Complexation of Green and Red Kaede Fluorescent Protein Chromophores by a Zwitterion to Probe Electrostatic and Induction Field Effects.

The photophysics of green fluorescent protein (GFP) and red Kaede fluorescent protein (rKFP) are defined by the intrinsic properties of the light-absorbing chromophore and its interaction with the protein binding pocket. This work deploys photodissociation action spectroscopy to probe the absorption profiles for a series of synthetic GFP and rKFP chromophores as the bare anions and as complexes with the betaine zwitterion, which is assumed as a model for dipole microsolvation. Electronic structure calculations and energy decomposition analysis using Symmetry-Adapted Perturbation Theory are used to characterize gas-phase structures and complex cohesion forces. The calculations reveal a preponderance for coordination of betaine to the phenoxide deprotonation site predominantly through electrostatic forces. Calculations using the STEOM-DLPNO-CCSD method are able to reproduce absolute and relative vertical excitation energies for the bare anions and anion-betaine complexes. On the other hand, treatment of the betaine molecule with a point-charge model, in which the charges are computed from some common electron density population analysis schemes, show that just electrostatic and point-charge induction interactions are unable to account for the betaine-induced spectral shift. The present methodology could be applied to investigate cluster forces and optical properties in other gas-phase ion-zwitterion complexes.

Action spectroscopy of the isolated red Kaede fluorescent protein chromophore.

Incorporation of fluorescent proteins into biochemical systems has revolutionized the field of bioimaging. In a bottom-up approach, understanding the photophysics of fluorescent proteins requires detailed investigations of the light-absorbing chromophore, which can be achieved by studying the chromophore in isolation. This paper reports a photodissociation action spectroscopy study on the deprotonated anion of the red Kaede fluorescent protein chromophore, demonstrating that at least three isomers-assigned to deprotomers-are generated in the gas phase. Deprotomer-selected action spectra are recorded over the S1 ← S0 band using an instrument with differential mobility spectrometry coupled with photodissociation spectroscopy. The spectrum for the principal phenoxide deprotomer spans the 480-660 nm range with a maximum response at ≈610 nm. The imidazolate deprotomer has a blue-shifted action spectrum with a maximum response at ≈545 nm. The action spectra are consistent with excited state coupled-cluster calculations of excitation wavelengths for the deprotomers. A third gas-phase species with a distinct action spectrum is tentatively assigned to an imidazole tautomer of the principal phenoxide deprotomer. This study highlights the need for isomer-selective methods when studying the photophysics of biochromophores possessing several deprotonation sites.

Asymmetric Oxidation of Enol Derivatives to α-Alkoxy Carbonyls Using Iminium Salt Catalysts: A Synthetic and Computational Study.

We report herein the first examples of asymmetric oxidation of enol ether and ester substrates using iminium salt organocatalysis, affording moderate to excellent enantioselectivities of up to 98% ee for tetralone-derived substrates in the α-hydroxyketone products. A comprehensive density functional theory study was undertaken to interpret the competing diastereoisomeric transition states in this example in order to identify the origins of enantioselectivity. The calculations, performed at the B3LYP/6-31G(D) level of theory, gave good agreement with the experimental results, in terms of the magnitude of the effects under the specified reaction conditions, and in terms of the preferential formation of the ( R)-enantiomer. Just one of the 30 characterized transition states dominates the enantioselectivity, which is attributed to the adoption of an orientation relative to stereochemical features of the chiral controlling element that combines a CH-π interaction between a CH2 group in the substrate and one of the aromatic rings of the biaryl section of the chiral auxiliary with a good alignment of the acetoxy group with the other biaryl ring, and places the smallest substituent on the alkene (a hydrogen atom) in the most sterically hindered position.

One- to Two-Exciton Transitions in Perylene Bisimide Dimer Revealed by Two-Dimensional Electronic Spectroscopy.

The excited-state energy levels of molecular dimers and aggregates play a critical role in their photophysical behavior and an understanding of the photodynamics in such structures is important for developing applications such as photovoltaics and optoelectronic devices. Here, exciton transitions in two different covalently bound PBI dimers are studied by two-dimensional electronic spectroscopy (2DES), a powerful spectroscopic method, providing the most complete picture of vibronic transitions in molecular systems. The data are accurately reproduced using the equation of motion-phase matching approach. The unambiguous presence of one-exciton to two-exciton transitions are captured in our results and described in terms of a molecular exciton energy level scheme based on the Kasha model. Furthermore, the results are supported by comparative measurements with the PBI monomer and another dimer in which the interchromophore distance is increased.

