Rapid Isomerization Research Articles

Photoregulation of Molecular Motors using Photochromic Nucleotide Analogue

Photochromic molecules can be interconverted between two quite different states with different spectroscopic properties using two different wavelength lights. Therefore, the photochromic molecules have received much attention because of their potential application to optical switches, bio-nanodevices, and molecular machines. Azobenzene is a typical photochromic molecule that undergoes rapid and reversible isomerization between the cis and trans-isomer in response to ultraviolet (UV) and visible (VIS) light irradiation, respectively. Previously, we have succeeded to control microtubule dependent ATPase activity of kinesin in which functional region was modified with azobenzene derivative photo-reversibly. Moreover, we synthesized photochromic ATP analogue PABITP composed of azobenzene to regulate ATP driven motor proteins. PABITP induced microtubule gliding for kinesin and the gliding velocity altered correlating to cis-trans photo-isomerization of azobenzene moiety. For myosin, although PABITP was hydrolyzed as a substrate for ATPase and induced conformational change of myosin motor domain, PABITP did not drive actin gliding. In this study, we designed and synthesized a novel photochromic nucleotide analogue composed of photochromic fulgimide moiety in order to regulate ATP driven molecular motors, myosin and kinesin photo-reversibly. The photochromic ATP analogue, Fulgimide-Tri-Phosphate (FITP) exhibited ring-opening and ring-closing photo-isomerization upon UV and VIS light irradiations. FITP worked as a substrate of skeletal muscle myosin and induced dissociation of acto-myosin in a different manner upon UV and VIS light irradiations. We also examined the interaction of FITP with kinesin and its effect for the polymerization of tubulin to microtubules.

Olefin Isomerization and Hydrosilylation Catalysis by Lewis Acidic Organofluorophosphonium Salts

Organofluorophosphonium salts of the formula [(C6F5)(3-x)Ph(x)PF][B(C6F5)4] (x = 0, 1) exhibit Lewis acidity derived from a low-lying σ* orbital at P opposite F. This acidity is evidenced by the reactions of these salts with olefins, which catalyze the rapid isomerization of 1-hexene to 2-hexene, the cationic polymerization of isobutylene, and the Friedel-Crafts-type dimerization of 1,1-diphenylethylene. In the presence of hydrosilanes, olefins and alkynes undergo efficient hydrosilylation catalysis to the alkylsilanes. Experimental and computational considerations of the mechanism are consistent with the sequential activation and 1,2-addition of hydrosilane across the unsaturated C-C bonds.

Propargyloxy- and allenyloxymethylferrocenes: Synthesis and oligomerization

Propargyloxymethylferrocene has been synthesized by the modified reaction of available N,N-dimethylaminomethylferrocene with propargyl alcohol in the presence of dichloroethane and NaOH, the yield reaching 99.6%. The rapid prototropic isomerization of propargyloxymethylferrocene in the superbase system t-BuOK/DMSO (room temperature, 4 min) leads to previously unknown allenyloxymethylferrocene in 80% yield. Preliminary cationic and radical polymerizations of both monomers have been studied and novel oligomers with the polyene chain and pendant ferrocenyl moieties containing soluble and insoluble fractions have been synthesized in 83–88% total yield. All the oligomers exhibit semiconducting and paramagnetic properties and stable up to 300–340 °C.

Palladium‐Catalyzed Selective Anti‐Markovnikov Oxidation of Allylic Esters

An aldol alternative: The palladium(II)-catalyzed anti-Markovnikov oxidation of allylic esters to aldehydes at room temperature provides a viable alternative to valuable cross aldol products. High regioselectivity towards the aldehyde product was achieved using the ester protecting group for the allylic alcohol. Rapid isomerization and the much higher rate of oxidation of the branched isomer result in the same product forming from both linear and branched allylic esters.

