 cm −1 . The corresponding complex structure is obtained. Rapid non-radiative decay induced by the Ar atom has been observed for the 0 0 0 level of the C state complex. Vibrational predissociation is identified for the (010) and (002) levels, 377 and 560 cm −1 above the band origin.

Mutations in leucine-rich repeat kinase-2 (LRRK2) are the most common genetic cause of Parkinson’s disease (PD). The most frequent kinase-enhancing mutation is the G2019S residing in the kinase activation domain. This opens up a promising therapeutic avenue for drug discovery targeting the kinase activity of LRRK2 in PD. Several LRRK2 inhibitors have been reported to date. Here, we report a selective, brain penetrant LRRK2 inhibitor and demonstrate by a competition pulldown assay in vivo target engagement in mice.

The anti-AIDS drug rilpivirine undergoes conformational changes to bind HIV-1 reverse transcriptase and retain potency against drug-resistance mutations. Our discovery that water molecules play an essential role in the drug binding is reported. Femtosecond experiments and theory expose molecular level dynamics of rilpivirine bound to HIV-1 reverse transcriptase. The two nitrile substituents (-CN), one on each arm of the drug, have vibrational spectra consistent with their protein environments being similar in crystals and in solutions. Two-dimensional vibrational-echo spectroscopy reveals a dry environment for one nitrile while unexpectedly the other is hydrogen-bonded to a mobile water molecule, not identified in earlier X-ray structures. Ultrafast nitrile-water dynamics are confirmed by simulations. A higher (1.51 Å) resolution X-ray structure indeed reveals a water-drug interaction network. Maintenance of a crucial anchoring hydrogen bond, despite the enlargement and structural variation of the binding pocket, may help retain the potency of rilpivirine against the pocket mutations.

Fluorescence excitation spectra of jet‐cooled van der Waals complexes of the planar aromatic hydrocarbon perylene with the n‐alkanes pentane, hexane, octane, and decane show in each case a single structural form. Rotational coherence transients observed for these species, spaced by 2.5–4.4 ns, are consistent with the n‐alkane chain oriented parallel to the long axis of perylene and placed 3.6 A above its surface. In contrast, the 1‐chloropentane and 1‐fluoropentane complexes of perylene both exhibit three conformational isomers (α,β,γ) in the electronic ground state. Rotational coherence experiments have measured the structures of these different species in the S1 state, via 000 excitation, proving the existence of three distinct isomers in each case. Dispersed emission spectra following vibronic excitation at 355 cm−1 (A10) indicate in each case that the γ structure relaxes to the α form. Knowledge of the structures of the different forms provides a basis to identify the photoisomerization trajectories. ...

Rotational coherence experiments using picosecond time-correlated single-photon counting spectroscopy have measured the polarized fluorescence time profiles of van der Waals aggregates of perylene with up to three cyclopropane molecules. Three structural isomers have been confirmed at the 2:1 level of aggregation, one of which contains a contacted cyclopropane dimer. The other two are symmetrically distinct isomers of a (1|1) species. The 3:1 aggregate also exhibits two resolvable structures. The structures of all complexes studied are shown to be generated by the off-center adsorption site of the cyclopropane molecule, evidently dictated by short-range repulsive forces.

Van der Waals complexes of perylene with 1-chlorobutane, prepared under supersonic jet conditions, exist in three distinct isomeric forms distinguishable by their spectral red-shifts of 265,238 and 184 cm −1 . Following vibronic excitation at 355 cm −1 , two of these isomers yield similarly shaped and broadened fluorescence bands of ≈ 180 cm −1 width, suggesting a rapid isomerization. A third isomer is not coupled, showing a distinct spectrum even for a vibrational energy of 700 cm −1 . Structure determinations of all three isomers are reported using rotational coherence spectroscopy. The existence is demonstrated of three remarkably different binding sites for 1-chlorobutane on the perylene surface.

Picosecond time‐resolved polarized fluorescence experiments involving time‐correlated single‐photon counting have studied rotational coherence phenomena of hydrogen‐bonded and other molecular aggregates in their lowest excited singlet states. The experiments are supported by detailed simulations. Using the molecule 2,5‐diphenyl‐1,3,4‐oxadiazole (PPD) as a host species, experiments have compared van der Waals aggregates with Ar1,2, (CH4)1,2, (H2O)1,2, (CH3OH)1,2, (C2H5OH)1, and (1‐C3H7OH)1. Bare PPD, and the argon and methane aggregates all exhibit prominent J‐type recurrences. The resulting sums of rotational constants (B+C) are consistent with center‐of‐mass‐bound, three‐dimensional structures, having out‐of‐plane distances for the attached species of 3.3–3.5 A. The 1:2 aggregates involving argon and methane exhibit additive spectral shifts and nearly additive rotational recurrence times. This shows that the sites for addition of consecutive species are equivalent.Calculations of rotational constants con...