Discovery of Small Molecule WWP2 Ubiquitin Ligase Inhibitors.

We have screened small molecule libraries specifically for inhibitors that target WWP2, an E3 ubiquitin ligase associated with tumour outgrowth and spread. Selected hits demonstrated dose-dependent WWP2 inhibition, low micromolar IC50 values, and inhibition of PTEN substrate-specific ubiquitination. Binding to WWP2 was confirmed by ligand-based NMR spectroscopy. Furthermore, we used a combination of STD NMR, the recently developed DEEP-STD NMR approach, and docking calculations, to propose for the first time an NMR-validated 3D molecular model of a WWP2-inhibitor complex. These first generation WWP2 inhibitors provide a molecular framework for informing organic synthetic approaches to improve activity and selectivity.

Novel binaphthyl and biphenyl α- and ß-amino acids and esters: organocatalysis of asymmetric Diels-Alder reactions. A combined synthetic and computational study.

Asymmetric catalysis of the Diels-Alder reaction between cyclopentadiene and cinnamaldehydes has been studied using as catalysts a range of novel α- and ß-aminoacids and aminoesters with binaphthyl and biphenyl backbones, providing enantioselectivities of up to 62% ee. B3LYP/6-31G* calculations, including free energy corrections, have been carried out on a binaphthyl catalyst example to identify transition state structures and to aid in the identification of major enantiomers. The calculated product ratios agree well with the experimental data; the transition states identified involve preferential approach of cyclopentene along a trajectory adjacent to the acid/ester group. The four lowest energy transition states display a stabilizing dipolar interaction between the carbonyl group oxygen atom and a terminal proton of the diene unit.

Formal Total Synthesis of (+)-C9-Deoxyomuralide from l-Leucine Using a Double Sacrificial Chirality Transfer Approach.

Formal stereocontrolled syntheses of (±)- and (+)-C9-deoxyomuralide is reported, constituting one of the shortest routes to the full carbon skeleton reported to date.

"Janus" Calixarenes: Double-Sided Molecular Linkers for Facile, Multianchor Point, Multifunctional, Surface Modification.

We herein report the synthesis of novel "Janus" calix[4]arenes bearing four "molecular tethering" functional groups on either the upper or lower rims of the calixarene. These enable facile multipoint covalent attachment to electrode surfaces with monolayer coverage. The other rim of the calixarenes bear either four azide or four ethynyl functional groups, which are easily modified by the copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC), either pre- or postsurface modification, enabling these conical, nanocavity reactor sites to be decorated with a wide range of substrates to impart desired chemical properties. Redox active species decorating the peripheral rim are shown to be electrically connected by the calixarene to the electrode surface in either "up" or "down" orientations of the calixarene.

New biphenyl iminium salt catalysts for highly enantioselective asymmetric epoxidation: role of additional substitution and dihedral angle.

New biaryl iminium salt catalysts for enantioselective alkene epoxidation containing additional substitution in the heterocyclic ring are reported. The effects upon conformation and enantioselectivity of this additional substitution, and the influence of dihedral angle in these systems, has been investigated using a synthetic approach supported by density functional theory. Enantioselectivities of up to 97% ee were observed.

Atropo- and Diastereoselective Construction of Tetracyclic Biphenylazepinium Salts Derived from Aminoalcohols: Use as Catalysts in Enantioselective Asymmetric Epoxidation.

A range of new biphenylazepinium salt organocatalysts effective for asymmetric epoxidation has been developed incorporating an additional substituted oxazolidine ring, and providing improved enantiocontrol in alkene epoxidation over the parent structure. Starting from enantiomerically pure aminoalcohols, tetracyclic iminium salts were obtained as single diastereoisomers through an atroposelective oxazolidine formation.

A gold-gold oil microtrench electrode for liquid-liquid anion transfer voltammetry.

Two flat gold electrodes are placed vis-à-vis with an epoxy spacer layer that is etched out to give a ca. 100 µm-deep electrochemically active trench. A water-insoluble oil phase, here the redox system N,N-diethyl-N'N'-didodecyl-phenylenediamine (DDPD) in 4-(3-phenylpropyl)-pyridine (PPP), is immobilized into the trench to allow anion transfer upon oxidation of DDPD (oil) to DDP⁺ (oil). In "mono-potentiostatic mode" quantitative transfer/expulsion of anions into the trench oil phase occurs. However, in "bi-potentiostatic mode" feedback currents dominated by rapid plate-to-plate diffusion normal to the electrode surfaces are observed. Comparison of "normal" diffusion and "lateral" diffusion shows that the rate of diffusion-migration charge transport across the oil film is anion hydrophobicity dependent.