Palladium‐Catalyzed Selective Anti‐Markovnikov Oxidation of Allylic Esters

The palladium(II)-catalyzed oxidation of alkenes to carbonyl compounds, usually referred to as the Wacker or Wacker– Tsuji reaction, is arguably one of the best-known reactions catalyzed by palladium. It is an important catalytic process industrially, for the production of ethanal, and synthetically, for the conversion of olefins to ketones. The oxidation of terminal alkenes typically proceeds with selective formation of methylketones. The anti-Markovnikov (AM) Wacker oxidation of terminal olefins to aldehydes remains, however, a major challenge. Under certain conditions, AM selectivity is obtained with styrenes, Michael-type acceptor alkenes and certain olefins, such as 2-vinyl-furanosides, bearing a directing functional group. Indeed, high aldehyde selectivity in the catalytic oxidation of phthalimide-protected allylic amines was reported by our group to yield a key intermediate in the preparation of b-amino acids. On the other hand, Sigman and co-workers have reported the regioselective oxidation of protected allylic amines controlled by various palladium catalysts to yield the corresponding methyl ketones. In 1986, Pd-catalyzed aldehyde selective oxidation of styrene with O2 and CuCl in tBuOH at 30 8C was reported by Feringa. Later, Wenzel reported good selectivity (6:1) for aldehyde formation from allyl acetate (56% combined yield of aldehyde and ketone), in tBuOHwith PdCl2/CH3CN/CuCl/ NaCl at 50 8C. More recently, the aldehyde-selective oxidation of styrenes was reported by Grubbs and co-workers using the catalyst [PdCl2(CH3CN)2], p-benzoquinone as oxidant, and tBuOH as solvent at 85 8C. However, a more general anti-Markovnikov alkene oxidation of non-aryl alkenes under mild conditions remains a challenge, despite the tremendous value in extending this reaction to other substrate classes, in particular allylic alcohols and esters. b-Hydroxy aldehydes are usually prepared by the crossaldol reaction between aldehydes or an aldehyde and a ketone. The direct catalytic formation of an aldehyde by selective attack at the terminal carbon of an a-olefin would be a highly valuable alternative. However, the selective antiMarkovnikov oxidation of allylic alcohols into b-hydroxy aldehydes has proven to be very difficult owing to formation of the ketone products and competing olefin isomerization. Herein, we demonstrate the aldehyde-selective catalytic oxidation of ester-protected allylic alcohols with as low as 0.5 mol% of [PdCl2(PhCN)2] and p-benzoquinone (BQ) as oxidant in tBuOH under ambient conditions. Importantly, the same anti-Markovnikov oxidation products were obtained selectively from both branched and linear allylic esters (Scheme 1), owing to rapid isomerization between allylic esters under the reaction conditions (see below).

Light-Induced Rearrangement of Thioether-Substituted Phosphanide Ligands: Scope and Limitations of a Remarkable Isomerization

Treatment of the thioether-substituted secondary phosphanes R(2)PH(C6H4-2-SR(1)) [R(2) = (Me3Si)2CH, R(1) = Me (1PH), iPr (2PH), Ph (3PH); R(2) = tBu, R(1) = Me (4PH); R(2) = Ph, R(1) = Me (5PH)] with nBuLi yields the corresponding lithium phosphanides, which were isolated as their THF (1-5Pa) and tmeda (1-5Pb) adducts. Solid-state structures were obtained for the adducts [R(2)P(C6H4-2-SR(1))]Li(L)n [R(2) = (Me3Si)2CH, R(1) = nPr, (L)n = tmeda (2Pb); R(2) = (Me3Si)2CH, R(1) = Ph, (L)n = tmeda (3Pb); R(2) = Ph, R(1) = Me, (L)n = (THF)1.33 (5Pa); R(2) = Ph, R(1) = Me, (L)n = ([12]crown-4)2 (5Pc)]. Treatment of 1PH with either PhCH2Na or PhCH2K yields the heavier alkali metal complexes [{(Me3Si)2CH}P(C6H4-2-SMe)]M(THF)n [M = Na (1Pd), K (1Pe)]. With the exception of 2Pa and 2Pb, photolysis of these complexes with white light proceeds rapidly to give the thiolate species [R(2)P(R(1))(C6H4-2-S)]M(L)n [M = Li, L = THF (1Sa, 3Sa-5Sa); M = Li, L = tmeda (1Sb, 3Sb-5Sb); M = Na, L = THF (1Sd); M = K, L = THF (1Se)] as the sole products. The compounds 3Sa and 4Sa may be desolvated to give the cyclic oligomers [[{(Me3Si)2CH}P(Ph)(C6H4-2-S)]Li]6 ((3S)6) and [[tBuP(Me)(C6H4-2-S)]Li]8 ((4S)8), respectively. A mechanistic study reveals that the phosphanide-thiolate rearrangement proceeds by intramolecular nucleophilic attack of the phosphanide center at the carbon atom of the substituent at sulfur. For 2Pa/2Pb, competing intramolecular β-deprotonation of the n-propyl substituent results in the elimination of propene and the formation of the phosphanide-thiolate dianion [{(Me3Si)2CH}P(C6H4-2-S)](2-).

