Dynamics change of phoborhodopsin and transducer by activation: study using D75N mutant of the receptor by site-directed solid-state 13C NMR

Pharaonis phoborhodopsin (ppR or sensory rhodopsin II) is a negative phototaxis receptor of Natronomonas pharaonis, and forms a complex, which transmits the photosignal into cytoplasm, with its cognate transducer (pHtrII). We examined a possible local dynamics change of ppR and its D75N mutant complexed with pHtrII, using solid-state (13)C NMR of [3-(13)C]Ala- and [1-(13)C]Val-labeled preparations. We distinguished Ala C(beta) (13)C signals of relatively static stem (Ala221) in the C-terminus of the receptors from those of flexible tip (Ala228, 234, 236 and 238), utilizing a mutant with truncated C-terminus. The local fluctuation frequency at the C-terminal tip was appreciably decreased when ppR was bound to pHtrII, while it was increased when D75N, that mimics the signaling state because of disrupted salt bridge between C and G helices prerequisite for the signal transfer, was bound to pHtrII. This signal change may be considered with the larger dissociation constant of the complex between pHtrII and M-state of ppR. At the same time, it turned out that fluctuation frequency of cytoplasmic portion of pHtrII is lowered when ppR is replaced by D75N in the complex with pHtrII. This means that the C-terminal tip partly participates in binding with the linker region of pHtrII in the dark, but this portion might be released at the signaling state leading to mutual association of the two transducers in the cytoplasmic regions within the ppR/pHtrII complex.     

Branched supramolecular polymers formed by bifunctional cyclodextrin derivatives

Branched supramolecular polymers have been prepared from the mixture of 3-cinnamamide-α-CD (1) and 3-N^α-cinnamamidehexancarbonyl-N^?-cinnamamide-lysinamide-α-CD (3) and from the mixture of 3-cinnamamidehexanamide-α-CD (2) and 3. Compounds 1 and 2 formed a linear supramolecular polymer, whereas compound 3 having two guest moieties formed a hyperbranched supramolecular polymer. Physical properties of these supramolecular polymers were studied by viscosity measurements in aqueous solutions. When compound 3 was added to the solution of compound 2, the η_sp/C value of the mixture of 2 and 3 was found to be much higher than that of compound 2. These results indicate that compound 3 functions as a branching moiety to increase the viscosity. Supramolecular polymers consisting of compound 2 or 3 did not show the viscosity increase, whereas the mixture of 2 and 3 gave highly viscous solutions and formed fibers from the concentrated aqueous solutions. It is caused by the branching of linear supramolecular polymers with compound 3 and hydrophobic and/or hydrogen bonding interactions between supramolecular polymers.     

Hidden electronic excited state of enhanced green fluorescent protein

Precise two-photon absorption spectra of the green fluorescent protein (GFP) and the mutants sapphire-GFP (T203I) and enhanced GFP (S65T/F64L), as well as a model compound for the chromophore, 4'-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI) were measured by multiplex two-photon absorption spectroscopy. The observed TPA bands of the anionic forms of enhanced GFP and HBDI were significantly shifted to the higher energy compared with the lowest-energy bands in one-photon absorption spectra. This result indicated the existence of a hidden electronic excited state in the vicinity of the lowest excited singlet (S1) state of the anionic form of the GFP chromophore, which is the origin of the blue shift of the two-photon absorption spectra as well as two-photon fluorescence excitation spectra.     

Subdiffraction imaging through the selective donut-mode depletion of thermally stable photoswitchable fluorophores: numerical analysis and application to the fluorescent protein Dronpa

The fast and reversible on/off switching of the fluorescence emission of the GFP-like fluorescent protein Dronpa has attracted considerable interest for applications in subdiffraction imaging. In this paper we study the use of a donut-mode beam in combination with two more overlapping laser beams to increase the imaging resolution through selective switching to the nonfluorescent photoswitched state. We devise and run a series of numerical simulations to determine suitable photophysical parameters of prospective, thermally stable photoswitchable molecules, in terms of photoswitching quantum yields, fatigue resistance, and possible presence of transient nonfluorescent states. Many of our findings are applicable to other measurements that make use of donut beams, and these guidelines can be used in the synthesis and screening of novel photoswitchable compounds. We experimentally demonstrate the possibility of obtaining increased resolution by making use of the efficient and thermally stable Dronpa photoswitching, using equipment that is commonly available.     