Photophysics of the red-form Kaede chromophore.

The green fluorescent protein (GFP) drove revolutionary progress in bioimaging. Photoconvertible fluorescent proteins (PCFPs) are an important branch of the FP family, of which Kaede is the prototype. Uniquely, PCFPs can be permanently switched from green to red emitting forms on UV irradiation, facilitating applications in site-specific photolabelling and protein tracking. Optimisation and exploitation of FPs requires understanding of the photophysical and photochemical behaviour of the chromophore. Accordingly, the principal GFP chromophore has been the subject of intense experimental and theoretical investigation. In contrast, the photophysics of the red emitting PCFP chromophore are largely unstudied. Here we present a detailed investigation of the excited-state properties of the Kaede chromophore in solution, utilising steady state measurements, ultrafast time-resolved electronic and vibrational spectroscopies, and electronic structure theory. Its excited state dynamics are very different to those of the parent GFP. Most remarkably, the PCFP chromophore has highly complex wavelength-dependent fluorescence decays and a mean lifetime an order of magnitude longer than the GFP chromophore. Transient electronic and vibrational spectroscopies suggest that these dynamics arise from a range of excited-state conformers that are spectrally and kinetically distinct but chemically similar. These conformers are populated directly by excitation of a broad thermal distribution of ground state structures about a single conformer, suggesting an excited-state potential surface with several minima. Temperature-dependence confirms the existence of barriers on the excited-state surface and reveals the radiationless decay mechanism to be internal conversion. These experimental observations are consistent with a model assuming a simple ground state potential energy surface accessing a complex excited state possessing multiple minima.

Liquid-liquid ion transport junctions based on paired gold electrodes in generator-collector mode.

Simultaneous electrochemically driven double anion transfer across liquid-liquid interfaces is demonstrated at a gold-gold junction electrode. In the presence of two closely spaced electrodes (generator and collector), anion uptake into the organic phase (oxidation) and anion expulsion into the aqueous phase (reduction) can be combined to result in a generator-collector anion transport system across the liquid-liquid interface. In this report we are employing a paired gold junction grown by electro-deposition to ca. 5 microm gap size with the N,N-diethyl-N',N'-didodecyl-phenylene-diamine water immiscible redox liquid immobilized into the gap to demonstrate simultaneous perchlorate anion uptake and expulsion. The effects of redox liquid volume and scan rate on the magnitude of currents and two mechanistic pathways for ion transport are discussed in the context of micro-electrophoretic processes.

Formal synthesis of (+)-lactacystin from l-serine.

A formal, stereocontrolled synthesis of lactacystin has been completed from t-Bu-O-l-serine, providing the key intermediate 13, also useful for the generation of a range of C-9 analogues.

A new twist in the photophysics of the GFP chromophore: a volume-conserving molecular torsion couple.

The simple structure of the chromophore of the green fluorescent protein (GFP), a phenol and an imidazolone ring linked by a methyne bridge, supports an exceptionally diverse range of excited state phenomena. Here we describe experimentally and theoretically the photochemistry of a novel sterically crowded nonplanar derivative of the GFP chromophore. It undergoes an excited state isomerization reaction accompanied by an exceptionally fast (sub 100 fs) excited state decay. The decay dynamics are essentially independent of solvent polarity and viscosity. Excited state structural dynamics are probed by high level quantum chemical calculations revealing that the fast decay is due to a conical intersection characterized by a twist of the rings and pyramidalization of the methyne bridge carbon. The intersection can be accessed without a barrier from the pre-twisted Franck-Condon structure, and the lack of viscosity dependence is due to the fact that the rings twist in the same direction, giving rise to a volume-conserving decay coordinate. Moreover, the rotation of the phenyl, methyl and imidazolone groups is coupled in the sterically crowded structure, with the methyl group translating the rotation of one ring to the next. As a consequence, the excited state dynamics can be viewed as a torsional couple, where the absorbed photon energy leads to conversion of the out-of-plane orientation from one ring to the other in a volume conserving fashion. A similar modification of the range of methyne dyes may provide a new family of devices for molecular machines, specifically torsional couples.