Flexibility Analysis of the Microtubules Polymerized in the Presence of Photochromic Nucleotide Analogues

Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis- and trans-form in response to ultraviolet (UV) and visible (VIS) light irradiation, respectively. Previously, we have incorporated azobenzene derivative into functional region of ATP driven motor protein and succeeded to control microtubule dependent ATPase activity reversibly by photo-irradiation. The results suggested that the photo-control system using photochromic molecules might be applicable to other functional biomolecules.It is known that tubulin utilize GTP to polymerize and the GTP is entrapped within the GTP binding site of tubulin. And the microtubules composed of GDP bound tubulin easily depolymerized. Interestingly, it was demonstrated the structure of the GTP analogue GMP-CPP bound microtubule is squigglier configuration than that of regular GTP bond microtubule. In the present study, we have employed photochromic nucleotide analogue, phenylazobenzoyl-iminoethyl-tri-Pphosphate (PABITP) in order to photo-control flexibility of microtubules. The PABITP bound microtubules showed much more flexible shape change during gliding on the in-vitro motility assay than regular GTP bound microtubule. The effect of photo-isomerization of PABITP entrapped within the site of microtubules induced by UV and VIS light irradiation for the flexibility of microtubules was also examined.

Aggregation-induced phosphorescence of iridium(iii) complexes with 2,2′-bipyridine-acylhydrazone and their highly selective recognition to Cu2+

Two cationic cyclometallated iridium(III) complexes with 2,2'-bipyridine-acylhydrazone were synthesized and characterized by spectroscopic and photophysical measurements. They exhibit remarkable aggregation-induced phosphorescent emission (AIPE) phenomenon which is caused by the restriction of rapid isomerization of the C=N bond in the acylhydrazone moiety and are supported by TD-DFT studies. They also act as a significant 'off-on' luminescent switch for Cu(2+) which works as both catalyzer and oxidant in the sensing process, resulting in hydrolysis and cyclization products which are highly emissive. The sensing properties of iridium(III) complexes are also investigated by ESI-MS spectrometry and (1)H NMR spectroscopy.

Capturing reaction paths and intermediates in Cre- loxP recombination using single-molecule fluorescence

Site-specific recombination plays key roles in microbe biology and is exploited extensively to manipulate the genomes of higher organisms. Cre is a well studied site-specific recombinase, responsible for establishment and maintenance of the P1 bacteriophage genome in bacteria. During recombination, Cre forms a synaptic complex between two 34-bp DNA sequences called loxP after which a pair of strand exchanges forms a Holliday junction (HJ) intermediate; HJ isomerization then allows a second pair of strand exchanges and thus formation of the final recombinant product. Despite extensive work on the Cre-loxP system, many of its mechanisms have remained unclear, mainly due to the transient nature of complexes formed and the ensemble averaging inherent to most biochemical work. Here, we address these limitations by introducing tethered fluorophore motion (TFM), a method that monitors large-scale DNA motions through reports of the diffusional freedom of a single fluorophore. We combine TFM with Förster resonance energy transfer (FRET) and simultaneously observe both large- and small-scale conformational changes within single DNA molecules. Using TFM-FRET, we observed individual recombination reactions in real time and analyzed their kinetics. Recombination was initiated predominantly by exchange of the "bottom-strands" of the DNA substrate. In productive complexes we used FRET distributions to infer rapid isomerization of the HJ intermediates and that a rate-limiting step occurs after this isomerization. We also observed two nonproductive synaptic complexes, one of which was structurally distinct from conformations in crystals. After recombination, the product synaptic complex was extremely stable and refractory to subsequent rounds of recombination.