Transglutaminase-mediated synthesis of a DNA-(enzyme)n probe for highly sensitive DNA detection

A new synthetic strategy for DNA-enzyme conjugates with a novel architecture was explored using a natural cross-linking catalyst, microbial transglutaminase (MTG). A glutamine-donor substrate peptide of MTG was introduced at the 5-position on the pyrimidine of deoxyuridine triphosphate to prepare a DNA strand with multiple glutamine-donor sites by polymerase chain reaction (PCR). A substrate peptide that contained an MTG-reactive lysine residue was fused to the N terminus of a thermostable alkaline phoshatase from Pyrococcus furiosus (PfuAP) by genetic engineering. By combining enzymatically the substrate moieties of MTG introduced to the DNA template and the recombinant enzyme, a DNA-(enzyme)(n) conjugate with 1:n stoichiometry was successfully obtained. The enzyme/DNA ratio of the conjugate increased as the benzyloxycarbonyl-L-glutaminylglycine (Z-QG) moiety increased in the DNA template. The potential utility of the new conjugate decorated with signaling enzymes was validated in a dot blot hybridization assay. The DNA-(enzyme)(n) probe could clearly detect 10(4) copies of the target nucleic acid with the complementary sequence under harsh hybridization conditions, thereby enabling a simple detection procedure without cumbersome bound/free processes associated with a conventional hapten-antibody reaction-based DNA-detection system.     

Efficient solid-phase synthesis of cyclic RGD peptides under controlled microwave heating

Cyclic RGD peptides are potent antagonists for the α_vβ₃ integrin receptor. In this Letter, microwave-assisted solid-phase synthesis of cyclic RGD peptides is described. In a coupling reaction between Fmoc-Arg(Pbf)-OH and high-loading H-Gly-Trt(2-Cl) resin, multiple coupling reactions were required for completion under the conventional HBTU activation. We found that the use of COMU, a new coupling reagent, under microwave heating to 50 °C accelerated the reaction even inside the resin. This method was applicable to the synthesis of linear pentapeptides, H-Asp(OtBu)-Xxx-Yyy-Arg(Pbf)-Gly-OH (Xxx = d-Phe(p-Br) or d-Tyr, Yyy = Lys(Boc) or MeVal). Cyclization of these peptides followed by deprotection gave the desired cyclic RGD peptides with high purity.     

Magneto-optical properties and exciton dynamics in diluted magnetic semiconductor nanostructures

Nanostructures of diluted magnetic semiconductors were fabricated to study novel magneto-optical properties that are derived from quantum confined band electrons interacting with magnetic ions. Quantum dots (QDs) of Cd_0.97Mn_0.03Se were grown by the self-organization on a ZnSe substrate layer. QDs of Zn_0.69Cd_0.23Mn_0.08Se and quantum wires (QWRs) of Cd_0.92Mn_0.08Se and Zn_0.69Cd_0.23Mn_0.08Se were fabricated by the electron beam lithography. A single quantum well (QW) of ZnTe/Zn_0.97Mn_0.03Te and double QWs of Cd_0.95Mn_0.05Te–CdTe were grown by molecular beam epitaxy. Magneto-optical properties and the formation and relaxation dynamics of excitons were investigated by ultrafast time-resolved photoluminescence (PL) spectroscopy. Excitons in these nanostructures were affected by the low-dimensional confinement effects and the interaction with magnetic ion spins. The exciton luminescence of the Cd_0.97Mn_0.03Se QDs shows the confined exciton energy due to the dot size of 4–6 nm and also shows marked increase of the exciton lifetime with increasing the magnetic field. The QDs of Zn_0.69Cd_0.23Mn_0.08Se fabricated by the electron beam lithography display narrow exciton PL spectrum due to the uniform shape of the dots. The exciton luminescence from the QWRs of Cd_0.92Mn_0.08Se and Zn_0.69Cd_0.23Mn_0.08Se shows the influence of the one-dimensional confinement effect for the exciton energy and the luminescence is linearly polarized parallel to the wire direction. The transient PL from the ZnTe/Zn_0.97Mn_0.03Te QWs displays, by the magnetic field, the level crossing of the exciton spin states of the nonmagnetic and magnetic layers and the spatial spin separation for the excitons. Cd_0.95Mn_0.05Te–CdTe double QWs show the injection of the spin polarized excitons from the magnetic well to the nonmagnetic QW.     