Spectral hole-burning experiments have measured the electronic spectra of aggregates of different deuterium-substituted isotopomers of methane (≤4:1) with perylene. At ≃2-cm -1 resolution, detail in the spectrum reveals at least two isomers each at the 1:1 level for the CH 4 , CH 3 D, and CH 2 D 2 complexes. Splitting observed for the CH 4 species (≃2 cm -1 ) suggests the presence of distinct nuclear spin isomers. The 1,1 aggregate contains two separate methane species, the motion of which is limited only by the perylene molecule. The detailed spectrum of this species differs radically from that of the other isomer (2,0), which contains a contacted methane dimer

Molecular dynamics simulations were performed for van der Waals clusters naphthalene⋅Arn, n=1 to 4. For all isomers and conformers of these clusters, dynamical quantities such as velocity autocorrelation functions, vibrational power spectra, and semiclassical electronic absorption spectra were calculated over a wide energy range, and averaged over a canonical distribution at temperatures in the range T=5 to 30 K. Electronic absorption spectra were calculated for the origin bands according to the semiclassical method [L. E. Fried and S. Mukamel, J. Chem. Phys. 96, 116 (1992)] and are compared with the corresponding experimental naphthalene⋅Arn R2PI spectra [T. Troxler and S. Leutwyler, J. Chem. Phys. 95, 4010 (1991)]. The appearance of distinct absorption bands due to specific isomers for a given cluster size, as observed experimentally, is well reproduced by the simulations. Comparison of calculated electronic shifts for different isomers allows clear assignments in the experimental spectra. Increasing th...

Rotational coherence spectroscopy involving polarized, time-correlated single-photon counting techniques has been used to measure the excited-state structures of hydrogen-bonded complexes between PPD and water at the 1:1 and 1:2 levels of aggregation. Structure determinations involved comparison of measured and simulated RCS traces using the measured B and C rotational constants. PPD/(H2O)1 reveals a singly hydrogen-bonded, in-plane structure with an N…HO distance of 2.95±0.1 A. The best structural estimate for the 1:2 complex, PPD/(H2O)2, consists of a water dimer, hydrogen bonded to one nitrogen atom of PPD.

Picosecond time-correlated single-photon counting spectroscopy has been used in rotational coherence measurements to determine the excited-state moments of inertia of p-cyclohexylaniline. This work complements earlier experiments involving single-color time-resolved fluorescence depletion spectroscopy, allowing the separation and identification of the rotational constants in the S 0 and S 1 states. Simulations have shown that the persistence of the K-type recurrences over several dozen cycles is consistent with a difference of ≃2% between the excited-state B and C constants

High throughput screening led to the identification of nicotinamide derivative 2 as a structurally novel mGluR5 antagonist. Optimization of the modular scaffold led to the discovery of 16m, a compound with high affinity for mGluR5 and excellent selectivity over other glutamate receptors. Compound 16m exhibits a favorable PK profile in rats, robust anxiolytic-like effects in three different animal models of fear and anxiety, as well as a good PK/PD correlation.

Oxygen concentration distributions in biological systems can be imaged by the phosphorescence quenching method in combination with two-photon laser scanning microscopy. In this paper, we identified the excitation regime in which the signal of a two-photon-enhanced phosphorescent probe (Finikova, O. S.; Lebedev, A. Y.; Aprelev, A.; Troxler, T.; Gao, F.; Garnacho, C.; Muro, S.; Hochstrasser, R. M.; Vinogradov, S. A. ChemPhysChem 2008, 9, 1673-1679) is dependent quadratically on the excitation power (quadratic regime), and performed simulations that relate the photophysical properties of the probe to the imaging resolution. Further, we characterized polymersomes as a method of probe encapsulation and delivery. Photophysical and oxygen sensing properties of the probe were found unchanged when the probe is encapsulated in polymersomes. Polymersomes were found capable of sustaining high probe concentrations, thereby serving to improve the signal-to-noise ratios for oxygen detection compared to the previously employed probe delivery methods. Imaging of polymersomes loaded with the probe was used as a test-bed for a new method.

Starting from non-peptidic sst1-selective somatostatin receptor antagonists, first compounds with mixed sst1/sst3 affinity were identified by directed structural modifications. Systematic optimization of these initial leads afforded novel, enantiomerically pure, highly potent and sst3-subtype selective somatostatin antagonists based on a (4S,4aS,8aR)-decahydroisoquinoline-4-carboxylic acid core moiety. These compounds can efficiently be synthesized and show promising PK properties in rodents.

A water-soluble triacetic acid cryptophane-A derivative (TAAC) was synthesized and determined by isothermal titration calorimetry (ITC) and fluorescence quenching assay to have a xenon association constant of 33,000 M−1 at 293 K, which is the largest value measured for any host molecule to date. Fluorescence lifetime measurements of TAAC in the presence of varying amounts of xenon indicated static quenching by the encapsulated xenon and the presence of a second non-xenon-binding conformer in solution. Acid-base titrations and aqueous NMR spectroscopy of TAAC and a previously synthesized tri-(triazole propionic acid) cryptophane-A derivative (TTPC) showed how solvation of the carboxylate anions can affect the aqueous behavior of the large, nonpolar cryptophane. Specifically, whereas only the crown-crown (CC) conformer of TTPC was observed, a crown-saddle (CS) conformer of TAAC was also detected in aqueous solution.

Structural simplification of the core moieties of obeline and ergoline somatostatin sst(1) receptor antagonists, followed by systematic optimization, led to the identification of novel, highly potent and selective sst(1) receptor antagonists. These achiral, non-peptidic compounds are easily prepared and show promising PK properties in rodents.