Synthesis and Activity of a Novel Autotaxin Inhibitor-Icodextrin Conjugate.

Autotaxin is an extracellular phospholipase D that catalyzes the hydrolysis of lysophosphatidyl choline (LPC) to generate the bioactive lipid lysophosphatidic acid (LPA). Autotaxin has been implicated in many pathological processes relevant to cancer. Intraperitoneal administration of an autotaxin inhibitor may benefit patients with ovarian cancer; however, low molecular mass compounds are known to be rapidly cleared from the peritoneal cavity. Icodextrin is a polymer that is already in clinical use because it is slowly eliminated from the peritoneal cavity. Herein we report conjugation of the autotaxin inhibitor HA155 to icodextrin. The conjugate inhibits autotaxin activity (IC50 = 0.86 ± 0.13 µg mL-1) and reduces cell migration. Conjugation of the inhibitor increased its solubility, decreased its membrane permeability, and improved its intraperitoneal retention in mice. These observations demonstrate the first application of icodextrin as a covalently-bonded drug delivery platform with potential use in the treatment of ovarian cancer.

The Effect of Conjugation on the Competition between Internal Conversion and Electron Detachment: A Comparison between Green Fluorescent and Red Kaede Protein Chromophores.

Kaede, an analogue of green fluorescent protein (GFP), is a green-to-red photoconvertible fluorescent protein used as an in vivo "optical highlighter" in bioimaging. The fluorescence quantum yield of the red Kaede protein is lower than that of GFP, suggesting that increasing the conjugation modifies the electronic relaxation pathway. Using a combination of anion photoelectron spectroscopy and electronic structure calculations, we find that the isolated red Kaede protein chromophore in the gas phase is deprotonated at the imidazole ring, unlike the GFP chromophore that is deprotonated at the phenol ring. We find evidence of an efficient electronic relaxation pathway from higher-lying electronically excited states to the S1 state of the red Kaede chromophore that is not accessible in the GFP chromophore. Rapid autodetachment from high-lying vibrational states of S1 is found to compete efficiently with internal conversion to the ground electronic state.

Dimethylsulfoniopropionate biosynthesis in marine bacteria and identification of the key gene in this process.

Dimethylsulfoniopropionate (DMSP) is one of the Earth's most abundant organosulfur molecules, a signalling molecule1, a key nutrient for marine microorganisms2,3 and the major precursor for gaseous dimethyl sulfide (DMS). DMS, another infochemical in signalling pathways4, is important in global sulfur cycling2 and affects the Earth's albedo, and potentially climate, via sulfate aerosol and cloud condensation nuclei production5,6. It was thought that only eukaryotes produce significant amounts of DMSP7-9, but here we demonstrate that many marine heterotrophic bacteria also produce DMSP, probably using the same methionine (Met) transamination pathway as macroalgae and phytoplankton10. We identify the first DMSP synthesis gene in any organism, dsyB, which encodes the key methyltransferase enzyme of this pathway and is a reliable reporter for bacterial DMSP synthesis in marine Alphaproteobacteria. DMSP production and dsyB transcription are upregulated by increased salinity, nitrogen limitation and lower temperatures in our model DMSP-producing bacterium Labrenzia aggregata LZB033. With significant numbers of dsyB homologues in marine metagenomes, we propose that bacteria probably make a significant contribution to oceanic DMSP production. Furthermore, because DMSP production is not solely associated with obligate phototrophs, the process need not be confined to the photic zones of marine environments and, as such, may have been underestimated.

Iminium salt catalysts for asymmetric epoxidation: the first high enantioselectivities.

A highly enantioselective iminium salt catalyst has been prepared and tested in the catalytic asymmetric epoxidation of unfunctionalized alkenes, giving up to 95% ee, the highest ee yet reported for iminium salt-catalyzed epoxidation. Catalyst loadings as low as 0.1 mol % may be used.

Short and versatile route to a key intermediate for lactacystin synthesis.

[reaction: see text] A key intermediate 14 for the synthesis of lactacystin 1 has been constructed in four steps and 33% overall yield. The key steps involve cyclization of a suitably functionalized glutamic acid derivative and concomitant alkylation of the resulting beta,beta-diketoester system, C-acylation of the cyclic alpha-amidoketone 9, and decarboxylbenzylation of 12. Alkylation of a related beta,beta-diketoester 5 was additionally achieved with several electrophiles.