Synthesis of two possible diastereomers of reticulatain-1

A convergent synthesis of two possible diastereomers of reticulatain-1 (1a and 1b) was accomplished. Comparison of the specific optical rotations of 1a and 1b did not allow for the strict determination of the absolute configuration. However, bis-(R)-MTPA esters of 1a and 1b showed a clear difference in chemical shifts in the ¹H NMR spectra. If the bis-(R)-MTPA ester of natural reticulatain-1 (1) is available, the absolute configuration of 1 will be determined. Inhibitory action of these compounds was examined with bovine heart mitochondrial complex I. Both compounds showed almost the same activity.     

Novel catalytic hydrogenolysis of silyl enol ethers by the use of acidic ruthenium dihydrogen complexes

Treatment of 1-trimethylsilyloxy-1-cyclohexene (1a) in the presence of a catalytic amount of the acidic dihydrogen complex [RuCl(η²-H₂)(dppe)₂]OTf (4a) [dppe=1,2-bis(diphenylphosphino)ethane, OTf=OSO₂CF₃] (10 mol.%) under 1 atm of H₂ in anhydrous ClCD₂CD₂Cl at 50 °C for 8 h afforded cyclohexanone (3a) and Me₃SiH in quantitative NMR yields. Silyl enol ethers such as 1-triethylsilyloxy-1-cyclohexene (1b), 1-t-butyldimethylsilyloxy-1-cyclohexene (1c), and other trimethylsilylethers (1d, 1e, and 1f) reacted similarly with H₂ to afford the corresponding ketones and trialkylsilanes. The direct proton transfer from H₂ to the trimethylsilyl enol ethers (1a and 1d-1f) was confirmed by the experiments employing D₂ gas, where α-monodeuterated ketones (3a′ and 3d′-3f′) were obtained in high yields. The enantioselective protonation of prochiral silyl enol ethers with 1 atm of H₂ by employing [RuCl(η²-H₂)((S)-BINAP)₂]OTf (4e) [BINAP=2,2′-bis(diphenylphosphino)-1,1′-binaphthyl] and [RuCl(η²-H₂)((R, R)-CHIRAPHOS)₂]OTf (4f) [CHIRAPHOS=2,3-bis(diphenylphosphino)butane] showed that no enantioselectivity was observed in either catalytic or stoichiometric protonation reactions under various reaction conditions. The reaction of [RuHCl(dppe)₂] (5a) with one equivalent of Me₃SiOTf under 1 atm of H₂ produced rapidly 4a, concurrent with the formation of Me₃SiH. Based on these studies, the mechanism for this novel hydrogenolysis of silyl enol ethers is proposed which involves heterolytic cleavage of the coordinated H₂ on the ruthenium atom caused by the nucleophilic attack of the oxygen atom of enol ethers to give ketones and Me₃SiOTf, and the subsequent reaction of the resultant complex 5a with Me₃SiOTf under 1 atm of H₂ to regenerate the original dihydrogen complex 4a. On the other hand, the stoichiometric reaction of a lithium enolate 6e with one equivalent of 4e at −78 °C in CH₂Cl₂ under 1 atm of H₂ afforded 2-methyl-1-tetralone (3e) with 75% ee (S) in >95% yield, together with the formation of [RuHCl((S)-BINAP)₂] (5e).     

Design, synthesis, and 1,3-dipolar cycloaddition of (5R)- [and (5S)]-5,6-dihydro-5-phenyl-2H-1,4-oxazin-2-one N-oxides as chiral (E)-geometry-fixed alpha-alkoxycarbonylnitrones

Optically pure (5R)- [and (5S)]-5,6-dihydro-5-phenyl-2H-1, 4-oxazin-2-one N-oxides [(5R)- and (5S)-2] were designed as chiral (E)-geometry-fixed alpha-alkoxycarbonylnitrones 1. The nitrones (5R)- and (5S)-2 were synthesized by three-step oxidation of (R)- and (S)-phenylglycinols [(R)- and (S)-3], condensation of the resulting (R)- and (S)-2-hydroxylamino-2-phenylethanols [(R)- and (S)-5] with glyoxylic acid, and cyclization of the intermediary nitrones (R)- and (S)-6b. The nitrone (5R)-2reacted with olefins 7-14 under mild conditions to afford the corresponding cycloadducts 15-22 as the main products via the least sterically demanding exo modes. Cycloadduct 30 obtained from (5S)-2 and cyclopentadiene was effectively elaborated to (1S,4S, 5R)-4-benzyloxycarbonylamino-2-oxabicyclo[3.3.0]oct-7-en-3-one (28), the key synthetic intermediate of carbocyclic polyoxin C.