Mechanistic Features of Isomerizing Alkoxycarbonylation of Methyl Oleate

The weakly coordinated triflate complex [(P^P)Pd(OTf)](+)(OTf)(-) (1) (P^P = 1,3-bis(di-tert-butylphosphino)propane) is a suitable reactive precursor for mechanistic studies of the isomerizing alkoxcarbonylation of methyl oleate. Addition of CH(3)OH or CD(3)OD to 1 forms the hydride species [(P^P)PdH(CH(3)OH)](+)(OTf)(-) (2-CH(3)OH) or the deuteride [(P^P)PdD(CD(3)OD)](+)(OTf)(-) (2(D)-CD(3)OD), respectively. Further reaction with pyridine cleanly affords the stable and isolable hydride [(P^P)PdH(pyridine)](+)(OTf)(-) (2-pyr). This complex yields the hydride fragment free of methanol by abstraction of pyridine with BF(3)·OEt(2), and thus provides an entry to mechanistic observations including intermediates reactive toward methanol. Exposure of methyl oleate (100 equiv) to 2(D)-CD(3)OD resulted in rapid isomerization to the thermodynamic isomer distribution, 94.3% of internal olefins, 5.5% of α,β-unsaturated ester and <0.2% of terminal olefin. Reaction of 2-pyr/BF(3)·OEt(2) with a stoichiometric amount of 1-(13)C-labeled 1-octene at -80 °C yields a 50:50 mixture of the linear alkyls [(P^P)Pd(13)CH(2)(CH(2))(6)CH(3)](+) and [(P^P)PdCH(2)(CH(2))(6)(13)CH(3)](+) (4a and 4b). Further reaction with (13)CO yields the linear acyls [(P^P)Pd(13)C(═O)(12/13)CH(2)(CH(2))(6)(12/13)CH(3)(L)](+) (5-L; L = solvent or (13)CO). Reaction of 2-pyr/BF(3)·OEt(2) with a stoichiometric amount of methyl oleate at -80 °C also resulted in fast isomerization to form a linear alkyl species [(P^P)PdCH(2)(CH(2))(16)C(═O)OCH(3)](+) (6) and a branched alkyl stabilized by coordination of the ester carbonyl group as a four membered chelate [(P^P)PdCH{(CH(2))(15)CH(3)}C(═O)OCH(3)](+) (7). Addition of carbon monoxide (2.5 equiv) at -80 °C resulted in insertion to form the linear acyl carbonyl [(P^P)PdC(═O)(CH(2))(17)C(═O)OCH(3)(CO)](+) (8-CO) and the five-membered chelate [(P^P)PdC(═O)CH{(CH(2))(15)CH(3)}C(═O)OCH(3)](+) (9). Exposure of 8-CO and 9 to (13)CO at -50 °C results in gradual incorporation of the (13)C label. Reversibility of 7 + CO ⇄ 9 is also evidenced by ΔG = -2.9 kcal mol(-1) and ΔG(‡) = 12.5 kcal mol(-1) from DFT studies. Addition of methanol at -80 °C results in methanolysis of 8-L (L = solvent) to form the linear diester, 1,19-dimethylnonadecandioate, whereas 9 does not react and no branched diester is observed. DFT yields a barrier for methanolysis of ΔG(‡) = 29.7 kcal mol(-1) for the linear (8) vs ΔG(‡) = 37.7 kcal mol(-1) for the branched species (9).

Molecular switches and cages

Molecular switches can toggle between two (or more) stable states encoding different physical features. They allow complex systems to respond to changes in their environment defined by light intensity, pH, temperature or voltage. As such, molecular switches play a key role in biology and information technology and have become important components of advanced materials. Chemists have developed a large number of synthetic switches, complementing the many types found in nature. Given their prominence in biology, it is not surprisingly that photoswitches, which are actuated by ultraviolet, visible or infrared light, have been an especially productive field of study. Despite their long history, new types of synthetic photoswitches continue to emerge and “old friends”, such as azobenzenes are constantly improved and optimized. This is not only true with respect to their photophysical properties but also with regard to their synthetic accessibility. Indeed, as molecular switches are applied in ever-increasing quantities, the efficiency of their syntheses becomes a primary concern, requiring the development of new synthetic methods and strategies. The corresponding results, however, are often buried in the Supporting Information of papers that focus on functional aspects, which does not reflect the relative effort that goes into the design and synthesis of these molecules. Caged compounds are a subclass of (photo)switches that have special functional features. As opposed to “true” switches, they can only be turned ON and the abatement of their activity depends on secondary processes (such as diffusion from the active zone or, more importantly, re-uptake systems). In terms of their biological application, especially in neurobiology, they are much further developed than reversibly switchable molecules. For instance, two-photon activation, which allows for very precise localization, is fairly well established with caged compounds but in its infancy in the case of reversible photoswitches. The present Thematic Series of the Beilstein Journal of Organic Chemistry addresses the crucial role that chemistry, and particularly organic chemistry, can play in tuning the functional features of molecular switches and cages. These features include their absorption spectra, conductivity, geometry and bistability, as well as their polarity, solubility, efficacy, or catalytic activity. Photoswitches are covered extensively, ranging from diarylethenes (Pu, Wagenknecht) to dihydroazulenes/vinylheptafulvenes (Nielsen) and azobenzenes (Ruck-Braun, Hoppmann). Two reviews discuss cis-azobenzenes with rapid thermal isomerization kinetics (Velasco), and highlight the azobenzene moiety as one of the smallest light-driven molecular motors conceivable (Merino). Issues of bistability are also addressed in an account on shape-persistent, optically active macrocycles (Pasini). Molecular switches that respond to changes in pH are covered as well (Haberhauer and Aprahamian), reflecting their importance in biology and in materials science. Finally, switching is highlighted as a synthetic strategy for building advanced materials that are useful in photovoltaics (Matile). Chemists, by nature, like to control things and probably actuate macroscopic switches more often than the average person. We hope that this collection of papers will motivate some of our colleagues to also consider nanoscopic versions of switches (and cages). For those of us who deal with these molecules on a daily basis, this Thematic Series will hopefully provide further inspiration and an impetus to continue work in this fascinating field of research. Dirk Trauner Munich, June 2012

Effect of Cu2+ on the Stability of Xanthophylls in Acetone

Investigations were conducted on the stability of lutein, zeaxanthin, and β-cryptoxanthin during incubation with CuSO4·5H2O. For this purpose, a method for simultaneous determination of lutein, zeaxanthin, and β-cryptoxanthin stereoisomers by HPLC-DAD and APcI-MS was developed. (all-E)-Lutein, (all-E)-zeaxanthin, and (all-E)-β-cryptoxanthin were baseline separated and identified using chromatographic retention times; UV/Vis absorption spectra and positive mass spectra were compared with literature data. It was found that Cu2+ exhibited a rapid isomerization induction of (all-E)-xanthophylls to their (Z)-isomers exceeding 4/1 mass ratio of Cu2+/pigments, and after the incubation time exceeding 8 h, the concentration of the formation (Z)-xanthophylls decreased, indicating that the Cu2+ also exhibited isomerization of (Z)-isomers. The (13-Z)-lutein and (13′-Z)-lutein were identified as the major (Z)-isomers of (all-E)-lutein treated by Cu2+, and the (9-Z)-zeaxanthin was identified as the major isomerization product of (all-E)-zeaxanthin, and an unidentified (Z)-β-cryptoxanthin was also detected.

Force–Reactivity Property of a Single Monomer Is Sufficient To Predict the Micromechanical Behavior of Its Polymer

We demonstrate an accurate prediction of the micromechanical behavior of a single chain of cyclopropanated polybutadiene, which is governed by rapid isomerization of the cyclopropane moieties at ~1.2 nN, from the force-rate correlation of this reaction measured in a small series of increasingly strained macrocycles. The data demonstrate that a single physical quantity, force, uniquely defines the dynamics across length scales from >100 to <1 nm and that strain imposed through molecular design and that imposed by micromanipulation techniques have equivalent effects on the kinetics of a chemical reaction. This represents a new method of screening potential monomers for applications in stress-responsive materials that could also facilitate atomistic interpretations of single-molecule force experiments.

Coordination Effects on Electron Distributions for Rhodium Complexes of the Redox-Active Bis(3,5-di-tert-butyl-2-phenolate)amide Ligand

New rhodium complexes of bis(3,5-di-tert-butyl-2-phenol)amine ([ONO(cat)]H(3)) were synthesized, and their electronic properties were investigated. These compounds were prepared by combining [ONO(q)]K and [(cod)Rh(μ-Cl)](2) in the presence of an auxiliary donor ligand to yield complexes of the type [ONO]RhL(n) (n = 3, L = py (1); n = 2, L = PMe(3) (2a), L = PMe(2)Ph (2b), PMePh(2) (2c), PPh(3) (2d)). Single-crystal X-ray diffraction studies on [ONO]Rh(py)(3) (1) revealed a six-coordinate, octahedral rhodium complex. In the case of [ONO]Rh(PMe(3))(2) (2a), X-ray diffraction showed a five-coordinate, distorted square-pyramidal coordination environment around the rhodium center. While 1 is static on the NMR time scale, complexes 2a-d are fluxional, displaying both rapid isomerization of the square-pyramidal structure and exchange of coordinated and free phosphine ligands. UV-vis spectroscopy shows stark electronic differences between 1 and 2a-d. Whereas 1 displays a strong absorbance at 380 nm with a much weaker band at 585 nm in the absorption spectrum, complexes 2a-d display an intense (ε > 10(4) M(-1) cm(-1)), low-energy absorption band in the region 580-640 nm; however, in the cases of 2a and 2b, the addition of excess phosphine resulted in changes to the UV-vis spectrum indicating the formation of six-coordinate adducts [ONO]Rh(PMe(3))(3) (3a) and [ONO]Rh(PMe(2)Ph)(3) (3b), respectively. The experimental and DFT computational data for the six-coordinate complexes 1, 3a, and 3b are consistent with their formulation as classical, d(6), pseudo-octahedral, coordination complexes. In the five-coordinate complexes 2a-2d, π-bonding between the rhodium center and the [ONO] ligand leads to a high degree of covalency and metal-ligand electron distributions that are not accurately described by formal oxidation state assignments.

Asymmetry in activator ring opens up Sigma54 dependent transcription in bacteria

Bacterial transcription requires the recruitment of the core RNA‐polymerase (RNAP) to specific promoter by a σ factor. This usually leads to rapid isomerization from un‐melted DNA (closed) to a melted state (open), from which transcription can begin. Unlike this, σ54 cannot spontaneously isomerize from closed to open complex state. This requires the system to be remodeled by bacterial Enhancer Binding Proteins (bEBPs). They belong to AAA+ ATPases superfamily. These activators are known to act as rings, but how this architecture assists them to perform mechanical work on targets is unknown. Conformational changes occur in the ring during nucleotide binding and hydrolysis. Recent crystal structures have shown heterogeneous nucleotide occupancy in the ring and co‐operative extension of the surface loops, which are believed to contact and remodel the polymerase. Here we present variations in the bEBP structure in the context of closed complex, by transmission electron microscopy. They have shown persistence of ring asymmetry, in the form of a split ring and hints at two possible sequential hydrolysis mechanisms in which the gap propagates around the ring in a constant or varied position relative to the rest of the asymmetric complex. Thus, it helps us to advances our understanding of how bEBPs use ATP binding and hydrolysis to regulate gene expression in bacteria.This work has been supported by NIH.

ChemInform Abstract: Iron‐Catalyzed Isomerizations of Olefins.

AbstractA simple catalyst system that effects rapid isomerization of olefins at room temperature is described.

Peripheral endocannabinoid microdialysis: in vitro characterization and proof-of-concept in human subjects

In vivo endocannabinoid (EC) microdialysis has only seldom been performed, mostly in rodent brain tissue. Low solubility in aqueous media, adsorption to surfaces, and instability with co-present human serum albumin (HSA) are the major obstacles in EC microdialysis. The addition of hydroxypropyl-ß-cyclodextrine (HPCD) to the perfusion fluid has been previously described to facilitate lipid microdialysis, but the general biophysical properties of HPCD, especially with respect to peripheral EC microdialysis, have not been described before. We report on the characterization of EC microdialysis using an in vitro system using Ringer's solution with 10% HPCD as the perfusion fluid and with fatty acid-free HSA as the matrix fluid. The endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2AG) were measured using LC-MS/MS. AEA was stable in the perfusion and matrix fluids, whereas 2AG was only stable in the perfusion fluid. In the matrix fluid, 2AG underwent rapid isomerization to 1-arachidonoyl glycerol. A relative recovery of 3.5% for AEA was found with 10% HPCD in the perfusion fluid and a flow rate of 1 μL/min. For 2AG, a similar relative recovery of 3.5% was estimated. Since 2AG was found unstable in the matrix fluid, a reliable calculation of the relative recovery rates was not possible. Delivery and recovery experiments revealed unequal inward and outward EC transport across the microdialysis membrane. Contrary to usual microdialysis findings, we observed increasing recovery rates for AEA with increasing flow rates. Long equilibration times of several hours were necessary to obtain constant relative recovery rates. In a proof-of-concept study in humans, we collected AEA from subcutaneous abdominal adipose tissue employing the described methodology. Our study suggests that the microdialysis technique is not suitable for the exact quantification of tissue EC concentrations, but it allows for their rough estimation.

Stereo-Selective Synthesis of 5-Norbornene-2-&amp;lt;i&amp;gt;exo&amp;lt;/i&amp;gt;-carboxylic Acid—Rapid Isomerization and Kinetically Selective Hydrolysis

Simple and efficient stereo-selective synthesis of exo-5-norbornene-2-carboxylic acid (NBCA) is reported. Preliminary studies on base promoted isomerization of methyl 5-norbornene-2-carboxylate (MNBC) revealed that rapid isomerization was accomplished with sodium tert-butoxide (tBuONa), and the exo-content at the equilibrium was ca. 60%. The hydrolyses of endo-rich MNBC (endo/exo = 80/20) under various conditions were carried out. The exo selectivity for resulting NBCA was improved when the hydrolysis was conducted with equimolar water at room temperature in the presence of the stronger base (tBuONa) (endo/exo: 18/82). Whereas the use of excess amount of water led to rapid and non-selective hydrolysis affording high endo content of the product. The plausible reaction mechanism involving rapid equilibrium of thermodynamic isomerization and kinetically preferred hydrolysis of exo ester is proposed.

Formation of Myosin-Photochromic ATP Analogue-Phosphate Analogue Ternary Complexes that Transit Reversibly Among Different ATPase Transient States by Light Irradiation

Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis- and trans-forms in response to ultraviolet (UV) and visible (VIS) light irradiation, respectively. Previously, we have cross-linked reactive cysteine SH1 and SH2 of skeletal muscle myosin subfragment-1(S1) with the sulfhydryl-reactive bifunctional azobenzene derivative, azobenzene-dimaleimide (ABDM) and succeeded to induce lever arm swinging reversibly by photo-irradiation. The results suggested that it might be possible to control or drive motor proteins by light irradiation. In the present study, we have employed photochromic ATP analogues that change its structure reversibly by light irradiation in order to photo-regulate function of myosin. Phenylazobenzoyl-iminoethyl-Tri-Phosphate (PABITP) have been designed and synthesized. PABITP was hydrolyzed by skeletal muscle myosin subfragment-1 (S1) and induced dissociation of acto-myosin. Formations of S1-PABIDP-Pi analogues (AlF4-, BeFn) ternary complexes that may mimic different transient states in ATPase cycle were examined. The formations of the ternary complexes were monitored by competing with fluorescent ADP analogue Mant-ADP. Trans PABITP formed the ternary complexes with AlF4- and BeFn. Interestingly, formation of S1-PABIDP-BeFn was saturated at 50%. It is known that there are two species of BeFn, which have different number of fluorine. One of them forms M∗•ATP state and the other forms M∗∗•ADP•Pi state. As S1-ADP-AlF4- ternary complex mimic only M∗∗•ADP•Pi state. Therefore, trans PABIDP forms predominantly ternary complexes mimics M∗∗•ADP•Pi with BeFn and AlF4-. The photo-isomerization to cis PABIDP by UV irradiation decreased the stability of the ternary complexes. The results suggest that trans PABITP may mimic transient state of M∗∗ADP-Pi state and isomerization to cis PABITP induced transition to other state in ATPase cycle.

Photochemistry of 2-diphenylmethoxyacetophenone. Direct detection of a long-lived enol from a Norrish Type II photoreaction

Photolysis of 2-diphenylmethoxyacetophenone (1) in deaerated acetonitrile at room temperature is expected to proceed via Norrish Type II reaction to generate benzophenone (2) and acetophenone (4) as the main isolated photoproducts. Although the acetophenone enol intermediate (3) is generally considered a putative intermediate that is a precursor of acetophenone, there are few reports of the direct spectroscopic detection of 3 when a Type II reaction is conducted in solution at room temperature. Presumably, rapid enol-ketone isomerisation under the reaction conditions causes 3 to have a very short lifetime that lowers its concentration below limits detectable by standard organic spectroscopic methods such as (1)H NMR. We report that 3 is readily observed at room temperature by (1)H NMR spectroscopy and showed a remarkably long lifetime of almost 1 h under our reaction conditions. It was found that the acetophenone enols from the classic Norrish Type II reactions of valerophenone and butyrophenone could also be readily detected by (1)H NMR in acetonitrile at room temperature, but that their lifetimes were similar (tens of minutes) from both precursors, but were considerably shorter than that of 1. The reason for the differences in the lifetime of the acetophenone enol is probably due to small amounts of adventitious catalysts that arise during the photolysis.